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ABSTRACT: In this paper, we have evaluated the diagnostic utility of three antigenic regions of the Mycoplasma pneumoniae P1, P30, and MPN456 gene products in order to replace the soluble, whole-cell bacterial extract in serological assays. Antigenic regions, being previously identified as B-cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering. Paired serum samples from 47 patients with M. pneumoniae infection and from 39 subjects with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection were included. Immunoglobulin G (IgG) antibodies against epitopes carried by recombinant antigens were measured by performing recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Rec-ELISA results were compared to those obtained by a commercial assay using the whole-cell Mycoplasma antigen. Our study demonstrates that all IgG Rec-ELISAs using recombinant antigens have better sensitivity with respect to the commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays. The use of recombinant antigens is effective in distinguishing M. pneumoniae-infected patients from uninfected individuals and shows that immunoassays based on recombinant antigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases.
European Journal of Clinical Microbiology 11/2010; 29(11):1377-86. · 2.86 Impact Factor
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ABSTRACT: The aim of this comparative study was to investigate the clinical usefulness of the measurement of Toxoplasma gondii IgG avidity in the postnatal diagnosis of congenital toxoplasmosis. IgG avidity values in serum samples from infants with congenital infection were compared with those in samples from uninfected infants, all born to mothers with toxoplasmosis acquired during gestation. This analysis revealed that IgG avidity values soon after birth reflected maternal values in the large majority of the samples. Low or borderline IgG avidity values were systematically found in the cohort of congenitally infected subjects. After birth, IgG avidity values slowly increased over time for up to 2 years in congenitally infected subjects. On the contrary, IgG avidity values in the uninfected infants remained stable over time. The presence of low IgG avidity in a newborn can be considered a marker of maternal seroconversion in the second or third trimester of gestation and, as a consequence, an indicator of risk for congenital toxoplasmosis. An IgG avidity assay can be easily carried out with antibodies eluted from dried blood spots (Guthrie cards), providing an opportunity to retrospectively evaluate the risk of congenital infection in special clinical circumstances, for example when suspicion of congenital infection arises during late infancy.
European Journal of Clinical Microbiology 12/2004; 23(11):825-30. · 2.86 Impact Factor
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ABSTRACT: Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).
International Journal for Parasitology 01/2002; 31(14):1659-68. · 3.39 Impact Factor
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O Minenkova, N Gargano,
A De Tomassi,
F Bellintani,
A Pucci,
P Fortugno,
E Fuscaldi,
A Pessi,
M Rapicetta,
M Miceli,
P Iudicone,
R Cortese,
F Felici,
P Monaci
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ABSTRACT: We screened phage libraries using sera from noninfected individuals and patients infected by hepatitis C virus (HCV). By applying different selection and maturation strategies, we identified a wide collection of efficient phage-borne ligands for HCV-specific antibodies. The selected ligands retained their antigenic properties when expressed as multimeric synthetic peptides. Peptides that mimic several immunodominant epitopes of the virus were used to develop a novel type of diagnostic assay which efficiently detects antibodies to HCV in serum. This type of analysis provides a conclusive diagnosis for many patients identified as indeterminate according to presently available serological assays.
European Journal of Biochemistry 10/2001; 268(17):4758-68. · 3.58 Impact Factor
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Analytical Biochemistry 10/2000; 284(2):412-5. · 3.00 Impact Factor
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ABSTRACT: A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
European Journal of Biochemistry 06/1995; 230(1):322-8. · 3.58 Impact Factor
