Are the enzyme immunoassays for antibodies to pneumococcal capsular polysaccharides serotype specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.     

Are the Enzyme Immunoassays for Antibodies to Pneumococcal Capsular Polysaccharides Serotype Specific?

The specificity of antibody binding to pneumococcal capsular polysaccharides (Pnc PSs) measured by enzyme immunoassay (EIA) was studied by inhibition of antibody binding by homologous and heterologous PSs. We found extensive cross-reactivity of antibody binding to type 6B, 19F, and 23F PSs but not to type 14 PS, even after treatment with cell wall PS (CPS). The cross-reactive antibody was highly prevalent in sera of infants and adults with naturally acquired antibody, but not in sera of infants and adults immunized with pneumococcal vaccines. However, a type 11A antibody response was seen after vaccination with heterologous PSs. Monoclonal antibodies prepared against a type 6B PS-tetanus toxoid conjugate recognized also other than the specific type of PS in the EIA, implying the possible existence of a cross-reactive epitope. Remarkable differences in specificity among type 6B PS preparations from different manufacturers were found. Moreover, different lots of type 11A PS from the same manufacturer showed differences in specificity. The results suggest that some Pnc PS preparations may contain cross-reactive epitopes or impurities, other than CPS, that are common to many types of Pnc PS. The specificity of antibodies, especially in sera from nonimmunized subjects, measured by EIA can be questioned.     

Use of recombinant epitopes to study the heterogeneous nature of the autoantibodies against thyroid peroxidase in autoimmune thyroid disease.

Microsomal antigen is often recognized by the sera from patients with autoimmune thyroid disease (AITD). Human thyroid peroxidase (hTPO) is the main component of this antigen. In a previous study, we expressed hTPO cDNA as fusion proteins in prokaryotic vector; we thereby defined seven antigenic peptides by using two rabbit polyclonal anti-hTPO antibodies. In the present study we used the seven epitopes and three widened peptides to define the reactivity pattern of 61 sera from patients with AITD. Thirty-eight of them reacted against at least one of the seven hTPO-restricted epitopes; 14 were negative against the seven determinants but recognized one or two of the extended peptides. Thus, the antibody response against hTPO appeared to be highly heterogeneous in AITD patient sera. Moreover, we demonstrated that the immunodetection of the hTPO on Western blotting with deoxycholate solubilized microsomes can be perfectly correlated with the recognition of one of the epitopes in the region 554-735.     

Anti-native and recombinant myeloperoxidase monoclonals and human autoantibodies

Myeloperoxidase (MPO) is one of the main antigen targets of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic vasculitides. It has been suggested that anti-MPO antibodies may recognize a single epitope on recombinant MPO. If confirmed on native MPO, this might allow specific therapeutic intervention with anti-idiotypic MoAbs to prevent antibody–antigen interaction which is thought to cause activation of neutrophils and vasculitis. We searched for restriction in the epitope recognition profile in 50 patients with anti-MPO autoantibodies, using both native and recombinant MPO. Mouse monoclonals were purified and tested in competition assays. At least four epitopes were identified on native MPO using these monoclonals and only two were conserved on recombinant MPO. We found that human MPO autoantibody response was not restricted to a single epitope on native MPO, as all sera tested did not show the same profile in competitive studies with monoclonals. Furthermure, 30% of human anti-native MPO sera failed to recognize rMPO.     

Anti-myeloperoxidase antibodies in sera from patients with propylthiouracil-induced vasculitis might recognize restricted epitopes on myeloperoxidase molecule

Myeloperoxidase (MPO) is one of the major target antigens of antineutrophil cytoplasmic antibodies (ANCA) in primary systemic vasculitis. It is known that propylthiouracil (PTU) could induce MPO-ANCA-positive vasculitis. The production of anti-MPO antibodies in patients with PTU-induced vasculitis may be different from that in patients with primary microscopic polyangiitis (MPA). One possible reason for this may be differences in epitope recognition. The aim of this study is to compare the epitopes of antibodies to MPO in sera from patients with PTU-induced vasculitis (n = 10) and MPA (n = 10). The sera were collected and used to inhibit monoclonal antibodies against human MPO (3D8 and 6B9) and affinity purified, horseradish peroxidase conjugated human anti-MPO antibodies (Pab1-HRP, Pab2-HRP) in a competitive inhibition enzyme-linked immunosorbent assay (ELISA) system using soluble human MPO as solid phase ligand. The Pab1-HRP and Pab2-HRP were affinity purified from plasma exchanges of a patient with PTU-induced vasculitis and a patient with MPA, respectively. The inhibition rates were evaluated and compared between the PTU and primary MPA groups. In the PTU group all 10 sera could inhibit 3D8: the average inhibition rate was 44.7% +/- 5.0%; 9/10 sera could inhibit 6B9: the average inhibition rate was 35.6% +/- 6.0%. However, in the MPA group all 10 sera could inhibit 3D8 and 6B9; the average inhibition rates were 68.4% +/- 16.1% (P < 0.01) and 62.2% +/- 17.2% (P < 0.01), respectively. Sera in both the PTU and MPA groups could inhibit Pab1-HRP and the inhibition rates were 81.4% +/- 9.4%versus 86.6% +/- 17.2% (P > 0.05). However, the average inhibition rate for Pab2-HRP in the MPA group was significantly higher than that in the PTU group (76.3% +/- 7.8%versus 58.9% +/- 15.5%, P < 0.01). We conclude that anti-MPO antibodies from patients with PTU-induced vasculitis and from patients with primary MPA could recognize more than one epitope on the native MPO molecule. Although the epitopes overlapped between the two groups, the epitopes of anti-MPO antibodies from patients with PTU-induced vasculitis might be more restricted.     

Identification of amino acids critical for IgE-binding to sequential epitopes of bovine kappa-casein and the similarity of these epitopes to the corresponding human kappa-casein sequence

Background:  The delineation of allergenic (i.e. IgE-binding) epitopes in cow's milk proteins and the amino acids (AAs) critical for IgE-binding is necessary to understand better the structural properties of an allergen and to develop more efficacious immunotherapeutic reagents. Furthermore, this information may enable us to understand better cross-sensitivity between different allergens.
Methods:  Eleven peptides, 10–14 AAs in length, representing the IgE-binding epitopes of κ-casein were synthesized on a derivatized cellulose membrane with single AA substitutions at each position. Membranes were incubated with pooled sera from 15 milk-allergic patients and individual sera from 10 of the patients included in the pool.
Results:  For 10/11 allergenic peptides, one to five different single AA substitutions resulted in elimination of IgE-binding of pooled patient sera. Overall at least one mutated peptide could be found for these 10 IgE-binding sites that resulted in a reduction of IgE-binding in at least 80% of the patients who recognized the native protein. Furthermore, the IgE-binding region at AA104–112 on bovine κ-casein showed a high degree of similarity with the human κ-casein, respectively, including the AAs critical for IgE-binding.
Conclusion:  This finding suggests that critical AAs should be assessed with both pooled and individual patient sera to account for the B-cell epitope heterogeneity between patients, with cow's milk allergy. In addition, we identified two potentially cross-reactive peptides between bovine and human caseins of unknown clinical relevance.     

Epitope mapping of anti-proteinase 3 and anti-myeloperoxidase antibodies.

Anti-proteinase 3 (PR3) and anti-myeloperoxidase (MPO) autoantibodies are present in many patients with Wegener's granulomatosis (WG) and microscopic polyarteritis. The aim of this study was to determine whether these antibodies bound to linear peptide sequences on their target antigens. If common linear epitopes were demonstrated, then these could be manufactured and used in diagnostic ELISAs for anti-PR3 and anti-MPO antibodies. In addition, any homology between these epitopes and bacterial or viral sequences might implicate those microorganisms in the development of these antibodies and the pathogenesis of the associated diseases. The presence of linear epitopes on PR3 and MPO was suggested by the binding of the corresponding autoantibodies to these proteins after they had been reduced with beta-mercaptoethanol (beta-ME) and denatured with SDS or boiling, and digested with proteases. Four of the 22 sera with anti-PR3 antibodies bound to PR3 in Western blots after treatment with SDS, beta-ME and boiling for 5 min. Thermal denaturation reduced the amount of binding more than other forms of denaturation. One serum with anti-PR3 antibodies bound to Lys-C and Glu-C-digested PR3 in dot blots. Linear epitopes could not be further defined by their binding in an ELISA using overlapping peptides corresponding to the PR3 molecule because of non-specific binding. Three of the five sera with anti-MPO antibodies bound to MPO in Western blots after treatment with SDS, beta-ME and boiling for 5 min. One serum with anti-MPO antibodies bound to Lys-C and Glu-C-digested MPO in dot blots. Again, linear epitopes could not be further defined using an ELISA with overlapping peptides because of non-specific binding. Some anti-PR3 and anti-MPO antibodies are likely to recognize linear epitopes, but these cannot be defined by use of a PIN ELISA system.     

Anti-myeloperoxidase autoantibodies react with native but not denatured myeloperoxidase.

We wondered whether anti-myeloperoxidase (MPO) autoantibodies (MPO-ANCA) found in patients with systemic vasculitis react with a conformational epitope or epitopes on the MPO molecule. Sera from 15 human MPO-ANCA, and a polyclonal and a monoclonal anti-MPO antibodies were reacted with MPO in native and denatured states. Human MPO-ANCA and mouse monoclonal anti-MPO reacted with native MPO, and a 120-kD band representing the MPO hologenzyme, but not with denatured MPO fragments; however, MPO-ANCA and mouse anti-MPO did not demonstrate competitive inhibition of binding to MPO. Polyclonal rabbit anti-MPO reacted with both native and denatured MPO. All MPO-ANCA tested showed the same patterns of reactivity with native and denatured MPO in dot blot and Western blot analyses. Both polyclonal and monoclonal anti-MPO antibodies inhibited MPO's protein iodination by over 90%, whereas MPO-ANCA IgGs, normal IgGs and disease control IgGs did not. These data suggest that (i) MPO-ANCA interact with a conformational epitope on the MPO molecule; and (ii) MPO-ANCA from different patients have similar reactivity with native versus denatured MPO.     

Acquired resistance to type II collagen-induced arthritis in rhesus monkeys is reflected by a T cell low-responsiveness to the antigen.

The use of serological tests for the diagnosis of HIV infection has revealed that some non-infected persons have antibodies that react with HIV-1 gag proteins. Here, the sera of three non-infected subjects reacting with p17 and 11 non-infected subjects reacting with p24 were investigated, using an enzyme immunoassay (EIA) with six recombinant gag antigens and Western blot analysis of proteolytic peptides of two of these gag antigens. The results indicate that whereas all p17-reactive sera could react with an unique epitope, individual p24-reactive sera recognize different epitopes. Investigations by EIA also demonstrated the role of sequences located far from the epitopes in making these epitopes accessible to the antibodies or in providing them with an antigenic conformation. In addition to the 14 subjects mentioned above, another subject was shown to have antibodies reacting with the p9 (NC) gag protein. Several proteins are known as having homology with HIV-1 gag proteins. Their possible role in eliciting cross-reactive antibodies is discussed.     

Comparison of a monoclonal antibody-blocking enzyme-linked immunoassay and a strip immunoblot assay for identifying type-specific herpes simplex virus type 2 serological responses

Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1- and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.     

Human Autoantibodies Against C1q: Lack of Cross-Reactivity with the Collectins Mannan-Binding Protein, Lung Surfactant Protein A and Bovine Conglutinin

The collectins, a group of humoral C-type lectins, have globular and collagen-like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen-like region of C1q (anti-C1qCLR) might cross-react with collectins, such as mannan-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin (BK). Anti-C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti-C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross-absorption and elution experiments combined with antibody detection by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross-reactive anti-C1qCLR antibodies. However, one serum with HUVS type anti-C1qCLR antibodies contained anti-MBP antibodies that were cross-reactive with SP-A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti-C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen-like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.     

Anti-human thyroid peroxidase and anti-human thyroglobulin antibodies present no cross-reactivity on recombinant peptides.

Thyroglobulin (Tg) and thyroid peroxidase (TPO) are two antigens largely recognized by the sera from patients with autoimmune thyroid disease (AITD). Recently, the complete mapping of both antigens was established with rabbit polyclonal antibodies by the use of recombinant proteins expressed in prokaryotic vector. Several investigators have argued for the existence of a cross-reactivity of some hetero- and autologous antibodies versus these two proteins. In the present study, using rabbit polyclonal antibody, mouse polyclonal antibody and autoimmune antibody (aAb), we observed no common epitope on human Tg (hTg) and human TPO (hTPO).     

Circulating Myeloperoxidase May Cause False Negative Findings in the Analysis of Myeloperoxidase Antibodies in Systemic Vasculitis

In systemic vasculitis reliable detection of myeloperoxidase antibodies (MPO-Abs) is of great clinical importance in the diagnosis and follow-up of patients. We have studied whether circulating myeloperoxidase (MPO) could have an effect on MPO-Ab findings. Serum MPO and MPO-Abs were measured in 50 healthy individuals, 35 patients and in the follow-up samples from two patients with Wegener's granulomatosis.
Heating the sera at 56°C for 30min reduced the concentration of immunoreactive MPO both in control and patient sera. In 71% of the patient sera heating made initially negative MPO-Abs detectable. In a few cases with severe vasculitis the antibody findings remained totally negative. These results, together with the data from the follow-up samples from two patients with Wegener's granulomatosis, revealed that the serological diagnosis of vasculitis may be considerably delayed if only native samples are analysed for MPO-Abs. These findings are of considerable clinical significance for the interpretation of MPO-Ab results.
Circulating myeloperoxidase affects MPO-Ab measurements, causing false negative findings in MPO-Ab assays. Therefore, it is recommended to denaturate circulating MPO by heating the sera before the analysis of MPO-Abs and to re-evaluate the cut off-values.     

Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses

Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1- and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.     

Mutational analysis of immunoglobulin E-binding epitopes of β-casein and β-lactoglobulin showed a heterogeneous pattern of critical amino acids between individual patients and pooled sera

BACKGROUND: For immunotherapeutic approaches, 'critical' amino acids (AAs) within allergenic epitopes are replaced with alternate AAs to eliminate IgE antibody binding. OBJECTIVE: To determine the critical AAs for IgE binding in beta-casein and beta-lactoglobulin (BLG). METHODS: Peptides of 10-14 AAs in length were synthesized on a derivatized cellulose membrane with single AA substitutions (alanine or glycine) at each position. Membranes were incubated with a pool of sera from 15 cow's milk-allergic patients and individual sera from six of the 15 patients. In cases where no decrease in binding occurred with a single AA substitution, peptides with two AA substitutions were generated and labelled. RESULTS: Using pooled patient sera, single AA substitutions led to complete elimination of binding to six of 11 peptides for beta-casein and to all six peptides for BLG. Substituting two AAs led to an elimination of binding to four of the remaining five beta-casein epitopes. However, in three of the 11 modified beta-casein peptides and five of the six BLG peptides, no decrease in IgE binding occurred in at least one individual patient. For these patients, critical AAs other than those defined by the patient serum pool were identified, indicating a heterogeneous pattern of IgE recognition. CONCLUSION: These results indicate that AAs critical for IgE binding are more heterogeneous than initially defined by pooled milk-allergic patient sera. For future immunotherapeutic interventions with mutated peptides, critical AAs should also be identified with individual patient sera to account for heterogeneity of IgE binding between patients.     

Comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for JC virus and BK virus

A comparison of antibody titers to JC virus (JCV) or BK virus (BKV) was made by hemagglutination inhibition (HI) and enzyme immunoassay (EIA) with 114 human plasma samples. Antibody titers to JCV or BKV determined by HI were lower than those determined by EIA. Nevertheless, as HI titers increased so did EIA titers. When antibody data were compared by the Spearman rank correlation test, highly significant correlations were found between HI and EIA titers. Results obtained by plotting EIA antibody titers for JCV against those for BKV generally showed a reciprocal relationship, i.e., samples with high antibody titers to JCV had lower antibody titers to BKV and vice versa. Some samples, however, had antibody titers to both viruses. Of the samples tested, 25.4% (25 of 114) had HI and EIA antibody titers to JCV and BKV which were identical or closely related. This is not the scenario one would expect for cross-reactive epitopes shared by the two viruses, but one suggesting that these samples were from individuals who had experienced infections by both viruses. Adsorption with concentrated JCV or BKV antigen of sera with high antibody titers to both JCV and BKV and testing by JCV and BKV EIA gave results which support this conclusion. Although 52.6% (51 of 97) of the samples from the Japanese population tested had very high antibody titers (>/=40,960) to either JCV or BKV, none of the samples were found by a dot blot immunoassay to have antibodies which cross-reacted with simian virus 40. The results from this study, in agreement with those of others, suggest that humans infected by JCV or BKV produce antibodies to species-specific epitopes on their VP1 capsid protein, which is associated with hemagglutination and cellular binding.     

Recognition of B-Cell Epitopes of the 65 kDa HSP in Behçet's Disease

B-cell epitopes of the mycobacterial 65 kDa heat shock protein (HSP) were mapped in sera from patients with Behçet's Disease (BD). A series of 47 overlapping synthetic peptides (15^ers) derived from the sequence of the Mycobacterium tuberculosis 65 kDa HSP was used in ELISA. Significant increases in IgA and IgG antibody levels were observed with peptides 111–125, 154–172 and 311–326 in sera from BD, compared with those from controls. Homologous peptides derived from the sequence of the human mitochondrial 60 kDa HSP were then examined. Peptides 136–150 and 336–351 showed comparable results to the homologous mycobacterial peptides 111–125 and 311–326, respectively. The B-cell epitopes defined in this investigation overlap with the T-cell epitopes the authors have previously reported in BD. Inhibition studies are consistent with the view that antibodies to each of the three B-cell epitope peptides represent a small proportion of the total B-cell epitope repertoire elicited by the 65 or 60 kD HSP. Sequential antibody studies suggest that IgA and IgG antibody titres to one or all three peptides tested may increase during exacerbations of ocular disease. The functional role of these antibodies needs to be determined, but the peptides may be involved in the immunopathogenesis of BD as they can induce experimental uveitis in Lewis rats, which is a principal manifestation of BD.     

Comparative studies on neutralisation of primary HIV-1 isolates by human sera and rabbit anti-V3 peptide sera.

IgG binding to V3 peptides and serum neutralising responses were studied in four HIV-1 infected individuals with progressive disease over a period of 31-70 months. The 18-20 mer peptides comprised residues 299-317 (numbering of HIV1 MN) in the N-terminal half of the V3 loop of the envelope glycoprotein gp120 and were derived from the sequences of autologous, as well as heterologous isolates. All four individuals studied lacked anti-V3 IgG binding to at least one autologous V3 sequence. V3 peptides to which autologous sera lacked binding IgG were all immunogenic in rabbits and induced antisera that were broadly cross-reactive by EIA and broadly cross-neutralising to primary HIV-1 isolates. This indicates that the peptides are immunogenic per se and that the respective human hosts have selective defects in recognising the corresponding V3 sequences. Despite the absence of antibody binding to autologous V3 peptides, the human sera had neutralising antibodies to autologous (three out of four cases), as well as heterologous isolates (all cases). Moreover, in vitro exposure of the patients' isolates to autologous neutralising serum or the homologous rabbit antiserum selected for variants with amino acid substitutions close to the crown of the V3 loop or in regions outside the sequence corresponding to peptides used for immunisation. The amino acid exchanges affected V3 positions known to be antigenic and which are also prone to change successively in infected persons. It is likely that neutralising antibodies recognise both linear and conformational epitopes in the V3 loop. Apparently, there are several, but restricted, numbers of ways for this structure to change its conformation and thereby give rise to neutralisation resistant viruses.     

Identification of antibody epitopes in the CB-11 peptide of bovine type II collagen recognized by sera from arthritis-susceptible and -resistant rhesus monkeys.

Sera from eight rhesus monkeys that had been immunized with native bovine type II collagen were tested for antibodies to cyanogen bromide peptides (CB peptides) of type II collagen by Western blotting. The monkeys produced IgG antibodies to a number of different CB peptides, with five out of eight animals producing antibodies to the CB-11 peptide (four arthritic, one non-arthritic). Antibody epitopes on the CB-11 peptide of bovine type II collagen recognized by these sera were investigated by epitope mapping. Peptides (8-mers overlapping by seven amino acids) representing the CB-11 region were synthesised and the sera screened for binding to these peptides to determine areas of high IgG antibody binding to this region of type II collagen. The profiles obtained were not identical, though there were some epitopes that were commonly recognized. Antibodies to one epitope, also present in human type II collagen, were found only in the sera of two animals with the severest arthritis. The technique of epitope mapping has successfully identified a number of epitopes within the CB-11 peptide of type II collagen recognized by antibodies from bovine type II collagen-immunized monkeys. Studies on the relevance of responses to the identified epitopes can now be undertaken.     

The effect of dithiotreitol on thyroid peroxidase and microsomal antigen epitopes recognized by auto and monoclonal antibodies.

The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).