Journal of Immunoassay and Immunochemistry (J IMMUNOASS IMMUNOCH)

Publications in this journal

• Article: Immunoassay of modified forms of human low density lipoprotein in isolated circulating immune complexes.

  Virella G, Colglazier J, Chassereau C, Hunt KJ, Baker NL, Lopes-Virella MF
  Journal of Immunoassay and Immunochemistry 01/2013; 34(1):1788-1791.
• Article: DEVELOPMENT AND EVALUATION OF PORCINE CYSTICERCOSIS QUICKELISA™ IN TRITURUS® EIA ANALYZER

  Sukwan Handali, Sowmya Pattabhi, Yeuk-Mui Lee, Maria Silva-Ibanez, Victor A. Kovalenko, Andrew E. Levin, Armando E. Gonzalez, Jacquelin M. Roberts, Hector H. Garcia, Robert H. Gilman, Kathy Hancock, Victor C. W. Tsang
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  ABSTRACT: We evaluated three diagnostic antigens (recombinant GP50, recombinant T24H, and synthetic Ts18var1) for cysticercosis and found that all three performed well in detecting cysticercosis in humans and pigs in several assay formats. These antigens were adapted to a new antibody detection format (QuickELISA). With one single incubation step which involves all reactants except the enzyme substrate, the QuickELISA is particularly suited for automation. We formatted the QuickELISA for the Triturus EIA analyzer for testing large numbers of samples. We found that in QuickELISA formats rGP50 and rT24H have better sensitivity and specificity than sTs18var1 for detecting porcine cysticercosis.
  Journal of Immunoassay and Immunochemistry 01/2010; 31(1):60-70.
• Article: Antigen heterologous enzyme linked immunosorbent assay for the measurement of estrone-3-glucuronide.

  S Nara, Tulsidas G Shrivastav, H Singh
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  ABSTRACT: We report a novel antigen heterologous enzyme linked immunosorbent assay for the direct estimation of estrone-3-glucuronide (E1-3-G) in diluted human urine. The differential behavior of antibody towards the labeled and unlabelled analyte in heterologous systems forms the basis of the present assay. Antiserum was raised in New Zealand white rabbits using estrone-3-glucuronide-bovine serum albumin (E1-3-G-BSA) as immunogen and nandrolone-17-HS (N-17-HS) coupled to horseradish peroxidase (HRP) with and without urea bridge ((N-17-HS-Urea-HRP/N-17-HS-HRP) as an enzyme labeled reagent. To the E1-3-G antibody coated wells, 50 microL standard or appropriately diluted (1:20) female urine samples were added along with nandrolone-17-HS-HRP/N-17-HS-Urea-HRP conjugate (100 microL) and were incubated at room temperature (RT) for 1 hour. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H(2)O(2)) as substrate. The cross-reaction of E1-3-G antiserum with C(18), C(19), C(21), C(27) steroids was less than 0.1% in both assays. Incorporation of urea bridge in the enzyme conjugate has decreased the effective displacement dose, i.e., ED(50) from 20 ng/ml to as low as 2 ng/mL. The sensitivity of the assay using N-17-HS-HRP and N-17-HS-Urea-HRP was 0.6 ng/mL and 0.4 ng/mL, respectively. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 4.7-9.0% and 5.1-6.4%, respectively. The estrone glucuronide level was also determined in female urine samples of 26 and 28 days cycle depicting a prominent peak corresponding to the preovulatory phase. The urinary E1-3-G values correlated well with those obtained by heat denaturation of urine samples r=0.94 (n=27).
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):80-90.
• Article: Label-free piezoelectric immunosensor for rapid assay of Escherichia coli.

  Miroslav Pohanka, Petr Skládal, Oto Pavlis
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  ABSTRACT: A piezoelectric sensor with immobilized polyclonal antibody was developed as a label-free assay for the model bacterium, Escherichia coli. The polyclonal antibody was prepared from mice BALB/c and covalently immobilized on the sensing gold electrode of the piezoelectric quartz crystal. The biosensor was able to detect E. coli in the range of 10(6)-10(9) CFU/mL; signal of the negative control was not statistically relevant in the selected range. Samples could be analyzed in four minutes and one measuring cycle including regeneration was completed within ten minutes. Repeatability of the developed method is discussed; the signal obtained from three different biosensors was 12.9+/-0.4 Hz for the sample containing 10(8) CFU/mL.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):70-9.
• Article: Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

  That T Ngo, Dyer Narinesingh
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  ABSTRACT: The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5-fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N-(2-hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol-O,O'-bis(2-aminoethyl)-N,N,N'N'-tetra acetate(EGTA). On an equal-molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):105-15.
• Article: Establishment of hybrid cell lines producing monoclonal antibodies to a synthetic peptide from the E1 region of the hepatitis C virus.

  Ashraf A Tabll, Samy B Khalil, Reem M El-Shenawy, Gamal Esmat, Amr Helmy, Abdel Fattah Attallah, Mostafa K El-Awady
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  ABSTRACT: We aimed at establishing hybridoma cells secreting monoclonal antibodies (mAbs) against E1 synthetic peptide of HCV. BALB/c mice were immunized with HCV E1-synthetic peptide (GHRMAWDMM) and its spleenocytes were fused with the P3NS1 myeloma cell line. Two highly reactive and specific mAbs (10C7 IgG2b mAb, and 10B2 IgG1 mAb) were generated. The target HCV E1 antigen was identified at approximately 38 kDa in serum of infected individuals. A newly developed ELISA detected the target antigen in 90% of sera from HCV RNA infected individuals with a specificity of 84%. So, the generated mAbs may provide promising probes for serodiagnosis of HCV infection.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):91-104.
• Article: Development of a lateral flow assay for rapid detection of bovine antibody to Anaplasma marginale.

  K Nielsen, W L Yu, L Kelly, R Bermudez, T Renteria, A Dajer, E Gutierrez, J Williams, J Algire, S Torioni de Eschaide
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  ABSTRACT: A rapid lateral flow assay for detection of bovine antibody to Anaplasma marginale was developed. The assay used a recombinant peptide of major surface protein 5 as the antigen and a monoclonal antibody specific for bovine IgG(1) conjugated with colloidal gold beads for detection. Serum and anticoagulated blood samples were obtained from cattle in an area where anaplasmosis was endemic. The samples were selected based on positive identification of the organism in blood smears. The unclotted blood samples were used for PCR determination of the presence of A. marginale while the sera were tested by a commercial competitive enzyme immunoassay (CELISA) and by the lateral flow assay (LFA). Similar samples, collected at a Canadian sales barn, were tested by the CELISA and LFA and 10% were tested by PCR for the presence of A. marginale nucleic acid. In addition, stored serum samples from a second endemic area were tested by CELISA and LFA. Of the 114 smear positive samples, all were positive by CELISA and LFA. All samples were also positive by PCR. Samples from Canadian sources (n=524) were negative in the CELISA but 11 sera gave false positive reactions in the LFA. All samples tested were PCR negative. Of 113 samples from herds with anaplasmosis, 53 were positive in the CELISA and 50 were LFA positive.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):10-8.
• Article: Novel tools for functional analysis of CD11c: activation-specific, activation-independent, and activating antibodies.

  Chanchal Sadhu, Lee Hendrickson, Ken O Dick, Tamara G Potter, Donald E Staunton
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  ABSTRACT: Functions and binding properties of four CD11c-specific mAbs are described here. The mAb 496B stimulated, while 496K inhibited ligand binding of CD11c. The stimulatory mAb, 496B, as well as the inhibitory mAbs BU15 and 496 K appear to act allosterically, as they do not bind the CD11c I domain. The mAb 3.9 bound preferentially to activated forms of CD11c and the binding was divalent cation dependent. CD11c binding to 3.9 recapitulates many of the integrin-ligand interactions. Our data suggest that 3.9 is a competitive antagonist, BU15 and 496K are allosteric antagonists, and 496B is an allosteric agonist of CD11c. These mAbs provide a set of tools to study the functions of the dendritic cell marker, CD11c.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):42-57.
• Article: Detection of Salmonella typhimurium in raw meats using in-house prepared monoclonal antibody coated magnetic beads and PCR assay of the fimA gene.

  Angela N Moreira, Fabricio R Conceição, Rita de Cássia S Conceição, Roberta J Ramos, José B Carvalhal, Odir A Dellagostin, José A G Aleixo
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  ABSTRACT: A method for detection of Salmonella Typhimurium in meat samples that uses in-house monoclonal antibody (MAb) coated magnetic beads for immunomagnetic separation (IMS) associated with PCR amplification of the gene fimA was developed. An internal amplification control (IAC) of the PCR reaction was constructed. The fimA PCR has shown 100% sensitivity and specificity when tested with various bacteria. The detection limit of the IMS-PCR method, using a post-enrichment in BHI broth for 6 h between IMS and PCR, was 1-10 CFU/mL. The method proved to be rapid (27 hrs), highly sensitive (1-10 CFU/25 g), and specific for detection of S. Typhimurium from experimentally contaminated pork and chicken meat samples.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):58-69.
• Article: Comparison between chicken and rabbit antibody based particle enhanced cystatin C reagents for immunoturbidimetry.

  Lars-Olof Hansson, Mats Flodin, Tom Nilsen, Karin Caldwell, Karin Fromell, Kathrin Sunde, Anders Larsson
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  ABSTRACT: We have compared three commercial particle enhanced cystatin C reagents. One of the reagents utilizes chicken antibodies and the other two reagents are rabbit antibody based. We show that the chicken antibody based reagent yields a higher delta absorbance when reacting with the antigen. IgY coupled to latex particles show a strong scatter response even at high antigen concentrations in contrast to the steep decline in scatter previously reported for IgY antibodies in solution. The reagent also showed a low CV for duplicate samples. Laying hens thus seems as an interesting source of antibodies for particle-enhanced immunoassays.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):1-9.
• Article: Establishment and characterization of monoclonal and polyclonal antibodies against human intestinal fatty acid-binding protein (I-FABP) using synthetic regional peptides and recombinant I-FABP.

  Satoshi Kajiura, Tetsuya Yashiki, Hiroyuki Funaoka, Yasuhiko Ohkaru, Ken Nishikura, Tatsuo Kanda, Yoichi Ajioka, Michihiro Igarashi, Katsuyoshi Hatakeyama, Hiroshi Fujii
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  ABSTRACT: We have succeeded in raising highly specific anti-human intestinal fatty acid-binding protein (I-FABP) monoclonal antibodies by immunizing animals with three synthetic regional peptides, i.e., the amino terminal (RP-1: N-acetylated 1-19-cysteine), middle portion (RP-2: cysteinyl-91-107) and carboxylic terminal (RP-3: cysteinyl-121-131) regions of human I-FABP, and the whole I-FABP molecule as antigens. We also raised a polyclonal antibody by immunizing with a recombinant (r) I-FABP. To ascertain the specificity of these antibodies for human I-FABP, the immunological reactivity of each was examined by a binding assay using rI-FABP, partially purified native I-FABP and related proteins such as liver-type (L)-FABP, heart-type (H)-FABP, as well as the regional peptides as reactants, and by Western blot analysis. In addition, the expression and distribution of I-FABP in the human gastrointestinal tract were investigated by an immunohistochemical technique using a carboxylic terminal region-specific monoclonal antibody, 8F9, and a polyclonal antibody, DN-R2. Our results indicated that both the monoclonal and polyclonal antibodies established in this study were highly specific for I-FABP, but not for L-FABP and H-FABP. Especially, the monoclonal antibodies raised against the regional peptides, showed regional specificity for the I-FABP molecule. Immunoreactivity of I-FABP was demonstrated in the mucosal epithelium of the jejunum and ileum by immunohistochemical staining, and the immunoreactivity was based on the presence of the whole I-FABP molecule but not the presence of any precursors or degradation products containing a carboxylic terminal fragment. It is concluded that some of these monoclonal and polyclonal antibodies, such as 8F9, 4205, and DN-R2, will be suitable for use in research on the immunochemistry and clinical chemistry of I-FABP because those antibodies can recognize both types of native and denatured I-FABP. In order to detect I-FABP in blood samples, it is essential to use this type of antibody, reactive to native type of I-FABP. It is anticipated that, in the near future, such a method for measuring I-FABP will be developed as a useful tool for diagnosing intestinal ischemia by using some of these antibodies.
  Journal of Immunoassay and Immunochemistry 02/2008; 29(1):19-41.
• Article: Monoclonal antibodies generated against excretory/secretory antigens of mammalian stage larvae of the lymphatic filarial parasite Wuchereria bancrofti.

  K Athisaya Mary, S L Hoti, K P Paily
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  ABSTRACT: Monoclonal antibodies (Mabs) against excretory/secretary (e/s) antigens of fourth stage (L4) larvae of Wuchereria bancrofti were raised and screened for their specificity and sensitivity and evaluated for their potential in detecting homologous e/s antigens in human blood samples. Five Mabs were obtained and, among them, Mab A7 showed high reactivity against e/s antigens of L4 and crude somatic antigens of microfilariae (mf) of W. bancrofti, and infective stage (L3) and adult stage larvae of Brugia malayi. It reacted strongly with sera of Mastomys coucha harbouring L4 stage of B. malayi moderately against sera of the animal having later stages of the parasite. But, it exhibited a low and negligible reactivity against the crude antigens of Setaria cervi and Ascaris lumbricoides, respectively. Another Mab, A6, showed very high reactivity against mf antigens of W. bancrofti and B. malayi and a moderate reactivity against antigens of S. cervi and A. lumbricoides. The two Mabs were tested for their reactivity against filarial antigens in human sera, whose microfilaraemic status was determined by membrane filtration of 1 mL blood sample collected during night. When Mab A7 was tested, 7 out of 22 serum samples (32.0%) from amicrofilaraemic normal individuals from filariasis endemic areas showed positive reactions for filarial antigens, indicating the presence of early stage (L4) of the parasite in them. It also reacted with 84% (n=19) mf positive samples and 11% of non endemic normal serum samples (n=17). Mab A6 showed high reactivity with 86% (n=26) of mf positive serum samples, but did not react with non-endemic normal serum samples (n=17). The results, thus, indicate that the Mab A7 has potential in the detection of e/s antigens of L4 stage larvae of filarial parasites in humans, enabling early diagnosis of filariasis. Mab A6 could be used in the diagnosis of patent infection with microfilaraemia. Western blotting with Mab A7 reacted with the 29.0 kDa protein band of L4 e/s antigens of W. bancrofti.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(4):343-57.
• Article: Detection of anti-Brucella antibodies in llama (Llama glama).

  K Nielsen, P Smith, W L Yu
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  ABSTRACT: Seven llamas were immunized with killed Brucella abortus S1119.3 cells and bled sequentially, resulting in 64 samples. An eighth llama was kept as a negative control. In addition, 299 llama and 2075 alpaca sera, submitted for diagnostic testing, were included. Sera from all llamas were tested by the buffered antigen plate agglutination test, the complement fixation test, and the indirect enzyme immunoassays using smooth and rough lipopolysaccharides. A competitive enzyme immunoassay and fluorescence polarization assays were also performed. The sensitivity values for llama sera ranged from 92.2% to 100% and the specificity values from 89.6% to 100%. No alpacas were immunized. The specificity values for alpaca sera ranged from 94.8% to 100% specific although some sera gave an 'agglutination like' reaction after about 10 minutes of incubation. The complement fixation test could not be used, as 31% of the sera were anticomplementary and 4% were false positive.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(1):61-6.
• Article: Comparison of two commercial immunoassays for the detection of anti-rubella IgM and IgG in pediatric patients.

  L C Oliveira, T S Okay, K H Kawasato, L P Lima
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  ABSTRACT: The only threatening modality of rubella is the Congenital Rubella Syndrome that affects fetuses of women who acquire infection during early pregnancy. Laboratory diagnosis is based on serological parameters. We compared anti-rubella IgM and IgG detection of two commercial immunoassay kits (Abbott and Roche). Although we observed an agreement of 97.8% for IgM and 95.7% for IgG when the categories positive, negative and indeterminate were considered, mean titers of IgG and the absorbance/cut off of IgM were statistically different for both kits, thus corroborating the idea that serological results depend very much on the methodology and must be carefully interpreted.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(3):297-306.
• Article: A sensitive direct ELISA for detection of prostaglandin E2.

  Nuntana Aroonrerk, Apichart Suksamrarn, Kanyawim Kirtikara
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  ABSTRACT: In order to improve the indirect ELISA for detection of PGE(2), a modified direct ELISA technique was developed to measure PGE(2) in cell culture supernatants. An evaluation of three types of coating buffer showed that PGE(2) was adsorbed efficiently to the solid phase using the gelatin phosphate buffer. The sensitivity of the assay was increased by employing polyclonal rabbit anti-PGE(2) antibody dilution of 1/100 and 1% skimmed milk as a blocking solution, with the detection limit of 7.8-500 ng/well. The within-run and between-run coefficients of variation (CV) ranges were 3.2-3.7% and 3.4-3.8%, respectively. A linear standard curve was observed over the range of 0.078-5 microg/mL with a coefficient of determination (r(2)) of 0.99. Our results indicated that the developed direct ELISA was sensitive and suitable for a quick determination of PGE(2) levels from cell culture supernatants.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(4):319-30.
• Article: Assessment of chromogen suitability in ELISA for the detection of anaplasmosis and trypanosomosis.

  Armando Reyna-Bello, Mariana C Eleizalde, Adriana M Silva
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  ABSTRACT: Two different ELISAs were routinely performed in our laboratory to detect bovine trypanosomosis and anaplasmosis. The ELISA test for trypanosomosis involved the adsorption of a soluble fraction of parasites as the antigen; and, the ELISA for anaplasmosis was performed with a purified recombinant protein MSP5r adsorbed to the plate. With the purpose of assessing the merit of ABTS and TMB, we compared the absorbance obtained from positive and negative control sera from both assays. The results obtained, suggest that TMB is more adequate for recombinant antigens and that ABTS is preferred when partially purified antigenic extracts are used in the ELISA test.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(1):1-11.
• Article: Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes.

  Guang-Ping Ruan, Li Ma, Xiao-Jing Meng, Min-Jie Meng, Xiao-Ning Wang, Ying Lin, Zheng-Qiang Wu, Xiao-Wei He, Ju-Fang Wang, Yong Zhu
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  ABSTRACT: HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(1):35-45.
• Article: Camel lactoferrin markedly inhibits hepatitis C virus genotype 4 infection of human peripheral blood leukocytes.

  El-Rashdy M Redwan, Ashraf Tabll
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  ABSTRACT: Hepatitis C virus (HCV) is a serious worldwide health risk and, to date, no effective treatments to prevent progression to chronic infection have been discovered. To combat the disease, Egyptian patients often use traditional medicines, for instance, camel milk, which contains lactoferrin. Currently, lactoferrin is one of the primary biopharmaceutical drug candidates against HCV infection. Camel lactoferrin (cLf) purification and biochemical and immunological characterization have shown its similarity to human and bovine lactoferrin, and crossreacts with the anti-human lactoferrin antibody. Incubation of human leukocytes with cLf then infected with HCV did not prevent the HCV entry into the cells, while the direct interaction between the HCV and cLf leads to a complete virus entry inhibition after seven days incubation. Our results suggest that the cLf may be one of the camel milk components having antiviral activity. In conclusion, we have demonstrated the potential for cLf to inhibit HCV entry into human leukocytes with more efficiency than human or bovine lactoferrin.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(3):267-77.
• Article: Fluorogenic hand-held immunoassay for the identification of ricin: rapid analyte measurement platform.

  R E Fulton, H G Thompson
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  ABSTRACT: Rapid analyte measurement platform (RAMPtrade mark) fluorogenic hand-held immunoassays (HHAs) were evaluated for inclusivity/sensitivity, exclusivity/specificity, sample matrix effects, ruggedness/stability, and reproducibility in detection of ricin (RCA(60)), a potential biological threat agent. The limit of detection of HHAs for RCA(60) was 14 ng/mL or approximately 140 pg/test. HHAs were inclusive in detection of ricin RCA(60), RCA(120), ricin A chain, and ricin B chain and exclusive in discrimination of RCA(60) from other toxins. None of the sample matrices tested affected assay performance. RAMPtrade mark ratios were tolerant of increases in sample volume, however, a decrease in sample volume of 25% resulted in significantly increased readings. RAMPtrade mark readings were highly reproducible lot-to-lot, cartridge-to-cartridge, day-to-day, and reader-to-reader.
  Journal of Immunoassay and Immunochemistry 02/2007; 28(3):227-41.