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Demystified ...: Monoclonal antibodies

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Abstract

Monoclonal antibodies are essential tools for many molecular immunology investigations. In particular, when used in combination with techniques such as epitope mapping and molecular modelling, monoclonal antibodies enable the antigenic profiling and visualisation of macromolecular surfaces. In addition, monoclonal antibodies have become key components in a vast array of clinical laboratory diagnostic tests. Their wide application in detecting and identifying serum analytes, cell markers, and pathogenic agents has largely arisen through the exquisite specificity of these unique reagents. Furthermore, the continuous culture of hybridoma cells that produce these antibodies offers the potential of an unlimited supply of reagent. In essence, when compared with the rather limited supply of polyclonal antibody reagents, the feature of a continuous supply enables the standardisation of both the reagent and the assay technique. Clearly, polyclonal and monoclonal antibodies have their advantages and disadvantages in terms of generation, cost, and overall applications. Ultimately, monoclonal antibodies are only produced when necessary because their production is time consuming and frustrating, although greatly rewarding (at least most of the time!). This is especially apparent when a monoclonal antibody can be applied successfully in a routine pathology laboratory or can aid in the clinical diagnosis and treatment of patients. In this article, the generation and application of monoclonal antibodies are demystified to enable greater understanding and hopefully formulate novel ideas for clinicians and scientists alike.

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... Reference virus and antigen preparation Sabin oral vaccine strain stock as sabin I (LSc, 2ab) obtained from the Razi Vaccine and Serum Research Institute was propagated in MRC-5 (ATCC-CCL-171) in the absence of fetal bovine serum (FBS) for mice inoculation (12). After elimination of the monolayers, supernatant was clarified by ultracentrifugation (Hettich, East Westphalia-Lippe, Germany) and concentrated (25X) with PEG 6000 (6,13). Infection titer of the nonconcentrated and concentrated supernatant was calculated as 10 7.10 CCID 50 /ml and 10 8.67 CCID 50 /ml, respectively in Hela cell line (ATCC-CCL-2) by a validated method (14). ...
... Female BALB/c mice were inoculated subcutaneously with mixed and homogenized concentrated virus and an equal amount of complete Freund‫׳‬s adjuvant, followed by booster injections with an equal amount of incomplete Freund‫׳‬s adjuvant three and six weeks after the first injection. Three days before fusion, the same viral antigen was diluted in PBS (1:3, at pH 7.4) without adjuvant, and then injected directly into vein of the mouse with the highest antibody titer (13). ...
... The fused cells were distributed in a 96-well plate (1.4×10 5 /well) coated by feeder layer (macrophage cells collected from peritoneal fluids, following intraperitoneal inoculation of sodium glycolate) 72 hours before fusion. When the first hybridoma colon was detected, the media were replaced with hypoxanthine thymidine instead of hypoxanthine-aminopterinthymidine (Biotech Resources,1995-7) (13). Hybridoma screening and cloning Seven to 14 days after the fusion, the wells were observed microscopically for the presence of hybridoma colons. ...
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Background and Objectives: Poliomyelitis remains a major public health problem in developing countries, which signify the need for extensive diagnostic and prevention research. The aim of the present study was to design monoclonal antibodies (MAbs) against poliovirus type I with biomedical, diagnostic and therapeutic applications. Methods: B-cells were isolated from a mouse challenged with polio antigen injection. The B-cell were fused with myeloma tumor cells. After evaluation and screening of approximately 250 hybridoma colons by ELISA, 35 colons with the highest antibody titer and no cross-reactivity were selected and subsequently cloned by limiting dilution. Finally, three colons capable of secreting MAbs against epitopes of poliovirus type I were used for MAb production. Next, the MAbs were characterized by antibody assays, isotyping, epitope analysis (western blot), cross-reactivity test, stability test, sterility test and mycoplasma test. Results: The results indicated that the MAbs were of IgG1 kappa chain, had good stability and no cross-reactivity. In western blot, a band at 26 kDa which is associated to VP3 neutralization protein was observed. Conclusion: These serotype-specific MAbs can be potentially used for identification of type I poliovirus for research, diagnostic and prevention purposes. Keywords: Monoclonal antibody, Hybridoma, Poliomyelitis, Poliovirus.
... Affinity maturation occurs during this process -the genetic code for the variable region of the B cell rearranges, and antibodies that are unique for the antigen that originally contacted the B cell are subsequently produced (Abbas et al., 2012). The complementary determining regions of an antibody interact with an antigen at the site known as an epitope, or determinant, which usually consist of 6-8 amino acids (Nelson et al., 2000). The strength at which a single antibody binds to an epitope is defined as the affinity, and the overall strength of the antibody-antigen interaction is defined as the avidity (Abbas et al., 2012). ...
... While polyclonal antibodies are specific for multiple epitopes of an antigen, monoclonal antibodies have a unique specificity and have revolutionized antibody-based therapeutics, but production requires a more labor intensive and timeconsuming in vitro process for development. The basis and methods of monoclonal antibody production have been extensively described elsewhere (Nelson et al., 2000;Skinner et al., 2013;Strockbine et al., 1985), and will only be briefly discussed here. ...
... The primary method of monoclonal antibody production is via hybridoma technology, which involves the production of an immortal plasma cell in vitro (Byrne et al., 2009;Nelson et al., 2000). Plasma cells from the bone marrow, primary lymph nodes and spleen of an immunized host can be recovered and fused with immortal myeloma cells, but murine-derived splenocytes are used for the vast majority of hybridoma fusions. ...
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Certain pathogenic Escherichia coli known as Shiga toxin (Stx)-producing E. coli (STEC) are a public health threat to the consumer, and are problematic for the food industry. Food products containing STEC are deemed unfit for human consumption, and STEC illnesses can cause hemolytic uremic syndrome (HUS), a disease affecting the kidneys in susceptible individuals. Optimizing detection methods in foods have been focused on more prompt and accurate analysis. This review addresses the role and applications of immuno-based assays for STEC detection in food systems. Immunoassay antibody capture systems and flow cytometry platforms have been implemented into several food-based detection systems. By applying antibodies that will interact with target microorganisms, immunoassays can be used to directly detect and quantify pathogens. Immuno-based protocols could potentially be further implemented into the food industry, limit the duration of the detection process and increase accuracy.
... Monoklonal antikorlar tek bir B hücre klonu tarafından oluşturulan, hedefledikleri yapılara bağlanarak hedefin bloklanmasını veya çeşitli mekanizmalarla elimine edilmesini sağlayan etkili moleküllerdir (1,2). Hedef yapılara karşı yüksek spesifisite ve afinite göstermeleri dolayısıyla etkinin sağlıklı hücreler yerine sadece hedef alınan hastalıklı hücrelerde görülmesini sağlarlar, bu sayede düşük yan etki gösterirler. ...
... Hedef yapılara karşı yüksek spesifisite ve afinite göstermeleri dolayısıyla etkinin sağlıklı hücreler yerine sadece hedef alınan hastalıklı hücrelerde görülmesini sağlarlar, bu sayede düşük yan etki gösterirler. Bu sebeple kanser gibi birçok hastalıkta avantaj sağlamaktadırlar ve günümüzde tanı, tedavi, biyogörüntüleme gibi çeşitli amaçlarla kullanılabilmektedirler (1,3). Günümüzde monoklonal etken maddeli ilaçlar çeşitli hastalıkların tedavisinde yaygın şekilde kullanılmaktadır (4). ...
... Sera were collected 7 days after the final challenge and stored at -20°C until use. For mAb generation, mice at the Leadgene Biomedical, Inc., facility were immunized with S1-RBD expressed in E. coli according to the hybridoma technique as previously described (26,27). In brief, the splenocytes were fused with mouse myeloma FO cells and sele cted by mo difi ed selec ted-me di um c ontain ing hypoxanthine-aminopterin-thymidine (Thermo Fisher Scientific, MA, USA), 15% fetal bovine sera (FBS) (HyClone, Logan, UT) and 2.5% HyBoost (Leadgene Biomedical Inc., Taiwan). ...
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus responsible for the ongoing COVID-19 pandemic. SARS-CoV-2 binds to the human cell receptor angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain in the S1 subunit of the spike protein (S1-RBD). The serum levels of autoantibodies against ACE2 are significantly higher in patients with COVID-19 than in controls and are associated with disease severity. However, the mechanisms through which these anti-ACE2 antibodies are induced during SARS-CoV-2 infection are unclear. In this study, we confirmed the increase in antibodies against ACE2 in patients with COVID-19 and found a positive correlation between the amounts of antibodies against ACE2 and S1-RBD. Moreover, antibody binding to ACE2 was significantly decreased in the sera of some COVID-19 patients after preadsorption of the sera with S1-RBD, which indicated that antibodies against S1-RBD can cross-react with ACE2. To confirm this possibility, two monoclonal antibodies (mAbs 127 and 150) which could bind to both S1-RBD and ACE2 were isolated from S1-RBD-immunized mice. Measurement of the binding affinities by Biacore showed these two mAbs bind to ACE2 much weaker than binding to S1-RBD. Epitope mapping using synthetic overlapping peptides and hydrogen deuterium exchange mass spectrometry (HDX-MS) revealed that the amino acid residues P463, F464, E465, R466, D467 and E471 of S1-RBD are critical for the recognition by mAbs 127 and 150. In addition, Western blotting analysis showed that these mAbs could recognize ACE2 only in native but not denatured form, indicating the ACE2 epitopes recognized by these mAbs were conformation-dependent. The protein–protein interaction between ACE2 and the higher affinity mAb 127 was analyzed by HDX-MS and visualized by negative-stain transmission electron microscopy imaging combined with antigen-antibody docking. Together, our results suggest that ACE2-cross-reactive anti-S1-RBD antibodies can be induced during SARS-CoV-2 infection due to potential antigenic cross-reactivity between S1-RBD and its receptor ACE2.
... The hybridoma technique has the potential to provide an inexhaustible supply of quality monospecific monoclonal antibodies (MAbs) 23 . MAbs can serve as useful diagnostic tools and act as probes for cellular and macromolecular investigations 24 . In the present study, the VP60 proteins of RHDV1 and RHDV2 were used as immunogens to prepare RHDV type-specific MAbs by hybridoma fusion. ...
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In 2010, a new rabbit hemorrhagic disease virus (RHDV) variant, designated RHDV2, was identified for the first time in Italy. Studies have shown that RHDV2 differs from RHDV1 (traditional RHDV) in terms of its antigenic profile and genetic characteristics. The VP60 protein of RHDV is a structural protein that plays important roles in viral replication, assembly, and immunogenicity. In this study, we immunized BALB/c mice with recombinant VP60 proteins from different RHDV subtypes. After three rounds of subcloning, type-specific positive hybridoma clones of RHDV1 and RHDV2 were further identified by an enzyme-linked immunosorbent assay, Western blotting, and an indirect immunofluorescence assay. Finally, three monoclonal antibodies (MAbs) (1D6, 1H2, and 3F2) that only recognize RHDV1, and four MAbs (1G2, 2C1, 3B7, and 5D6) that only recognize RHDV2 were identified. The epitopes recognized by these MAbs were mapped by Western blotting. Sequence analysis showed that the epitope sequences recognized by 1D6, 1H2, and 3F2 are highly conserved (98%) among RHDV1 strains, whereas the epitope sequences recognized by 1G2, 2C1, 3B7, and 5D6 are 100% conserved among RHDV2 strains. The high conservation of the epitope sequence showed that the screened MAbs were type-specific, and that they could distinguish different RHDV subtypes.
... 6-week-old BALB/C mice were immunized with 50 μg of the purified GST-fiber four times every 7 days. At day 3 following the fourth immunization, the spleen cells from one immunized mouse were fused with SP2/0 cells (Roche, Mannheim, Germany), as previously described (Nelson et al. 2000). The hybridoma cells secreting antibodies against the fiber protein of FAdV-8 were screened through immunofluorescence assay (IFA) using the cells infected with FAdV-8. ...
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In recent years, hepatitis-hydropericardium syndrome (HHS) and inclusion body hepatitis (IBH) caused by fowl adenovirus (FAdV) infection have resulted in significant economic losses to the poultry industry worldwide. Epidemiological analysis revealed that serotype FAdV-8 is one of the major pathogenic FAdVs currently prevalent in domestic flocks. Although the fiber protein of FAdV plays vital roles in viral infection and pathogenesis, the B cell epitope in the fiber protein is less known. In this study, two monoclonal antibodies (mAbs) specific to fiber protein of FAdV-8, designated as 4D9 and 5F10, were prepared. Although the mAb 4D9 and 5F10 could not neutralize FAdV-8 infection, 4D9 and 5F10 showed good activities of indirect immunofluorescence, western blot and immunoprecipitation. Epitope analysis revealed that mAb 5F10 recognized 187-219aa in the fiber whereas mAb 4D9 recognized 113-149aa in the fiber. Sequence analysis showed that the epitope recognized by mAb 5F10 was conserve across serotypes FAdV-7, 8a and 8b whereas that for mAb 4D9 was only conserve in FAdV-8b. The generation of mAbs specific to fiber of FAdV-8 and the identification of the novel B cell epitopes here lay the foundation for further studying the antigenicity of the fiber and developing specific diagnosis for FAdV-8.
... By using culture supernatant or a purified immunoglobulin preparation, further analysis of a possible antibody producing hybridoma are often made in terms of reactivity, specificity, and cross-reactivity [10]. ...
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Coronavirus disease 2019 (COVID-19) is a new pandemic disease. Here, we would like to attempt to explain how to prepare the vaccine for infected Covid-19 patients. Take the blood (serum) sample from recovered patients of the covid-19 and then isolate the antibody from the serum and purify with the tactic of IEC based purification and separation method. After that, identify the antibody which was helped to recover from Covid-19 by various methods. Hybridoma technology was used for supplying the multiple antibodies for vaccination. These vaccines are going to be used for mankind.
... In our current study, we newly produced nine different mAbs that recognized HPeV3-VP0 as an antigen. We then performed epitope mapping of our generated mAbs, as identification of the epitope is a key step in the characterization of monoclonal antibodies [34]. Based on the epitope analysis, the mAbs were able to recognize three different areas of HPeV3-VP0 and specify 12-14 aa length epitopes within the HPeV3-VP0. ...
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Human parechovirus type 3 (HPeV3) is an etiologic agent of respiratory diseases, meningitis, and sepsis-like illness in both infants and adults. Monoclonal antibodies (mAbs) can be a promising diagnostic tool for antigenic diseases such as virus infection, as they offer a high specificity toward a specific viral antigen. However, to date, there is no specific mAb available for the diagnosis of HPeV3 infection. In this study, we developed and characterized mAbs specific for HPeV3 capsid protein VP0. We used cell-free, wheat germ-synthesized viral VP0 protein for immunizing BALB/c mice to generate hybridomas. From the resultant hybridoma clones, we selected nine clones producing mAbs reactive to the HPeV3-VP0 antigen, based on enzyme-linked immunosorbent assay (ELISA). Epitope mapping showed that these mAbs recognized three distinct domains in HPeV3 VP0. Six mAbs recognized HPeV3 specifically and the other three mAbs showed cross-reactivity with other HPeVs. Using the HPeV3-specific mAbs, we then developed an ELISA for viral antigen detection that could be reliably used for laboratory diagnosis of HPeV3. This ELISA system exhibited no cross-reactivity with other related viruses. Our newly developed mAbs would, thus, provide a useful set of tools for future research and ensure HPeV3-specific diagnosis.
... Although the monoclonal antibody has excellent specificity and low background, it had weaker Ag-Ab conjugation than did the polyclonal antibodies. 27 As a result, we tried to use the two antibodies together as the coating antibody in this experiment to improve the linear range and the sensitivity. This proved successful, and the PL intensity increased with the PCT concentration in a linear manner (Figure 2), which indicated the advantages of both the polyclonal antibody and monoclonal antibody. ...
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In this study, we used a CdSe/ZnS core/shell quantum dot (QD) as a fluorescent probe and developed a quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) to quantitatively determine procalcitonin (PCT) levels in samples. The QD-antibody probe had a high fluorescent intensity and excellent stability, which met the needs of commercial fluorescent probe materials. Due to the excellent properties of clinical testing for PCT, this QD-FLISA method showed tremendous potential for use in in vitro diagnostic (IVD) kits.
... A type of immunotherapy called mAbs have gained much attention in the pharmaceutical industry in recent times [50]. An mAb is any antibody which has novel specificity and is derived from a single-cell B clone [51]. These therapeutic antibodies are primarily produced in mammalian host cell lines such as the NS0 murine myeloma cells, PER.C6 ® human cells and Chinese hamster ovary (CHO) cells. ...
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The new era of cellular immunotherapies has provided state-of-the-art and efficient strategies for the prevention and treatment of cancer and infectious diseases. Cellular immunotherapies are at the forefront of innovative medical care, including adoptive T cell therapies, cancer vaccines, NK cell therapies, and immune checkpoint inhibitors. The focus of this review is on cellular immunotherapies and their application in the lung, as respiratory diseases remain one of the main causes of death worldwide. The ongoing global pandemic has shed a new light on respiratory viruses, with a key area of concern being how to combat and control their infections. The focus of cellular immunotherapies has largely been on treating cancer and has had major successes in the past few years. However, recent preclinical and clinical studies using these immunotherapies for respiratory viral infections demonstrate promising potential. Therefore, in this review we explore the use of multiple cellular immunotherapies in treating viral respiratory infections, along with investigating several routes of administration with an emphasis on inhaled immunotherapies.
... Monoclonal antibodies (mAbs) are antibodies with single specificity generated from immunization of plasma B cells in vitro (Nelson et al., 2000). Due to their high selectivity and potency, mAbs can significantly improve therapeutic efficiency and reduce toxicity (Cui et al., 2017;Imai & Takaoka, 2006). ...
Article
Antibody disulfide bond reduction has been a challenging issue in monoclonal antibody manufacturing. It could lead to decrease of product purity and failure to meet targeted product profile and/or specifications. More importantly, disulfide bond reduction could also impact drug safety and efficacy. Scientists across industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategies and developing novel approaches to maintain high product quality. Additionally, in recent years as more complex molecules (such as bispecific and trispecific antibodies) emerge, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative challenging issue. Given the disulfide reduction challenges that biotech industry is facing, in this review, we provide a comprehensive scientific summary of the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and its potential remediated recovery based on published papers. First, this paper intends to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discusses disulfide bond reduction impact on product stability, associated analytical methods for disulfide bond reduction detection and characterization, process control strategies as well as their manufacturing implementation. In addition, brief perspectives on the development of future mitigation strategies are also reviewed, including platform alignment, mitigation strategy application for the emerging new modalities such as bispecific and trispecific antibodies as well as using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from the published papers. This article is protected by copyright. All rights reserved.
... There are some disadvantages associated with antibody therapy including high cost and the long generation process [206]. Overall, these studies show potential for the use of antibodies as therapeutics, but further research is needed in this area, particularly to study the risks and the effects associated with using these antibodies in humans. ...
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Herpes stromal keratitis (HSK) is a disease that commonly affects the cornea and external eye and is caused by Herpes Simplex Virus type 1 (HSV-1). This virus infects approximately 66% of people worldwide; however, only a small portion of these people will develop symptoms in their lifetime. There is no cure or vaccine available for HSV-1; however, there are treatments available that aim to control the inflammation caused by the virus and prevent its recurrence. While these treatments are beneficial to those suffering with HSK, there is a need for more effective treatments to minimise the need for topical steroids, which can have harmful effects, and to prevent bouts of disease reactivation, which can lead to progressive corneal scarring and visual impairment. This review details the current understanding of HSV-1 infection and discusses potential novel treatment options including microRNAs, TLRs, mAbs, and aptamers.
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Ribosome display utilizes formation of the mRNA-ribosome-polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA-ribosome-polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.
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The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD4, AG10, BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG10 and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG10 and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG10 and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG10 and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG10 and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.
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In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.
Chapter
Antibody based drugs have not only advanced the prevention and treatment of a number of life threatening diseases including cancer and arthritis, but have also provided the thrust for the continued success of the biopharmaceutical and pharmaceutical industry. Currently at least seven therapeutic antibodies are listed among the top ten revenue generating pharmaceutical products. These antibodies are generally monoclonal in origin and include, chimerised, humanised and human antibody types. Recently, new classes of antibody drugs are emerging and are expected to be among the next generation of block buster drugs within the Industry. These new classes include antibody conjugates, bispecific antibodies and antibody fragments. In this chapter, we will discuss the methods used for the production of antibodies in both hybridoma and non-hybridoma cells lines. The chapter will also focus on the successful use of these antibodies as therapeutics in cancer and rheumatoid arthritis.
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Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has caused severe economic losses in China. Gp85 protein is the main envelope protein and the most variable structural protein of ALV-J. It is also involved in virus neutralization. In this study, a specific monoclonal antibody, 4A3, was produced against the ALV-J gp85 protein. Immunofluorescence assays showed that 4A3 could react with different strains of ALV-J, including the British prototype isolate HPRS103, the American strains, an early Chinese broiler isolate, and layer isolates. A linear epitope on the gp85 protein was identified using a series of partially overlapping fragments spanning the gp85-encoding gene and subjecting them to western blot analysis. The results indicated that (134)AEAELRDFI(142) was the minimal linear epitope that could be recognized by mAb 4A3. Enzyme-linked immunosorbent assay (ELISA) revealed that chicken anti-ALV-J sera and mouse anti-ALV-J gp85 sera could also recognize the minimal linear epitope. Alignment analysis of amino acid sequences indicated that the epitope was highly conserved among 34 ALV-J strains. Furthermore, the epitope was not conserved among subgroup A and B of avian leukosis virus (ALV). Taken together, the mAb and the identified epitope may provide valuable tools for the development of new diagnostic methods for ALV-J.
Article
Adjuvants are added to a vaccine to enhance the antibody response to the antigen. A number of different materials have been used as adjuvants. In this study, mice were immunized with ovalbumin plus one of three adjuvants: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), or alum. Control mice received no vaccine. FCA is generally considered the strongest adjuvant and alum the weakest, but use of FCA is often avoided due to concerns over negative effects on the animals. All mice that received an adjuvant/antigen vaccination produced high levels of antibodies to ovalbumin, whereas no control mice did. Levels of anti-ovalbumin antibodies were not significantly different between groups that received FCA, FIA, or alum. Of the three adjuvants tested, alum is the most cost-effective and possibly the safest for the animal. This, coupled with the results of this study, suggest that it is the most practical of the three tested adjuvants, at least in conjunction with ovalbumin as an antigen. However, different antigens may require different adjuvants, and a general recommendation would require further testing.
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Dengue is the most prevalent human arboviral disease. The morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. Brazil is a hotspot for dengue, but no serological diagnostic test has been produced using Brazilian dengue virus isolates. This study aims to improve the development of immunodiagnostic methods for dengue virus (DENV) detection through the production and characterization of 22 monoclonal antibodies (mAbs) against Brazilian isolates of DENV-1, -2 and -3. The mAbs include IgG2bκ, IgG2aκ and IgG1κ isotypes, and most were raised against the envelope or the pre-membrane proteins of DENV. When the antibodies were tested against the four DENV serotypes, different reactivity patterns were identified: group-specific, subcomplex specific (DENV-1, -3 and -4 and DENV-2 and -3) and dengue serotype-specific (DENV-2 or -3). Additionally, some mAbs cross-reacted with yellow fever virus (YFV), West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV). None of the mAbs recognized the alphavirus Venezuelan equine encephalitis virus (VEEV). Furthermore, mAbs D3 424/8G, D1 606/A12/B9 and D1 695/12C/2H were used to develop a capture enzyme-linked immunosorbent assay (ELISA) for anti-dengue IgM detection in sera from patients with acute dengue. To our knowledge, these are the first monoclonal antibodies raised against Brazilian DENV isolates, and they may be of special interest in the development of diagnostic assays, as well as for basic research.
Chapter
In many laboratories, immunohistochemistry (IHC) has become a routine supplement to the classic morphologic approach of investigational pathology. The specificity of the IHC test is largely dependent on the ability of the primary antibody to bind to epitopes on the target antigen without cross-reacting to epitopes on off-target antigens. The development of a new IHC test begins with the optimization of each of the steps in the analytical phase (phase 2) of the IHC test including: antigen retrieval, blocking nonspecific activities, and the binding and selection of primary antibody. This chapter discusses the use of some intermediate filaments (IFs) in IHC of neoplastic diseases. Cancer of unknown primary site (CUPS) includes all cancers in which the tissue or organ of origin cannot be determined by clinicopathologic analysis. Following the human oncology approach for IHC to identify CUPS, the main tumor categories are carcinoma, melanoma, leukocytic tumors (including histiocytic) and sarcoma.
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A recent outbreak of hepatitis-hydropericardium syndrome caused by serotype 4 fowl adenovirus (FAdV-4) has resulted in significant economic losses to the poultry industry worldwide. However, little is known about the molecular pathogenesis of FAdV-4. In this study, a novel monoclonal antibody (mAb) targeting the fiber-2 protein of FAdV-4 was generated, mAb 3C2. Indirect immunofluorescence assay showed that mAb 3C2 neither reacted with serotype 8 fowl adenovirus (FAdV-8) nor reacted with the fiber-1 protein of FAdV-4; it specifically reacted with the fiber-2 protein of FAdV-4. Notably, mAb 3C2 could efficiently immunoprecipitate the fiber-2 protein in chicken liver cells either infected with FAdV-4 or transfected with pcDNA3.1-Fiber2. Moreover, mAb 3C2 demonstrated marked neutralizing activity against FAdV-4 and could efficiently inhibit the infection of FAdV-4 in vitro. Using truncated fiber-2 constructs, the epitope recognized by mAb 3C2 was determined to be located between amino acids 416-448 at the C-terminus of fiber-2. Our data not only provide a foundation for the establishment of a rapid fiber-2 peptide-based diagnostic assay for FAdV-4 but also highlight the critical role of the fiber-2 protein in mediating infection by FAdV-4. Furthermore, the epitope recognized by 3C2 might serve as a novel target for the development of a vaccine targeting FAdV-4.
Article
Background: Colorectal cancer (CRC) accounts for the third most common cancer-related death in human. Wnt growth factors signaling have an important role in the CRCs’ development. Frizzled family receptor 7 (FZD7) belongs to the 10-member Frizzled (FZD) family of receptors and has an important role in triggering Wnt signaling. Antibody-therapy is an important class of modern medicine. Human single chain antibodies, which are composed of VH-Linker-VL are small antigen-binding units with unique properties, which overcome monoclonal antibodies and are applied for certain cancer immunotargeting approaches. Objectives: This study aimed at determining the anti-tumor properties of 2 anti-FZD7 scFvs phage antibodies. Methods: Two specific anti-FZD7 scFv antibodies were assessed for their anti-cancer effects. Flow cytometry was used to investigate the binding ability of scFvs to SW-480 colorectal cancer cell line, and SKBR3 cells, as the negative FZD7 cell line. Cell proliferation assay was performed to evaluate the growth inhibition effects of the different concentrations of anti-FZD7scFv phage antibodies (1000 to 4000 phage/cell). Mann-Whitney U test was used to compare percentages of cell growth between treated and untreated cells. Results: Fluorescent activated cell sorting (FACS) analysis revealed that scFv I and IIboundto 62.4% and 63.7% of SW-480 cells, respectively, whereas 6.4% and 8.3% bound to SKBR-3 cells, as the negative control. Results of MTT assays on SW-480 demonstrated growth inhibition of 53%, 63%, and 83% for scFv I (3000 phage/cell) after 24, 48, and 72 hours, respectively, while scFv II (4000 phage/cell) treatment showed growth inhibition of 33%, 47%, and 63% for the same period of time, respectively. No significant inhibitory effect for scFvs on the cell growth of SKBR-3 cells was observed (P < 0.05). Conclusions: Targeted therapy using antibodies has been considered as a new cancer immunotherapy in the recent years. In this study we applied 2 anti-FZD7 antibodies to assess their effect on colorectal cancer cells. Results demonstrated that both scFvs bound to the SW-480 colorectal cancer cell expressing FZD7 receptor. Significant cell growth inhibition for both anti-Fdz7 scFvs after 72 hours of incubation with the optimum amounts of the antibodies was detected. The stronger potential of scFv I for inhibiting the growth of cancer cells was also obtained. The use of scFvs against colorectal cancer cells offers a therapeutic potential for these antibodies in colorectal cancer immunotherapy.
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During the past two decades, avian leukosis virus (ALV) caused tremendous economic losses to poultry industry in China. ALV-K as a newly found subgroup in recent years, which made the control and eradication of ALV more difficult as they were originated from the recombination of different subgroups. To date, specific rapid detection methods refer to ALV-K are still missing. Gp85 is the main structural protein of the virus, which mediates the invasion of host cells by the virus and determinates the classification of subgroups. In this study, we prepared a monoclonal antibody (Mab) named Km3 against Gp85 of ALV-K. Immunofluorescence assay showed that Km3 specifically recognized the strains of ALV-K rather than the strains of ALV-A or ALV-J. To explain the subgroups specificity of Km3, the epitope cognized by the Mab was identified by Western blotting using 15 overlapping fragments spanning the Gp85. Finally, the peptide ¹²⁹AFGPRSIDTLSDWSRPQ¹⁴⁵ was identified as the minimal linear epitope recognized by Km3. Alignment of Gp85 from different subgroups showed that the epitope was highly conserved among ALV-K strains, which was quite different from that of the strains from ALV -A, -B and -J. In conclusion, the Mab Km3 may serve as a useful reagent for ALV-K detection and diagnosis in the future.
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The novel SARS-CoV-2 infection has ripped through international health systems and protocols causing unprecedented mortality, morbidity and global trade deficits amounting to billions. Various monoclonal antibodies have been proposed for use in the treatment of COVID-19 infections. One such drug is LY-CoV555 which in an ongoing phase two trial study conducted by Chen P et al, showed to have an elimination of 99.97% of the viral RNA. The monoclonal antibody 47D11 discovered by Wang et al, binds to SARS-CoV-2. The 47D11 has been reconfigured into a human IgG1 isotope. It has shown that the 47D11 mAb effectively neutralizes the SARS-COV-2 virus. The stance and development however for the treatment of COVID-19 with monoclonal antibodies has shifted from a monotherapy to a so-called monoclonal antibody “cocktail” therapy. REGN-COV2 is such a cocktail developed with the use of two monoclonal antibodies REGN10987 and REGN10933 which have subsequently been named Imdevimab and Casirivimab. REGN-COV2 is currently under study in four phase 2 and 3 trial studies. These studies are multicentric in nature and are being conducted to evaluate the drug’s efficacy, dosing and clinical use as compared to the placebo. The mechanism of action of such monoclonal antibodies is related chiefly to the inhibition of the virus’s ability to perform its invasion and multiplication within the human body. The severity coupled with the sheer novelty of the SARSCoV-2 virus demands the use of newer therapies to both decrease the mortality and morbidity in patients suffering from the infection. The use of a combination of monoclonal antibodies is thereby well established and evident to both decrease the viral infection load, but is also useful in disrupting the virus’s life cycle and thus decreases the replication and viral shedding. It is therefore poignant that a combination of monoclonal antibodies, a “cocktail” therapy is employed so as to attack the virus at its various stages and thus this multifaceted approach may enhance the patient’s prognosis.
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Ribosomal protein S3 (rpS3), a member of 40S small ribosomal subunit, is a multifunctional protein with various extra-ribosomal functions including DNA repair endonuclease activity and is secreted from cancer cells. Therefore, antibodies with high specificity against rpS3 protein could be useful cancer biomarkers. In this study, polyclonal antibody (pAb) and monoclonal antibodies (mAbs) were raised against rpS3 protein and epitope mapping was performed for each antibody; the amino acid residues of rpS3 were scanned from amino acid 185 to 243 through peptide scanning to reveal the epitopes of each mAb. Results showed that pAb R2 has an epitope from amino acid 203 to 230, mAb M7 has an epitope from amino acid 213 to 221, and mAb M8 has an epitope from amino acid 197 to 219. Taken together, novel mAbs and pAb against rpS3 were raised and mapped against rpS3 with different specific epitopes.
Chapter
Animals play a vital role in our lives as they provide milk, meat, and other by-products for daily consumption. At the same time, their health directly/indirectly affects human beings as we both share a common environment. The One Health concept makes it imperative that the animals should remain healthy as it will have an impact on our health as well as economy. Animal disease diagnosis, their health monitoring, prevention, and control of diseases are important from this aspect. Tools for rapid diagnosis of animal diseases are crucial for early diagnosis and imposing control measures. Molecular techniques such as, genome sequencing, restriction fragment length polymorphism (RFLP), DNA microarray, PCR, and real-time PCR are some of the rapid techniques, but they need expertise and sophisticated labs. Conventional methods such as isolation of the pathogen, serological techniques, are laborious and time taking process. Therefore, to overcome the said limitations of conventional and molecular tools, biosensors are the better alternatives as they are rapid and can be used as pen-side diagnostic tests. They can provide test results in a few minutes under field conditions. In this chapter, we give an elaborative description of various types of biosensors and their utility in animal disease diagnostics and health monitoring.
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Matrix metalloproteinase-9 (MMP-9) degrades collagen and other cellular matrix proteins. After acute ischemic stroke, increased MMP-9 levels are correlated with hemorrhage, lack of reperfusion and stroke severity. Nevertheless, definitive data that MMP-9 itself causes poor outcomes in ischemic stroke are limited. In a model of experimental ischemic stroke with reperfusion, we examined whether ischemia and recombinant tissue plasminogen activator (r-tPA) therapy affected MMP-9 expression, and we used specific inhibitors to test if MMP-9 affects brain injury and recovery. After stroke, MMP-9 expression increased significantly in the ischemic vs. non-ischemic hemisphere of the brain (p<0.001). MMP-9 expression in the ischemic, but not the non-ischemic hemisphere, was further increased by r-tPA treatment (p<0.001). To determine whether MMP-9 expression contributed to stroke outcomes after r-tPA treatment, we tested three different antibody MMP-9 inhibitors. When compared to treatment with r-tPA and saline, treatment with r-tPA and MMP-9 antibody inhibitors significantly reduced brain hemorrhage by 11.3 to 38.6-fold (p<0.01), brain swelling by 2.8 to 4.3-fold (p<0.001) and brain infarction by 2.5 to 3.9-fold (p<0.0001). Similarly, when compared to treatment with r-tPA and saline, treatment with r-tPA and an MMP-9 antibody inhibitor significantly improved neurobehavioral outcomes (p<0.001), decreased weight loss (p<0.001) and prolonged survival (p<0.01). In summary, both prolonged ischemia and r-tPA selectively enhanced MMP-9 expression in the ischemic hemisphere. When administered with r-tPA, specific MMP-9 inhibitors markedly reduced brain hemorrhage, swelling, infarction, disability and death, which suggests that blocking the deleterious effects of MMP-9 may improve outcomes after ischemic stroke.
Preprint
Disulfide bond reduction has been a challenging issue in antibody manufacturing, as it leads to reduced product purity, failed product specifications and more importantly, impacting drug safety and efficacy. Scientists across industry have been examining the root causes and developing mitigation strategies to address the challenge. In recent years, with the development of high-titer mammalian cell culture processes to meet the rapidly growing demand for antibody biopharmaceuticals, disulfide bond reduction has been observed more frequently. Thus, it is necessary to continue evolving the disulfide reduction mitigation strategy and development of novel approaches to achieve high product quality. Additionally, in recent years as more complex molecules emerge such as bispecific and trispecific antibodies, the molecular heterogeneity due to incomplete formation of the interchain disulfide bonds becomes a more imperative issue. Given the disulfide reduction challenges that our industry are facing, in this review, we provide a comprehensive contemporary scientific insight into the root cause analysis of disulfide reduction during process development of antibody therapeutics, mitigation strategies and recovery based on our expertise in commercial and clinical manufacturing of biologics. First, this paper intended to highlight different aspects of the root cause for disulfide reduction. Secondly, to provide a broader understanding of the disulfide bond reduction in downstream process, this paper discussed disulfide bond reduction impact to product stability and process performance, analytical methods for detection and characterization, process control strategies and their manufacturing implementation. In addition, brief perspectives on development of future mitigation strategies will also be reviewed, including platform alignment, mitigation strategy application for bi- and tri-specific antibodies and using machine learning to identify molecule susceptibility of disulfide bond reduction. The data in this review are originated from both the published papers and our internal development work.
Chapter
While cytopathology is useful, efficient, and relatively noninvasive, diagnostic conundrums can arise when there is confounding overlap between the cytomorphology of certain cell types. It can also be challenging to distinguish among infectious agents on routine microscopic examination. Ancillary diagnostics, particularly immunocytochemistry and cytochemical staining, can overcome the confines of relying on conventional cytologic features and allow definitive diagnoses. This chapter addresses the general principles, practical applications, and diagnostic algorithms relevant to these two separate but complementary approaches to special staining. Particular emphasis is placed on the advantages, challenges, and caveats of these adjunct techniques.
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Avian leukosis virus (ALV) is an avian oncogenic retrovirus that can induce virus-associated neoplasia and causes great economic loss in poultry industry. It is known that the capsid antigen p27 is the group-specific antigen that is highly conserved among all ALV subgroups, and is the most abundant immunogenic viral protein. In the present study, five overlapping fragments (GST- p27-F1/2, GST- p27-F2-1/2/3) of ALV-p27 were subjected to Western blotting analysis using a monoclonal antibody (5D3) against ALV-p27 to identify the epitope. The result showed that the epitope recognized by 5D3 is located within 173-240 amino acid of the ALV-p27 protein. For precise mapping of this epitope, a set of overlapping peptides were synthesized. Indirect enzyme linked immunosorbent assay (ELISA) revealed that ¹⁹³CFRQKSQPDI²⁰² motif was the minimal fragment recognized by 5D3, so this motif represented a linear B-cell epitope of ALV-p27. Homology analysis indicated that 5D3 defined epitope is highly conserved among ALV strains. The identified epitope might be useful in clinical applications and as a tool for further study of the structure and function of ALV-p27.
Chapter
Macromolecule drugs particularly antibody drugs are very powerful therapies developing rapidly in the recent 20 years, providing hopes for many patients diagnosed with “incurable” diseases in the past. They also provide more effective and less side effects for many afflicting diseases, and greatly improve the survival rate and life quality of patients. In the last two decades, the proportion of US Food and Drug Administration (FDA) approved macromolecules and antibody drugs are increasing quickly, especially after the discovery of immune checkpoints. To crown all, the 2017 Nobel prize in physiology or medicine was given to immunotherapy. In this chapter, we would like to summarize the current situation of macromolecule and antibody drugs, and what effort scientists and pharmaceutical industry have made to discover and manufacture better antibody drugs.
Article
Many biotechnological applications require the simultaneous binding of affinity reagents to non-overlapping target epitopes, the most prominent example being sandwich immunoassays. Typically, affinity pairs are identified via post facto functional analysis of clones that were not selected for complementarity. Here, we developed the Rapid Affinity Pair Identification via Directed Selection (RAPIDS) process, which enables the efficient identification of affinity reagents that function together as complementary pairs, from in vitro libraries of ~10⁹ variants. We used RAPIDS to develop highly-specific affinity pairs against biomarkers of tuberculosis, Zika virus, and sepsis. Without additional trial-and-error screening, these affinity pairs exhibited utility in multiple assay formats. The RAPIDS process applies selective pressure to hundreds of thousands of potential affinity pairs to efficiently identify complementary pairs that bind to separate epitopes without binding to one another or non-targets, yielding diagnostic assays that are sensitive and specific by design.
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The gold standard to detect bladder cancer, cystoscopy, is an invasive procedure requiring ambulant hospitalization; thus presenting an obstacle for routine diagnosis. We aim to develop a non-invasive detection method as an alternative that selectively captures shed cancer cells in the patient’s urine via surface-immobilized anti-EpCAM antibody. However, the urine sample storage conditions prior to analysis affects the subsequent cancer cell capture rates by the device. In this report, we investigate the capture rates of HT1197 and HT1376 bladder cancer cells in different media (fresh and aged urine as well as PBS) and storage temperatures prior to analysis (37°C and 4°C) as well as in the presence of adjuvants in the medias (free antibodies and cell debris). Capture efficiencies decreased in as little as 1 hour of the sample being incubated at 37°C in all media studied here. Furthermore, cell debris played a strong part in reducing the capture efficiency. From the data, we conclude that storing the sample at 4°C resulted in the best capture efficiency if storage of more than one hour is required; giving valuable insights for this sensor’s translation from laboratory to real-world applications.
Chapter
Identifying antigen–antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen–antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen–antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.
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Campylobacter jejuni is a leading cause of foodborne illness worldwide, mainly due to consumption and handling of contaminated raw chicken. Rapid detection methods for C. jejuni are vital for monitoring contamination levels in chicken products and reducing human Campylobacteriosis cases. The ‘gold standard’ culture-based method of Campylobacter detection takes 3–5 days and is too slow to permit effective intervention. Immuno-based methods are faster, but usually necessitate use of animals or hybridoma technology to produce antibodies; making them difficult and expensive to produce. Here, we report the generation and characterization of recombinant single-chain variable fragment (scFv) antibodies specific for C. jejuni cells, and evaluation of one scFv antibody for an immunomagnetic separation-quantitative PCR (IMS-qPCR) method to rapidly, sensitively, and specifically detect low numbers of C. jejuni. An scFv antibody phage-display library was constructed using spleen mRNA derived from a rabbit immunized with gamma-irradiated C. jejuni cells. This library was screened by surface biopanning against C. jejuni whole cells. Enriched clones were analyzed by enzyme-linked immunosorbent assay (ELISA). Two scFv antibodies that strongly and specifically recognized C. jejuni cell were expressed in Escherichia coli. Western blot analysis showed that one antibody, scFv80, was expressed as a soluble protein and retained its specific and strong binding to C. jejuni cells. This recombinant monoclonal scFv antibody was purified and used to covalently coat paramagnetic beads to be used for IMS-qPCR. The IMS-qPCR method was able to specifically and sensitively detect C. jejuni in mixed cultures within 3 h. Electronic supplementary material The online version of this article (10.1007/s00253-018-8949-x) contains supplementary material, which is available to authorized users.
Chapter
Many point-of-care diagnostic tests rely on a pair of monoclonal antibodies that bind to two distinct epitopes of a molecule of interest. This protocol describes the identification and generation of such affinity pairs based on an easily produced small protein scaffold rcSso7d which can substitute monoclonal antibodies. These strong binding variants are identified from a large yeast display library. The approach described can be significantly faster than antibody generation and epitope binning, yielding affinity pairs synthesized in common bacterial protein synthesis strains, enabling the rapid generation of novel diagnostic tools.
Article
Recombinant monoclonal antibodies (Ab's) have widespread application as research tools, diagnostic reagents and as biotherapeutics. Whilst studying the cellular molecular switch protein m-ras, a recombinant monoclonal antibody to m-ras was generated for use as a research tool. Antibody genes from a single rabbit B cell secreting IgG to an m-ras specific peptide sequence were expressed in mammalian cells, and monoclonal rabbit IgG binding was characterized by ELISA and peptide array blotting. Although the monoclonal Ab was selected for specificity to m-ras peptide, it also bound to both recombinant full-length m-ras and h-ras proteins. The cross-reactive binding of the monoclonal Ab to h-ras was defined by peptide array blot revealing that the Ab showed preference for peptide sequences containing multiple positively charged amino acid residues. These data reinforce the concept of antibody multispecificity through multiple interactions of the Ab paratope with diverse polypeptides. They also emphasize the importance of immunogen and Ab selection processes when generating recombinant monoclonal Ab's.
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Dengue is the most common mosquito-transmitted viral infection for which an improved vaccine is still needed. Although nonstructural protein-1 (NS1) immunization can protect mice against dengue infection, molecular mimicry between NS1 and host proteins makes NS1-based vaccines challenging to develop. Based on the epitope recognized by the anti-NS1 monoclonal Ab (mAb) 33D2 which recognizes a conserved NS1 wing domain (NS1-WD) region but not host proteins, we synthesized a modified NS1-WD peptide to immunize mice. We found that both mAb 33D2 and modified NS1-WD peptide immune sera could induce complement-dependent lysis of dengue-infected but not un-infected cells in vitro. Furthermore, either active immunization with the modified NS1-WD peptide or passive transfer of mAb 33D2 efficiently protected mice against all serotypes of dengue virus infection. More importantly, dengue patients with more antibodies recognized the modified NS1-WD peptide had less severe disease. Thus, the modified NS1-WD peptide is a promising dengue vaccine candidate.
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Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.
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Murine monoclonal antibody 2B8 specifically recognizes the CD20 phosphoprotein expressed on the surface of normal B lymphocytes and B- cell lymphomas. The light- and heavy-chain variable regions of 2B8 were cloned, after amplification by the polymerase chain reaction, into a cDNA expression vector that contained human IgG1 heavy chain and human kappa-light chain constant regions. High-level expression of chimeric- 2B8 antibody (C2B8) was obtained in Chinese hamster ovary cells. Purified C2B8 exhibited antigen binding affinity and human-tissue reactivity similar to the native murine antibody. In vitro studies showed the ability of C2B8 to bind human C1q, mediate complement- dependent cell lysis of human B-lymphoid cell lines, and lyse human target cells through antibody-dependent cellular cytotoxicity. Infusion of macaque cynomolgus monkeys with doses ranging from 1.6 mg/kg to 6.4 mg/kg resulted in greater than 98% depletion of peripheral blood (PB) B cells and 40% to 70% depletion of lymph node B cells. Recovery of PB B cells usually started at 2 weeks after treatment and required 60 to greater than 90 days to reach normal levels. As much as 95% depletion of B cells in peripheral lymph nodes and bone marrow was observed following weekly injections of 16.8 mg/kg antibody. No toxicity was observed in any of the animals. These results offer the possibility of using an “immunologically active” chimeric anti-CD20 antibody as an alternative approach in the treatment of B-cell lymphoma.
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Prognostic factors can be useful for making decisions about which patients should receive adjuvant therapy, and predictive factors can be used to predict response or lack of response to a particular therapy. We review the standard factors that are available today for primary breast cancer, and we describe some of the new, potential prognostic and predictive factors that are currently under investigation.
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Of 221 patients with breast cancer of known epidermal growth factor receptor (EGFR) and oestrogen receptor (ER) status, 99 had developed recurrences during the period of follow-up (range 3-60 months, median 24 months). Of these, 72 received endocrine therapy as first-line treatment for relapse. Immunohistochemical assessment of c-erbB-2 protein product expression was made using paraffin-embedded tumour tissue from 65 of these 72 patients. Including patients whose disease remained stable for more than 6 months with those showing an objective response (CR or PR for more than 3 months), only one (7%) of 14 c-erbB-2 positive tumours responded to endocrine manipulation compared with 19 (37%) of 51 c-erbB-2 negative tumours (P less than 0.05). Coexpression of c-erbB-2 reduced the response rate of ER positive patients from 48% to 20% and of ER negative cases from 27% to 0% (P less than 0.01). EGFR and c-erbB-2 protein appeared to have additive effects in reducing the likelihood of response, and none of eight patients with EGFR positive, c-erbB-2 positive tumours derived benefit from endocrine therapy. The results of this study suggest that c-erbB-2 protein overexpression, a marker of poor prognosis in breast cancer, is associated with a lack of response to endocrine therapy on relapse, and particularly in combination with EGFR may be useful in directing therapeutic choices.
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Three hundred and one primary breast cancers from patients with tumor infiltrated lymph nodes were analyzed for the presence of HER-2/neu oncoprotein by two procedures: Western blot (WB) and immunohistochemistry (IHC). Overexpression of this protein was found by WB in 16.6% of the tumors, and by IHC in 16.3%. Concordance between the two methods was found in 95% of tumors (286/301). In 7 cases we found HER-2/neu by IHC but not by WB, while the opposite was found in the remaining 8 patients. This discrepancy was found mainly in samples with HER-2/neu values just above the cut points and were therefore close to the sensitivity limits of the procedures used here. This study helps to define the parameters that should be considered to evaluate the immunostaining for HER-2/neu as positive (i.e., membrane staining, IHC score of 2 or more). The results obtained by both techniques were correlated with several currently used prognostic factors. Higher HER-2/neu protein expression was found in tumors lacking estrogen or progesterone receptors, in tumors with high S-phase fraction and in patients with more than 3 positive lymph nodes. In contrast, no relationship was found between overexpression of this protein and tumor size, ploidy, or age of the patient. Patients with elevated HER-2/neu expression showed a significantly worse overall survival by both methods, IHC (p = 0.05) and WB (p = 0.001). In conclusion, there is very high agreement between IHC and WB when measuring expression of HER-2/neu and both techniques showed prognostic significance.
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Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.
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Fourteen monoclonal antibodies (MAbs) of putative specificity for human IgG allotypes and isoallotypes were evaluated for reactivity and specificity in 8 different assay systems. The study showed that the MAbs tested could be classified into 1 of 4 groups: those exhibiting allotypic specificity regardless of the assay system, allotypic specificity dependent on the assay system, isoallotypic specificity, and those showing neither allotypic nor isoallotypic specificity. These observations were presumably dependent on antigen presentation, epitope integrity and/or antibody multispecificity. For the G1m(a), G1m(f), G1m(z), G3m(g) and G3m(u) specificities, MAbs have been produced which can be used for routine typing purposes in defined haemagglutination and enzyme-linked immunosorbent assay systems. MAbs are also available that show 'non-g' isoallotypic specificity.
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To assess the practical prognostic value of c-erbB2, we performed a study on 942 invasive ductal carcinomas treated with primary surgery between 1980 and 1986 in our center. We evaluated its expression by immunohistochemistry in paraffin-embedded tissue using a polyclonal antipeptide antibody. Of 942 tumors, 229 (24%) showed a positive membrane staining. We observed a significant association between c-erbB2 and Scarff-Bloom-Richardson grading (p < 0.0001) and a negative correlation between c-erbB2 and both estrogen and progesterone receptors (p < 0.0001). In our analysis, with respect to overall survival (OS), relapse-free survival (RFS), and metastasis-free survival (MFS), c-erbB2 was statistically significant (p < or = 0.0001) for the whole group and the node-positive subgroup. In multivariate analysis, c-erbB2 appeared to be an independent variable for RFS and MFS in the node-negative group. However, in our hands, c-erbB2 had a poor prognostic value in comparison with the classical prognostic variables such as histological grade, nodal status (N), hormonal receptor status (estrogen and progesterone receptors), and tumor size, and it did not supersede the classical parameters.
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Overexpression of erbB-2 proto-oncogene has been found in 20%-30% of human breast carcinomas and in most cases correlates with poor clinical prognosis. Using antisense oligonucleotides targeted to the 5' cap region of erbB-2RNA, we were able to inhibit erbB-2 protein expression, proliferation, and anchorage-independent growth of breast cancer cells up to 90%. These effects were sequence specific and restricted to cells expressing elevated level of erbB-2 protein. These support the feasibility of using antisense erbB-2 oligonucleotides to inhibit the progression of erbB-2-overexpressing breast cancer cells.
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A series of 107 lymph node-negative (LNN) breast cancers was stained immunohistochemically with a combination of p53 and c-erb B-2. The immunohistochemical results were semiquantitated using a previously described system by Allred et al. p53 immunopositive cases were further screened for DNA mutations by the polymerase chain reaction-single-strand conformation polymorphism method (PCR-SSCP). Three representative cases showing mobility shifts were directly sequenced. One hundred of 103 invasive carcinomas were of no special type (infiltrating ductal carcinomas not otherwise specified). The three special type carcinomas included a tubular carcinoma, a classic infiltrating lobular carcinoma, and a mucinous carcinoma. Twenty-six patients (25.2%) had grade I carcinomas, and 77 patients (75%) had grade 2 or 3 carcinomas. There were four cases composed predominantly of ductal carcinoma in situ (DCIS) with foci of microinvasion. Twenty-seven of 107 patients (25%) died of disease. All those who died had grade 2 or 3 tumors. Univariate analysis showed that p53 and c-erb B-2 positivity (score > 6) were associated with a decreased overall survival (OS) (P = .0012 and P = .010, respectively), and a decreased disease-free survival (DFS) (P = .0009 and P = .027, respectively). The multivariate model selected these two variables as the best predictors of both OS and DFS (all P = or < .01). These results suggest that semiquantitative immunohistochemical analysis of p53 and c-erb B-2 provides prognostic information in LNN disease.
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Mutations in certain genes that regulate the cell cycle, such as p16 and p53, are frequently found in human cancers. However, tumor-specific mutations are uncommon in genes encoding cyclin E and the CDK inhibitor p27Kip1, two cell-cycle regulators that are also thought to contribute to tumor progression. It is now known that levels of both cyclin E and p27 can be controlled by posttranscriptional mechanisms, indicating that expression of these proteins can be altered by means other than simply mutation of their respective genes. Thus, changes in p27 and cyclin E protein levels in tumors might be more common than previously anticipated and may be indicators of tumor behavior.
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The B cell epitope mapping of La/SSB was performed using 20mer synthetic peptides overlapping by eight amino acids covering the whole sequence of the protein. IgG, purified from sera of five patients with systemic lupus erythematosus (SLE) and four sera from patients with primary Sjögren's syndrome (pSS) were tested against the overlapping synthetic peptides. Peptides highly reactive with purified IgG were those spanning the regions 145-164, 289-308, 301-320 and 349-368 of the La protein. Determination of the minimum required length of the antigenic determinants disclosed the following epitopes: 147HKAFKGSI154, 291NGNLQLRNKEVT302, 301VTWEVLEGEVEKEALKKI318 and 349GSGKGKVQFQGKKTKF364. Predicted features and molecular similarities of the defined epitopes were investigated using protein databases. The La epitope 147HKAFKGSI154 presented 83.3% similarity with the 139HKGFKGVD146 region of human myelin basic protein (MBP) and 72% similarity with the fragment YKNFKGTI of human DNA topoisomerase II. Peptides corresponding to these sequences cross-reacted with anti-La/SSB antibodies. Sixty-three sera with anti-La/SSB antibodies from patients with pSS or SLE, 35 sera without anti-La/SSB antibodies from patients with SS or SLE and 41 sera from age/sex-matched healthy blood donors were tested against biotinylated synthetic epitope analogues in order to determine their sensitivity and specificity for the detection of anti-La/SSB antibodies. Anti-La/SSB were detected with various frequencies ranging from 20% to epitope 147HKAFKGSI154 to 100% to epitope 349GSGKGKVQGKKTKF364. The overall sensitivity and specificity using all assays with the synthetic peptides were found to be 93.6% and 85.6%, respectively. In conclusion, antibodies to La/SSB constitute a heterogeneous population, directed against different linear B cell epitopes of the molecule. The epitope 147HKAFKGSI154 presents molecular similarity with fragments of two other autoantigens, i.e. human MBP and DNA topoisomerase II. Finally, synthetic epitope analogues exhibit high sensitivity and specificity for the detection of anti-La/SSB antibodies.
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The prognostic significance of Bcl-2 protein expression and bcl-2 gene rearrangement in diffuse large cell lymphomas (DLCL) is controversial. Bcl-2 protein expression prevents apoptosis and may have an important role in clinical drug resistance. The presence of a bcl-2 gene rearrangement in de novo DLCL suggests a possible follicle center cell origin and perhaps a distinct clinical behavior more akin to low-grade non-Hodgkin's lymphoma (NHL). The purpose of this study was to determine the impact of Bcl-2 protein expression and bcl-2 gene rearrangement (mbr and mcr) on survival of a cohort of patients with DLCL who were uniformly evaluated and treated with effective chemotherapy. Patients included the original MACOP-B cohort (n = 121) and the initial 18 patients treated with the VACOP-B regimen (total = 139). All patients had advanced-stage disease, were 16 to 70 years old, and corresponded to Working Formulation categories F, G, or H. No patients had prior treatment, discordant lymphoma, or human immunodeficiency virus seropositivity. Paraffin sections from diagnostic biopsies were analyzed for bcl-2 gene rearrangement including mbr and mcr breakpoints by polymerase chain reaction and Bcl-2 protein expression by immunohistochemistry. With a median follow-up of 81 months, overall (OS), disease-free (DFS), and relapse-free survival (RFS) were measured to determine the prognostic significance of these parameters. Analyzable DNA was present in 118 of 139 (85%) cases, with 14 demonstrating a bcl-2 rearrangement (11 mbr, 3 mcr). All 14 of these bcl-2 gene rearrangement-positive cases were found in the 102 patients with a B-cell immunophenotype, but the presence of this rearrangement had no significant influence on survival. Bcl-2 protein expression was interpretable in 116 of 139 (83%) cases, with immunopositivity detected in 54 of 116 (47%). Using a cut-off of greater than 10% Bcl-2 immunopositive tumor cells for analysis, positive Bcl-2 protein expression was seen in 28 of 116 (24%) patients and the presence of this expression correlated with decreased 8-year OS (34% v 60%, P < .01), DFS (32% v 66%, P < .001), and RFS (25% v 59%, P < .001). Bcl-2 protein expression remained significant in multivariate analysis that included the clinical international prognostic index factors and immunophenotype (P < .02). In conclusion, although bcl-2 gene rearrangement status could not be shown to have an impact on outcome, Bcl-2 protein expression is a strong significant predictor of OS, DFS, and RFS in DLCLs.
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IDEC-C2B8 is a chimeric monoclonal antibody (MoAb) directed against the B-cell-specific antigen CD20 expressed on non-Hodgkin's lymphomas (NHL). The MoAb mediates complement and antibody-dependent cell-mediated cytotoxicity and has direct antiproliferative effects against malignant B-cell lines in vitro. Phase I trials of single doses up to 500 mg/m2 and 4 weekly doses of 375 mg/m2 showed clinical responses with no dose-limiting toxicity. We conducted a phase II, multicenter study evaluating four weekly infusions of 375 mg/m2 IDEC-C2B8 in patients with relapsed low-grade or follicular NHL (Working Formulation groups A-D). Patients were monitored for adverse events, antibody pharmacokinetics, and clinical response. Thirty-seven patients with a median age of 58 years (range, 29 to 81 years) were treated. All patients had relapsed after chemotherapy (median of 2 prior regimens) and 54% had failed aggressive chemotherapy. Infusional side effects (grade 1-2) consisting of mild fever, chills, respiratory symptoms, and occasionally hypotension were observed mostly with the initial antibody infusion and were rare with subsequent doses. Peripheral blood B-cell depletion occurred rapidly, with recovery beginning 6 months posttreatment. There were no significant changes in mean IgG levels and infections were not increased over what would be expected in this population. Clinical remissions were observed in 17 patients (3 complete remissions and 14 partial remissions), yielding an intent to treat response rate of 46%. The onset of these tumor responses was as soon as 1 month posttreatment and reached a maximum by 4 months posttreatment. In the 17 responders, the median time to progression was 10.2 months (5 patients exceeding 20 months). Likelihood of tumor response was associated with a follicular histology, with the ability to sustain a high serum level of antibody after the first infusion, and with a longer duration of remission to prior chemotherapy. One patient developed a detectable but not quantifiable immune response to the antibody that had no clinical significance. IDEC-C2B8 in a dose of 375 mg/m2 weekly for 4 weeks has antitumor activity in patients with relapsed low-grade or follicular NHL. Results with this brief, outpatient treatment compare favorably with results with standard chemotherapy, and IDEC-C2B8 has a better safety profile. Further studies evaluating IDEC-C2B8 in other types of lymphoma either alone or combined with chemotherapy are warranted.
Article
The mouse anti-human CD3 mAb OKT3 is a potent immunosuppressive agent used for the treatment of acute transplant rejection. OKT3 therapy is associated with acute toxicity resulting from in vivo T cell activation and systemic cytokine release, and a human anti-mouse Ab response. T cell activation is thought to be triggered by CD3 cross-linking mediated by the Abs bridging T cells and Fc receptor-bearing cells. Recent studies in a mouse model indicate that anti-mouse CD3 Abs with low affinity for Fc receptors can achieve immunosuppression without T cell activation, toxicity, or an anti-Ab response. To obtain an analogous Ab to improve the current anti-human CD3 therapy, a humanized Ab with low affinity for Fc receptors is needed. In this study, we introduced mutations into the upper CH2 region of IgG2 and expressed the altered Fc as chimeric OKT3 Abs. Compared with chimeric OKT3 IgG1, IgG2, IgG3, and IgG4, the IgG2 mutants were less mitogenic to T cells, and they did not induce the release of TNF-alpha, IFN-gamma, or IL-2. In parallel, we observed no functional interaction of the IgG2 mutant Abs with K562 cells, which express the IgG2-binding Fc receptor on their surface. Despite no measurable T cell activation, the mutant Abs could still modulate the CD3 complex. When coupled to a humanized anti-CD3, the IgG2 variant may provide a drug with less acute toxicity and immunogenicity, but may still retain potent immunosuppressive properties.
Article
The objective of the current study is to investigate the relationship between bcl-2 and p53 expression and prognosis in prostatic carcinoma. One hundred and forty-six needle core biopsy specimens obtained before any treatment were used for immunohistochemical detection of bcl-2 and p53 positivity. The relationship between these proteins was assessed by serial sections. Associations with Gleason score, clinical stage and patient survival were studied. Additionally, multivariate analysis by Cox proportional hazard model was used to determine the prognostic significance of these proteins. Bcl-2 positivity was found in 20% and p53 in 27% of 146 prostatic carcinomas. Both bcl-2 and p53 positivity were found only in 5%. They were expressed almost reciprocally in the tumors. P53 positivity correlated with high Gleason grade tumors. Bcl-2 positivity correlated with high T stage. Both bcl-2 and p53 positivity correlated with poor survival and short progression-free period. Multivariate analysis revealed that bcl-2 positivity was an independent prognostic indicator (p = 0.001). Bcl-2 and p53 were almost independently expressed in prostatic cancer. Both correlated with malignant phenotypes of prostatic cancer. The combined data from this staining further improved the ability to predict the patient prognosis.
Article
p27 is an inhibitor of the cell cycle with potential tumor suppressor function. Decreased levels of p27 protein expression have been correlated with poor prognosis in patients with breast and colorectal carcinomas. Although as many as a third of patients with clinically localized prostate cancer will have relapse after radical prostatectomy, predicting who will have recurrence remains enigmatic. We examined the ability of p27 protein levels to predict outcome in patients with clinically localized disease who underwent radical prostatectomy. p27 protein expression was evaluated in 86 patients with clinical stage T1-2 prostate cancer who were treated with radical prostatectomy. Archived paraffin embedded specimens were sectioned and immunostained with p27 antibody, and scored by 2 independent observers in a blinded fashion. The absence or presence of p27 protein was then correlated with biochemical relapse in univariate and multivariate analyses. In a multivariate analysis that included age, preoperative prostate specific antigen, Gleason score and pathological stage p27 was a strong independent predictor of disease-free survival (p = 0.0184, risk ratio 3.04), second only to pathological stage (p = 0.0001, risk ratio 6.73). Even more strikingly, multivariate analysis demonstrated that p27 was the strongest predictor of biochemical recurrence (p = 0.0081, risk ratio 4.99) among factors studied in patients with pathological T2a-T3b disease. Absent or low levels of p27 protein expression appear to be an adverse prognostic factor in patients with clinically organ confined disease treated by radical prostatectomy. This marker appears to be especially useful in those patients in whom surgery is believed to be potentially curative, that is patients with pathological T2-T3b disease. Patients with low or absent p27 protein expression may be candidates for novel adjuvant therapies.
Article
A high number of activated cytotoxic T lymphocytes (CTL) in Hodgkin's disease (HD) biopsy specimens is related to an unfavorable clinical outcome, suggesting that resistance of the Hodgkin and Reed-Sternberg (H-RS) cells to CTL-mediated killing is an important pathogenic factor in HD. bcl-2 and defective p53 are known to inhibit apoptosis induced either by CTLs or by therapy. The purpose of this study was to use immunohistochemical techniques to analyze whether differences in expression of these proteins in H-RS cells in primary biopsy specimens from 78 patients with HD were related to clinical outcome and to assess the number of CTLs in those cells. Cases with H-RS cells mostly staining positive for bcl-2 but negative for p53 had a poor prognosis (55% 5-yr survival). In the group of patients whose H-RS cells had low positivity for both p53 and bcl-2, the 5-year survival was 90%. p53 expression in a high percentage of H-RS cells was invariably related to a 100% 5-year survival, irrespective of bcl-2 expression. Biopsy specimens from patients with a fatal clinical outcome, in which few H-RS cells expressed p53 and many H-RS cells expressed bcl-2, contained relatively many activated CTLs. These data demonstrate that the combination of expression of the apoptosis-regulating proteins p53 and bcl-2 in the H-RS cells can be used as a prognostic marker for HD, and they indicate that resistance to apoptosis of H-RS cells is an important pathogenic mechanism. Our data also support the hypothesis that in patients with a poor prognosis, apoptosis-resistant H-RS cells might be selected for by the presence of many activated CTLs.
Article
Cyclin E and the cyclin-dependent kinase inhibitor p27 are two important regulators of the G1-S transition modulating the activity of cyclin-dependent kinases. Aberrations in the cell cycle control are often observed in tumors and might even be mandatory in tumor development. To investigate the importance of cell-cycle defects in malignant lymphomas we have characterized the expression of cyclin E and p27 in 105 newly diagnosed lymphomas using immunohistochemistry. A significant, inverse correlation between p27 and cyclin E expression was observed (rs = −.24, P = .02) and both proteins correlated with the S-phase fraction (rs = −.35, P < .001 andrs = .45, P < .001, respectively). The inverse relationship between p27 expression and proliferation was abrogated in some lymphomas, suggesting that p27 downregulation can represent a genuine aberration. Survival analysis was performed in 105 patients with a median observation time of 86 months. Low p27 and high cyclin E expression were significantly associated with a poor prognosis (P = .0001 and .03, respectively). In a multivariate Cox analysis, p27 expression, stage, serum lactate dehydrogenase level, grade, and age were independent prognostic factors, in contrast to S-phase fraction and cyclin E expression. This is the first report showing that p27 expression in malignant lymphomas has independent prognostic significance, which necessitates future studies regarding its more precise biological role in lymphoid tumorogenesis. © 1998 by The American Society of Hematology.
Article
Rituximab, a chimeric monoclonal antibody that binds specifically to the CD20 antigen, induced objective responses in 50% of patients with low-grade or follicular B-cell lymphoma. Because most nonfollicular B-cell lymphomas also express the CD20 antigen, we conducted a phase II study to evaluate the efficacy and tolerability of this new agent in patients with more aggressive types of lymphoma. Patients with diffuse large B-cell lymphoma (DLCL), mantle cell lymphoma (MCL), or other intermediate- or high-grade B-cell lymphomas according to the Working Formulation were included in this prospective randomized phase II study if they were in first or second relapse, if they were refractory to initial therapy, if they progressed after a partial response to initial therapy, or if they were elderly (age >60 years) and not previously treated. The patients received 8 weekly infusions of rituximab at the dose of 375 mg/m2 in arm A or one infusion of 375 mg/m2 followed by 7 weekly infusions of 500 mg/m2 in arm B. Patients were evaluated 2 months after the last rituximab infusion. Fifty-four patients were randomized from 9 centers in Europe and Australia (28 in arm A and 26 in arm B). A total of 5 complete responses (CR) and 12 partial responses (PR) were observed among the 54 enrolled patients, with no difference between the two doses. In an intent-to-treat analysis, the CR rate was 9% (CI95%, 3% to 20%) and the PR rate was 22% (CI95%, 12% to 36%), for an overall response rate of 31% (CI95%, 20% to 46%