Peptide Enzyme-linked Immunosorbent Assay Research Articles

Serodiagnosis of Chagas' disease by enzyme-linked immunosorbent assay using two synthetic peptides as antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against Trypanosoma cruzi. Two synthetic T. cruzi peptides, TcD and PEP2, were used. The specificity and sensitivity of the peptide ELISA were determined with 260 serum samples from individuals living in an area in which Chagas' disease is endemic. ELISAs were performed with the peptides singly or in combination. The evaluation of these tests showed that 168 (93.8%) of 179 serum samples from T. cruzi-infected patients were positive when TcD peptide was used as antigen; 164 (91.6%) samples were positive with PEP2, and 178 (99.4%) samples were positive when the two peptides were combined. Thus, the sensitivity of the ELISA using the two peptides exceeded 99%. The specificity was evaluated by using a panel of 118 serum samples that included samples from 81 individuals living in an area of endemicity with negative serology for Chagas' disease and from 37 patients from areas in which T. cruzi was not endemic but with other pathologies, such as leishmaniasis, tuberculosis, and leprosy. Only two false-positive serum samples were found in this group of individuals, giving a test specificity of more than 98%. Because these peptides can be synthesized and are very stable at room temperature, the use of such reagents can improve the standardization and reproducibility of ELISAs for the serodiagnosis of T. cruzi infection.

Evaluation of the WHO human immunodeficiency virus (HIV) antibody testing strategy for the diagnosis of HIV infection

Objective: To evaluate a WHO testing strategy based on the use of two consecutive enzyme-linked immunosorbent assays (ELISA) as an alternative to ELISA followed by Western blotting (WB) for the serologic diagnosis of HIV infection. Study Design: The study was of 2069 consecutive serum specimens from patients suspected of HIV infection received for HIV diagnostic testing at the HIV laboratory, Muhimbili Medical Centre, Dar es Salaam. The strategy involved testing all sera with Behring indirect anti-HIV 1 + 2 peptide ELISA, followed by Wellcozyme anti-HIV-1 recombinant competitive ELISA on those sera reactive by the first ELISA. WB was done on a sample of the sera reactive on both ELISAs and on all those giving discordant results on the two ELISAs. Of the 2069 sera tested, 837 (40.5%) were negative on the first ELISA, 1172 (56.6%) were reactive on both ELISAs and 60 (2.9%) were initially reactive on the first test but not on the second assay. Results: Of the 1172 sera reactive on both ELISAs, 329 (28.1%) were tested by WB. The diagnostic accuracy of the WHO alternative testing strategy using WB confirmation as the ‘gold’ standard was as follows: sensitivity 99.4% (326/328), specificity 99.7%, (893/896), positive predictive value 99.1% (328/331) and negative predictive value 99.8% (893/895). Repeated testing by ELISA of the sera which initially gave discordant results on the two ELISAs increased the sensitivity to 100%. Three sera giving false positive reactions on both ELISAs became negative on both ELISAs after retesting. In order to achieve a specificity and a positive predictive value of 100%, it would have been necessary to subject all sera reacting on both ELISAs to retesting on one ELISA. Conclusions: A second ELISA based on different antigens and a different test principle compared with the first ELISA could be used as an alternative to the WB assay for confirmation of HIV antibodies. However, some modifications of the WHO strategy for diagnostic HIV antibody testing were required in order to maximize the diagnostic accuracy.

The use of peptide ELISA in determining malaria endemicity

A study was undertaken with the objective to find out whether a reliable transmission index could be established by a simple peptide ELISA. This would be important to evaluate the success of the bioenvironmental malaria control programmes implemented in endemic areas like the Haldwani and Shankargarh districts of Uttar Pradesh, India. We have estimated antimalarial antibodies against three synthetic peptides derived from blood stage antigens of Plasmodium falciparum and against P. falciparum crude antigen by enzyme-linked immunosorbent assay (ELISA), in children and adults. We have found that while all the peptides react with serum antibodies, as does the whole crude blood stage antigen, a nonapeptide from RESA antigen gave the most uniform results. Our results suggest that synthetic peptides, which are easy to obtain and can be fully characterized, may offer a simple way to measure malaria endemicity.

Selectivity of human T lymphotropic virus type-1 (HTLV-1) and HTLV-2 infection among different populations in Brazil.

A seroprevalence study for human T lymphotropic virus type-1 (HTLV-1) and HTLV-2 was conducted in Sao Paulo, Brazil among 2,312 individuals that included following groups: 1,148 volunteer blood donors, 37 patients with tropical spastic paraparesis (TSP), 53 with lymphoproliferative disorders, 171 with a history of multiple blood transfusions, 268 human immunodeficiency virus type-1 (HIV-1) seropositive subjects, and 635 Amazonian Indians. Antibodies to HTLV-1/2 were screened by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot and/or radioimmunoprecipitation. The differentiation of HTLV-1 and HTLV-2 was achieved using a synthetic recombinant peptide (rgp46) ELISA. We confirmed the presence of HTLV-1 infection in Brazil, both in blood donors (0.4%) and in patients exposed to blood transfusions (2.9%), as well as the occurrence of HTLV-1-associated TSP (11 patients, or 30% of all TSP cases) and adult T cell leukemia/lymphoma (two cases, or 3.5% of all hematologic malignancies). The HIV-1 infected individuals were shown to be coinfected (8.9%) with either HTLV-1 or HTLV-2. All HIV-1 and HTLV-2 coinfected individuals were intravenous drug abusers. In addition, we also demonstrated the presence of HTLV-2 (4.7%), and HTLV-1/2 (0.8%) in tribes of Amazonian Indians who lived in the eastern Amazon basin (southeastern State of Para). The selectivity of these retroviral infections in particular groups is emphasized, as well as the need for HTLV-1/2 screening of all blood donors in Brazil as a public health measure.

Isolation, partial characterization, and molecular cloning of a human colon adenocarcinoma cell-surface glycoprotein recognized by the C215 mouse monoclonal antibody.

The monoclonal antibody C215 (IgG2a) was obtained by the immunization of BALB/c mice with the human colon adenocarcinoma cell line COLO 205 and used in the targeting of colorectal carcinomas. The partial characterization and purification of the C215 target molecule from solubilized COLO 205 membranes indicated that it is an integral membrane glycoprotein of the non-mucin type. The denatured antigen appeared as a major 40-kDa form in Western blots after SDS-polyacrylamide gel electrophoresis and migrated as a monomeric 36-kDa species after the reductive cleavage of intramolecular disulfide bridges. Using a five-step procedure, the antigen was purified 4,300-fold from COLO 205 tumors raised in nude mice to a homogeneity of 95% when assessed by capillary electrophoresis. Removal of N-linked carbohydrate by peptide:N-glycosidase treatment did not affect the visualization of the purified antigen in immunoblots but resulted in a faster migration in the SDS gels. The amino acid sequence was partially determined. Seventeen contiguous NH2-terminal amino acids were identified and coincided exactly with residues 82-98 of the GA733-2 protein cloned by Szala et al. (Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewske, Z., Koprowski, H., and Linnenback, A. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3542-3546). Therefore, the predicted amino acid sequence of this protein was used to prepare overlapping synthetic peptides that cover the entire extracellular domain in order to identify the C215 epitope. A likely epitope, close to the NH2 terminus and corresponding to the first distinct hydrophilic stretch after the putative signal sequence, was identified in a peptide enzyme-linked immunosorbent assay. Moreover, GA733-2 cDNA was used for the cloning of the C215 protein from COLO 205 cells and the subsequent transfection to K36.16 mouse T cell leukemia cells. The transfected cells were C215 reactive in fluorescence-activated cell sorter analysis, and a 42 kDa band was visualized in Western blots under both non-reducing and reducing conditions. Our findings indicate a close relationship between the C215 antigen and other members of the GA-733 family, some of which are currently being used as targets in clinical trials with monoclonal antibodies. The mammalian expression system described here will enable further studies into the biological role of this protein and the construction of animal models in order to develop optimal therapeutic strategies.

Antigenic evidence of the presence of the aberrant HIV-1ANT70 virus in Cameroon and Gabon

The search for an effective vaccine against HIV-1 is of major importance. Some promising research has been conducted on the V3 loop peptides of HIV-1 in chimpanzees. In this article are reported the results of a research project on the prevalence of sera collected from HIV-1 positive people in Cameroon Gabon Ivory Coast and Kenya which are reactive to a genetically divergent HIV-1 V3 peptide isolate (ANT70) and peptides obtained from 3 other HIV-1 isolates. A total of 895 serum samples were tested using the V3-peptide enzyme-linked immunosorbent assay (ELISA). 40 HIV-1 seronegative sera 10 from each country were used as negative controls. Overall 88% (787/895) of the sera reacted with the V3 loop of MN peptide 246 (27%) sera reacted with the MAL peptide and 53% (471/895) reacted with the Z6 peptide component of the V3 loop. A total of 14 (14/895) sera (9 from Cameroon and 5 from Gabon) reacted with the ANT70 peptide ELISA. The authors suggest that their results should prompt further investigation in mapping antigenic diversity in Africa and that this knowledge is necessary in the development of an HIV-1 vaccine.

Immunoneutralization of heterodimeric FSH using FSH beta subunit as the immunogen.

Immunization with beta subunit of gonadotropin may elicit antibody formation to endogenous heterodimeric gonadotropin and result in reproductive unresponsiveness. The objectives of this study were to determine if antibodies produced in rats following immunization with human follicle stimulating hormone beta-subunit (hFSH-beta) could bind to and immunoneutralize heterodimeric FSH, and to elucidate the immunoneutralizing epitope. Mature female Sprague-Dawley rats received subcutaneous injections of 10 micrograms of hFSH-beta emulsified in complete Freund's adjuvant, while control animals received only adjuvant. Animals received 10 micrograms hFSH-beta booster injections emulsified in incomplete Freund's adjuvant 2, 4, 11, and 21.5 wk after the initial immunization. Immunization with hFSH-beta produced appreciable antibody titers against human FSH (hFSH) as measured in enzyme-linked immunosorbent assays (ELISA) of immunized rat sera. A more modest titer to rat (rFSH) and no antibody response to rat Luteinizing Hormone (rLH) was observed, thus confirming the specificity of the immune response. Titers against hFSH increased throughout the study. Rat anti-hFSH-beta sera was tested to determine its ability to inhibit binding (immunoneutralizing) of 125I-hFSH to the FSH receptor. Continued immunization resulted in all animals producing immunoneutralizing antibodies. Immunization of rats also resulted in disrupted estrous cycles, but only animals with subsequent titers high enough to completely block binding of FSH to its receptor failed to conceive. In order to assess the immunoneutralizing epitope, antisera were tested in a peptide ELISA. Peptides used in the ELISA corresponded to amino acids that spanned the entire hFSH beta sequence. It was found that antibodies from all rats immunized with hFSH beta bound to amino acids within hFSH-beta 33-53. Collectively, these data suggest that amino acids within hFSH-beta 33-53 are necessary but not sufficient to confer immunocontraception. Amino acids within this linear sequence appear in a variety of epitopes of hFSH-beta and hFSH, only some of which are immunoneutralizing. (Am J Reprod Immunol. 1993; 29:000-000.)

Structure-function relationships of HIV-1 reverse transcriptase determined using monoclonal antibodies.

The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is one of the main targets in approaches to the chemotherapy of AIDS. A detailed knowledge of structure-function relationships of this enzyme is a prerequisite for rational drug design. We have used monoclonal antibodies as tools to identify functionally important regions of the protein. The preparation of 23 murine monoclonal antibodies (mAb) against HIV-1 reverse transcriptase and their different effects on the enzyme are described. The interaction of purified mAbs with HIV-1 RT was demonstrated by enzyme-linked immunosorbent assay (ELISA), Western blots, and high performance liquid chromatography size exclusion chromatography. One of the antibodies also recognized recombinant HIV-2 RT. Antibody binding epitopes on HIV-1 RT were analyzed by immunoblotting using cyanogen bromide fragmented RT, C-terminally truncated mutants, and a peptide ELISA employing 15-mer synthetic overlapping peptides spanning nearly the complete polypeptide chain. The epitopes were mapped within three domains corresponding to amino acids 200-230, 300-428, and 528-560. Two mAbs show neutralizing properties on enzymatic functions of RT. One affects the polymerase activity and to a certain degree the RNase H activity of the enzyme, whereas the other inhibits the latter activity exclusively. mAb 28, which blocks the polymerase activity, interferes with the nucleotide binding region of RT, as shown by fluorescence spectroscopy using a labeled template/primer complex. By investigating the antibody effects on dimer formation of the heterodimeric enzyme, three domains corresponding to amino acids 230-300, 350-428, and residues around amino acid 540 involved in protein-protein interactions were localized.

Identification of broadly reactive continuous antigenic determinants of simian immunodeficiency virus glycoproteins.

The env polyprotein sequences of several simian immunodeficiency virus (SIV) isolates were analyzed using computer algorithms designed to predict immunologically reactive protein segments. Peptides corresponding to predicted epitopes were synthesized and employed in peptide enzyme-linked immunosorbent assay (ELISA) screening of serum panels from experimentally infected macaques, as well as naturally infected, asymptomatic mangabeys and African green monkeys. Several of the peptides are recognized by a high percentage of antisera from each panel of monkeys indicating that they represent group-specific antigenic determinants of SIV. Several type-specific determinants also were identified. These peptides may be a useful tool for studying the kinetics of SIV glycoprotein-specific immune responses produced by infected and vaccine-protected monkeys at the epitope level.

Mapping of B-cell epitopes on the polypeptide chain of the Epstein-Barr virus major envelope glycoprotein and candidate vaccine molecule gp340.

The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.

Human papillomavirus type 18 E6 and E7 antibodies in human sera: increased anti-E7 prevalence in cervical cancer patients

Antibody-reactive regions on the human papillomavirus type 18 (HPV-18) E6 and E7 proteins were identified with rabbit polyclonal anti-fusion protein sera by screening of an fd phage expression library containing subgenomic HPV-18 DNA fragments and by testing of overlapping decapeptides representing the E6 and E7 open reading frames. Peptides comprising the delineated regions (designated E6/1 to E6/4 and E7/1) were synthesized and used in an enzyme-linked immunosorbent assay (ELISA) to detect anti-HPV-18 antibodies in human sera. A total of 232 human serum samples (identical numbers of cervical cancer patients and age-matched controls) collected in Tanzania were tested. Similar prevalences (between 0.8 and 4.3%) of antibodies recognizing the different E6 peptides were found in the sera from tumor patients and controls. With a synthetic 28-mer peptide (designated pepE701) comprising the E7/1 region, a significant difference was found: 10 of 116 tumor serum samples but 0 of 116 control serum samples showed a specific reaction (P less than 0.001). This observation confirms earlier results with HPV-16 E7 fusion proteins (I. Jochmus-Kudielka, A. Schneider, R. Braun, R. Kimmig, U. Koldovsky, K. E. Schneweis, K. Seedorf, and L. Gissmann, J. Natl. Cancer Inst. 81:1698-1704, 1989). A lower prevalence of anti-HPV-18 E7 antibodies was observed when 188 human serum samples collected in Germany from tumor patients and controls were tested (3 of 94 positive in the cancer group; 0 of 94 positive in the control group). The type specificity of anti-HPV-18 E7 antibodies was demonstrated when the HPV type found by Southern hybridization in the cervical cancer biopsies was compared with seroreactivity: 4 of 8 serum samples obtained from HPV-18 DNA-positive but 0 of 16 serum samples from HPV-18 DNA-negative tumor patients reacted in the HPV-18 E7 ELISA. In addition, HPV-18-positive sera failed to react in a peptide ELISA with the homologous HPV-16 E7 region (M. Müller, H. Gausepohl, G. de Martinoff, R. Frank, R. Brasseur, and L. Gissmann, J. Gen. Virol. 71:2709-2717, 1990) and vice versa.

Epitope Mapping of Human Follicle Stimulating Hormone-α Using Monoclonal Antibody 3A Identifies a Potential Receptor Binding Sequence*

Five monoclonal antibodies (mabs) were generated (3A, 4B, 5F, 2E, 1E) by immunizing BALB/c mice with human (h) FSH. The mabs were used to relate antigenic structures (epitopes) to function (receptor binding). All five mabs could immunoneutralize (inhibit binding to receptor) hFSH and could be placed into two groups based on potency (degree of neutralization). Group I mabs (5F, 2E, 1E) were less potent than group II mabs (3A, 4B) even though group I mabs had a 2-fold higher average affinity constant than group II. Those data suggested that group II mabs recognize an epitope near or in the receptor binding site of hFSH. Immunoradiometric epitope cross-matching demonstrated that group I and group II mabs recognize different epitopes. Further characterization of 5F and 3A (representative of group I and group II, respectively) utilized an enzyme-linked immunosorbent assay (ELISA) and a RIA. In the ELISA, both mabs bound hFSH and hFSH alpha but not hFSH beta. In the RIA, 3A bound [125I]hFSH and [125I]hFSH alpha but not [125I]hFSH beta. In contrast, 5F bound only [125I]hFSH. hFSH effectively competed with [125I]hFSH for 5F and 3A. In contrast, hFSH alpha competed with [125I]hFSH for 5F but not for 3A even though 3A could bind hFSH alpha in the ELISA and the RIA. These results suggest that 3A and 5F recognize different epitopes. The epitope recognized by 3A is unique in that its conformation appears to be dependent on association with hFSH beta. Since 3A was a more potent inhibitor of receptor binding than 5F, its epitope specificity was characterized further by epitope mapping. This was accomplished utilizing a peptide ELISA and by affinity chromatography. The results from epitope mapping demonstrated that 3A recognizes sequences 61-78 and 73-92 with binding to 73-92 being 4-fold greater than to 61-78. Thus, the epitope comprised of sequence 73-92 (and to a lesser extent 61-78) appears to be important for receptor binding.

Immunoaccessible peptide sequences of the major outer membrane protein from Chlamydia trachomatis serovar C

The antigenicity of the major outer membrane protein of Chlamydia trachomatis serovar C was assessed by using overlapping hexapeptide homologs of serovar C major outer membrane protein and rabbit antisera in a peptide enzyme-linked immunosorbent assay. Five immunogenic sites were found distributed within variable sequences of the protein: four were immunodominant and three were surface exposed on native elementary bodies of serovar C. None was surface exposed on serovars H, I, and J.

A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.

Rapid and specific diagnosis of HIV-1 and HIV-2 infections

To identify cost-effective testing strategies for HIV-1 and HIV-2 infections, we evaluated different combinations of tests on serum specimens from 1134 consecutive patients attending tuberculosis treatment centers in Abidjan, Côte d'lvoire. Virus-specific whole-virus enzyme-linked immunosorbent assay (WVE), Western blot (WB) and synthetic peptide enzyme-linked immunosorbent assay (SPE) were used in sequential fashion to determine the true prevalence of infection; 27% were reactive to HIV-1, 5% to HIV-2, and 10% to both viruses. Of 239 specimens positive on WB for both HIV-1 and HIV-2, SPE diagnosed 38% as HIV-1-reactive and 16% as HIV-2-reactive, while 46% remained reactive to both viruses. Using WVE or one of two rapid (5-10 min) mixed (HIV-1 and HIV-2) antigen tests (RMATs) as a screening test, followed by SPE as a supplemental test, gave results with sensitivity of 97.3-99.2%, specificity of 99.5-99.7%, and positive predictive value for diagnosing HIV infection of 99.4-99.6%, with important savings in time and reagent costs. SPE allows more specific distinction between HIV-1 and HIV-2 infections than WB, and could replace it as a supplemental test in many settings. WB may be required for specimens reactive on screening tests but negative on SPE, until sensitivity of the SPE is further evaluated. A mixed antigen screening test followed by SPE seems to be an efficient testing strategy for diagnosing HIV-1 and HIV-2 infections.

Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products.

A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well-characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as might occur with antigens that are components of subcellular particles.

The Antibody Response to the HIV-1 Specific "Out" (vpu) Protein: Identification of an Immunodominant Epitope and Correlation of Antibody Detectability to Clinical Stages

Overlapping decapeptides based on the sequences of two human immunodeficiency virus type 1 (HIV-1) strains (HXB2 and ELI) were used to identify an immunodominant epitope of the nonstructural protein "out" (vpu) of the human immunodeficiency virus type 1. Of 29 HIV-1 antibody-positive sera, 6 reacted with decapeptides corresponding to the C-terminal amino acid sequence VEMGVEMGHHAPWDVDDL of the "out" (vpu) protein. This oligopeptide was synthesized by the solid phase method and used to develop an enzyme-linked immunosorbent assay (ELISA) for screening of 243 HIV-1-seropositive and 75 HIV-1-seronegative sera. It was found that 26% of the HIV-1 antibody-positive sera were reactive in the "out" (vpu) peptide ELISA, whereas none of the HIV-1-negative sera reacted with the oligopeptide. Correlation of reactivity of sera with the Walter Reed (WR) staging classification demonstrated that individuals classified WR 1 (36%) and WR 2 (42%) were more often reactive than patients classified WR 3-6 (11%).

Mapping of B-cell epitopes of the human hepatitis B virus X protein

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.

Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides

The antigenicity of the major outer membrane protein (MOMP) of Chlamydia trachomatis was comprehensively evaluated by using overlapping hexapeptide homologs of serovar B MOMP and polyclonal rabbit antisera in a peptide enzyme-linked immunosorbent assay. Of 367 hexapeptides, 152 showed reactivities with at least one antiserum. Seven hexapeptides located within variable domain (VD) IV (residues 288 to 316) were found to be most reactive in terms of their binding titer and frequency, suggesting that VD IV is the immunodominant region within the MOMP as detected by this assay. Peptide-reactive antibodies could also recognize corresponding epitopes on either viable or acetone-permeabilized organisms. The antigenic specificity and immunoaccessibility of epitopes located in VD IV were resolved by absorbing antisera with chlamydial elementary bodies. Six antigenic sites were found in this region and included a B-type-specific site (S1), four subserogroup-specific sites (S2 and S4 to 6), and one species-specific site (S3), each displaying varying degrees of surface exposures on elementary bodies from different C. trachomatis serovars.

A comparison of HIV-1 and HIV-2 infections in hospitalized patients in Abidjan, Côte dʼlvoire

In late 1988, a cross-sectional study of 1715 adult medical patients hospitalized in Abidjan, Côte d'Ivoire, west Africa, showed an overall prevalence of HIV infection of 46% in men and 28% in women. On the basis of specific testing by whole virus enzyme-linked immunosorbent assay (ELISA), Western blot and synthetic peptide ELISA, HIV-1 infection was found in 25%, HIV-2 infection in 4%, and reactivity to both viruses in 11% of male and female patients combined. People infected with HIV-2, as well as those who were reactive to both HIV-1 and HIV-2, had a frequency of AIDS-associated symptoms and signs similar to that in HIV-1-infected patients, and significantly greater than that in seronegative patients. The significance of dual reactivity, and the natural history and disease spectrum of HIV-2 infection, require further study. Synthetic peptide ELISA is valuable for specific serodiagnosis of HIV-1 and HIV-2 infections. Advanced HIV-2 infection in hospitalized patients in Abidjan is associated with the same symptoms and signs as HIV-1 infection.