Modification of the ELISA for the estimation of tetanus antitoxin in human sera

The use of indirect ELISA for the quantitation of tetanus toxin neutralizing antibodies in human sera is limited by marked overestimations in low titered sera. The reasons for the discrepancy between the results obtained by ELISA and by in vivo assay and modifications of the ELISA to overcome the problem were investigated. Catching ELISA and indirect ELISA using trays coated with the contaminant proteins in toxoid preparations indicated that antibodies to contaminants were only partly responsible for the discrepancy and the introduction of these modifications did not solve the problem. In ELISA competition experiments with toxin neutralizing monoclonal antibodies, the human immunoglobulins irrelevant in toxin neutralization, but detectable in indirect ELISA, were found to be difficult to inhibit in their binding to the solid antigen phase. These might represent antitoxins bound bivalently to the solid phase but with affinities in monovalent binding insufficient for toxin neutralization or other coupled antibodies due to conformational changes of the antigen. A competition ELISA with toxin in solution was therefore developed to assess selectively the antitoxin capable of binding the antigen in solution and by this approach the in vivo activities of even low titered sera were accurately predicted. This antigen competition ELISA may be easily introduced into routine tetanus serology and the principle may also be of value for the in vitro detection of functional antibodies to other antigens.     

Induction of neutralizing antibody in mice against poliovirus type II with monoclonal anti-idiotypic antibody

Syngeneic monoclonal anti-idiotope antibody Ab2,2-17C3SCC was raised against an idiotope on a protective monoclonal antibody with specificity for poliovirus type II. Ab2,2-17C3SCC detects a paratope-related interspecies IdX. Ab2,2-17C3SCC purified from supernatant fluids of hybridoma cells by protein A-Sepharose was injected into 4- to 6-wk-old BALB/c mice. The sera of the mice were screened for the expression of antibodies bearing the corresponding idiotope. Immunization of mice with Ab2,2-17C3SCC induced antibodies of complementary specificity. Furthermore, micro VN tests suggest that Ab2,2-17C3SCC can substitute for antigen in the induction of anti-polio neutralizing antibodies, and hence can function as a monoclonal anti-idiotypic vaccine.     

Toxoplasma gondii infection: analysis of serological response by 2-DE immunoblotting

The potency testing of Clostridium perfringens mono- and multicomponent veterinary vaccines is currently performed with the mouse neutralisation test (MNT) to estimate levels of C. perfringens beta- and epsilon-antitoxin levels in the sera of rabbits immunised with the vaccine. Two in vitro methods based on monoclonal antibodies (mAb) have been developed for the determination of specific antibodies against C. perfringens beta-toxin (capture ELISA) and epsilon-toxin (competitive ELISA) in these sera. Both test systems show high specificity and good reproducibility. These ELISA procedures were used in addition to the routine batch potency test in mice (MNT) to determine beta- and epsilon-antitoxin levels in 523 samples of rabbit serum. There was good agreement between the rank order of sera determined in vivo and the rank order determined in vitro. Linear regression analysis gave correlation coefficients of 0.88 for the capture ELISA and 0.41 for the competitive ELISA, with a significance level of P < 0.01 in both cases. Furthermore, a prevalidation study was carried out in four laboratories to evaluate the transferability of the ELISA procedures and the interlaboratory reproducibility of the results. Three coded serum samples were tested several times. The results indicated that both ELISA systems are suitable candidates to replace the MNT used for the potency testing of beta- and epsilon-toxoid in mono- and multicomponent veterinary vaccines. However, these assays still need to be validated in an international collaborative study.     

Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method.

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.     

抗HLJ1单克隆抗体的制备及抗原检测方法的建立

为制备抗人肝脏DnaJ-like蛋白(Human liver DnaJ-like protein, HLJ1)的单克隆抗体, 并建立免疫组化和双抗体夹心ELISA检测HLJ1的方法。采用淋巴细胞杂交瘤技术, 获得两株能稳定分泌抗HLJ1单克隆抗体的杂交瘤细胞株A4C7和 C4C8。经鉴定, 两株单抗的亚类均为IgG1, 并且效价高、特异性好。以单抗A4C7和C4C8作为一抗, 对人胎肝组织石蜡切片进行免疫组化染色, 结果表明, 两株单抗均为阳性染色, 且HLJ1主要定位于胎肝细胞的胞浆。选取A4C7进行HRP酶标记, 并以HRP- A4C7作为酶标抗体, 以C4C8作为包被抗体, 建立双抗体夹心ELISA方法, 并进行棋盘滴定确定抗体的最佳工作浓度。该检测方法的线性范围是15~750 ng/mL, 灵敏度下限达15 ng/mL, 特异性良好。所建立的免疫组化和双抗体夹心ELISA 法可用于快速、灵敏地检测组织及血清中的HLJ1蛋白, 为HLJ1的肿瘤相关性研究提供了有力的工具。     

Isolation, purification and characterization of surface antigens of the bovine filarial parasite Setaria digitata for the immunodiagnosis of bancroftian filariasis

The surface antigens of the bovine filarial parasite Setaria digitata were isolated by EDTA extraction and purified by affinity chromatography using sepharose bound human filarial (Wuchereria bancrofti) antibodies obtained from chronic human filarial sera. The purified and crude antigens were used in enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies in bancroftian filariasis. The purified antigen showed sensitive and specific reactions in ELISA for the detection of antibodies in filarial sera and showed least cross reactivity with other parasitic infections. The crude and purified antigens showed about 18 and 6 peptide bands respectively in SDS-PAGE and about 11 and 6 antigenic bands respectively in enzyme-linked immunoelectrotransfer blot (EITB). The purified antigen was observed to be glycoprotein in nature. It was possible to identify the stage-specific infection in human filariasis by using the crude and purified antigens in EITB.     

A poliovirus type 1 neutralization epitope is located within amino acid residues 93 to 104 of viral capsid polypeptide VP1.

Using nuclease Bal31, deletions were generated within the poliovirus type 1 cDNA sequences, coding for capsid polypeptide VP1, within plasmid pCW119. The fusion proteins expressed in Escherichia coli by the deleted plasmids reacted with rabbit immune sera directed against poliovirus capsid polypeptide VP1 (alpha VP1 antibodies). They also reacted with a poliovirus type 1 neutralizing monoclonal antibody C3, but reactivity was lost when the deletion extended up to VP1 amino acids 90-104. Computer analysis of the protein revealed a high local density of hydrophilic amino acid residues in the region of VP1 amino acids 93-103. A peptide representing the sequence of this region was chemically synthesized. Once coupled to keyhole limpet hemocyanin, this peptide was specifically immunoprecipitated by C3 antibodies. The peptide also inhibited the neutralization of poliovirus type 1 by C3 antibodies. We thus conclude that the neutralization epitope recognized by C3 is located within the region of amino acids 93-104 of capsid polypeptide VP1.     

Improved distribution of antigenic site specificity of poliovirus-neutralizing antibodies induced by a protease-cleaved immunogen in mice.

Previous studies showed that the distribution of antigenic site specificity of neutralizing antibodies to type 3 poliovirus obtained with the inactivated poliovirus vaccine can be deficient as compared with that obtained following poliovirus infection. This observation was shown by the relatively low capacity of sera from inactivated-poliovirus-vaccine-immunized persons to neutralize poliovirus cleaved at antigenic site 1. We investigated possibilities for improving the situation in a mouse model. Balb/c mice were immunized with intact or trypsin-cleaved type 3 poliovirus (Saukett strain). Sera from mice immunized with the intact virus readily neutralized the intact virus but neutralized the cleaved virus only rarely. In contrast, cleaved-virus-immunized mice produced antibodies that were able to neutralize the cleaved virus as well as the intact one. Mice immunized with a 100-fold-higher dose of the intact virus produced significant levels of antibodies to the cleaved virus, too. Somewhat surprisingly, mice immunized with high doses of the cleaved virus produced antibodies specific for the intact loop between beta sheets B and C of VP1 (virion protein 1), which should be cleaved in the immunogen. This was shown by a higher titer of antibodies to intact Saukett virus than to the corresponding cleaved virus, as well as to a type 1/type 3 hybrid poliovirus in which only the BC loop amino acids were derived from type 3 poliovirus. The cleavage-induced enhanced availability of antigenic determinants residing outside the BC loop was also shown by increased neutralization titers of monoclonal antibodies specific for some of these other determinants. These results indicate that by using a trypsin-cleaved type 3 poliovirus as a parenteral immunogen, it is possible to change the distribution of antigenic site specificities of neutralizing antibodies to resemble that following poliovirus infection.     

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.     

The development of an enzyme-linked immunosorbent assay for measuring the potency of vaccines containing Clostridium chauvoei antigens

Current standards (British Pharmacopeia (Veterinary) 1985) for vaccines containing Clostridium chauvoei require a potency test based on a challenge assay in guinea-pigs. Animal welfare and cost considerations favour the development of alternatives. Most veterinary clostridial vaccines are multi component, requiring assays in rabbits to test the potency of components other than C. chauvoei. We describe the application of an ELISA to measure the response to C. chauvoei vaccines in rabbits. The antigen is a sonicated extract of C. chauvoei strain CH4, intended to include a mixture of cellular and soluble antigens. The rabbit response to more than 70 vaccines containing C. chauvoei has been assessed against a reference serum which has been assigned an arbitrary potency of 100 units ml-1. The antibody titres of rabbit sera have been compared with the results of guinea-pig challenge potency tests on the same vaccines. The pass level in the guinea-pig potency test is equivalent to a rabbit ELISA titre of 50 units ml-1.     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.     

Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28,400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000-2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine.     

Immunodiagnosis of human dirofilariasis by enzyme-linked immunosorbent assay using recombinant DNA-derived fusion protein.

An enzyme-linked immunosorbent assay (ELISA) using antigenic beta-galactosidase-Dirofilaria immitis recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human dirofilariasis. D. immitis-infected human sera reacted strongly with FP that was immobilized with anti-beta-galactosidase monoclonal antibody on microplates. However, the FP did not react with sera from patients with other filariasis. In detection of anti-D. immitis IgG antibody. ELISA using FP was highly sensitive and specific compared to that using crude somatic antigen.     

Self-antigen tetramers discriminate between myelin autoantibodies to native or denatured protein

The role of autoantibodies in the pathogenesis of multiple sclerosis (MS) and other demyelinating diseases is controversial, in part because widely used western blotting and ELISA methods either do not permit the detection of conformation-sensitive antibodies or do not distinguish them from conformation-independent antibodies. We developed a sensitive assay based on self-assembling radiolabeled tetramers that allows discrimination of antibodies against folded or denatured myelin oligodendrocyte glycoprotein (MOG) by selective unfolding of the antigen domain. The tetramer radioimmunoassay (RIA) was more sensitive for MOG autoantibody detection than other methodologies, including monomer-based RIA, ELISA or fluorescent-activated cell sorting (FACS). Autoantibodies from individuals with acute disseminated encephalomyelitis (ADEM) selectively bound the folded MOG tetramer, whereas sera from mice with experimental autoimmune encephalomyelitis induced with MOG peptide immunoprecipitated only the unfolded tetramer. MOG-specific autoantibodies were identified in a subset of ADEM but only rarely in adult-onset MS cases, indicating that MOG is a more prominent target antigen in ADEM than MS.     

Detection of cross-reactive antibody to BLV p24 in sera of human patients infected with HTLV

For detection of antibody to bovine leukemia virus (BLV) major core protein of p24 and cross-reactive antibody in human patients infected with human T cell leukemia virus type I (HTLV-I), monoclonal antibody, D432 against BLV p24 was used by competitive binding enzyme-linked immunoadsorbed assay (ELISA). In sera from cattle with enzootic bovine leukosis (EBL) which were positive for BLV antibodies by immunodiffusion test, 109 out of 112 (97.3%) were positive for BLV p24 antibody by competitive binding ELISA. By using the same procedures, 21 samples from adult T cell leukemia (ATL) patients and healthy carriers with HTLV-I were tested for cross-reactive antibody to BLV p24. All 21 samples were positive for HTLV-I antibodies by immunofluorescence test and/or ELISA. By competitive binding ELISA using non-treated BLV antigens, none of these 21 samples inhibited the binding of the D432. When the BLV antigen was treated by several different denaturation procedures, several HTLV-I positive samples showed the inhibition of the D432 binding and the most effective treatment was by 2-mercaptoethanol (2-ME). Sixteen out of 21 samples showed the presence of cross-reactive antibody against 2-ME-treated BLV antigens. The cross-reactivity of human sample to BLV p24 antigen was further confirmed by Western blotting of the 2-ME-treated BLV antigens. None of the 28 samples from leukemia patients other than ATL which were negative for HTLV-I antibodies showed inhibition of the D432 by the competitive binding ELISA.     

Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay

Background

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.

Methodology/Principal Findings

We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer.

Conclusions/Significance

Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.     

Development of a Blocking ELISA for Detection of Serum Neutralizing Antibodies against Newly Emerged Duck Tembusu Virus

Background

Since April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The causative agent was identified as a duck Tembusu virus (DTMUV), a member of the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. DTMUV is highly contagious and spreads rapidly in many species of ducks. More than 10 million shelducks have been infected and approximately 1 million died in 2010. The disease remains a constant threat to the duck industry; however, it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world. The lack of reliable methods to detect the serum antibodies against DTMUV has limited our ability to conduct epidemiological investigations in various natural hosts and to evaluate the efficiency of vaccines to DTMUV.

Methodology/Principal Findings

A neutralizing monoclonal antibody (mAb) 1F5 binding specifically to the E protein was developed. Based on the mAb, a blocking enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neutralizing antibodies against DTMUV. The average value of percent inhibition (PI) of 350 duck serum samples obtained from DTMUV-free farms was 1.0% ±5.8% (mean ± SD). The selected cut-off PI values for negative and positive sera were 12.6% (mean +2SD) and 18.4% (mean +3SD), respectively. When compared with a serum neutralizing antibody test (SNT) using chicken embryonated eggs, the rate of coincidence was 70.6% between the blocking ELISA and SNT, based on the titration of 20 duck DTMUV-positive serum samples.

Conclusions/Significance

The blocking ELISA based on a neutralizing mAb allowed rapid, sensitive, and specific detection of neutralization-related antibodies against DTMUV.     

Methods for analysis of biologically functional antibodies to the HIV-1 gp120 principal neutralizing domain.

The humoral response to HIV-1 infection has been demonstrated by a variety of immunoassays utilizing viral proteins. While several assays detect HIV-1 infection with high sensitivity and great specificity, little progress has been made to develop immunoassays correlative with disease progression and viral transmission. Antibodies toward the V3 domain of HIV-1 envelope can prevent virus infection and block virus-mediated cell fusion in vitro. Such properties may be critical to the course of the disease. Furthermore, understanding the role of neutralizing antibodies against HIV-1 during infection in humans and generating biologically relevant neutralizing antibodies are paramount to developing an efficacious AIDS vaccine. In this study we explored peptide binding and neutralization assays and their relation to predicting disease progression and viral transmission. Biologically relevant polyclonal and monoclonal neutralizing antibodies that were derived from natural HIV-1 infection of humans, experimental infections of chimpanzees, and viral envelope protein peptide immunizations were characterized. Comparison of V3-specific monoclonal antibodies by antigen-limited ELISA and a quantitative HIV-1 neutralization assay demonstrated a less than optimal predictive relationship between binding and neutralization potency. On the other hand, polyclonal sera from goats immunized with V3-specific peptides derived from three different HIV-1 strains, as well as sera from other HIV-1-infected individuals demonstrated correlation between binding affinity and neutralization.     

Evaluation of an enzyme-linked immunosorbent assay based on binding inhibition for type-specific quantification of poliovirus neutralization-relevant antibodies

To detect neutralization-relevant antibodies against 3 types of poliovirus (PV) without using tissue cultures and live viruses, an enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody-binding inhibition was evaluated using sera from 80 vaccinated Japanese children and 60 Pakistani poliomyelitis patients. Compared with the neutralization test, the sensitivity of the inhibition ELISA was 100% (111/111) for detection of anti-PV1 antibody, 98.3% (118/120) for anti-PV2, and 96.5% (82/85) for anti-PV3, and the specificity was 93.1% (27/29), 100% (20/20), and 92.7% (51/55), respectively. Thus, the inhibition ELISA showed excellent potential as a seroepidemiologic tool in both vaccinated and naturally-infected populations.     

Immunogenic regions of the GA733-2 tumour-associated antigen recognised by autoantibodies of patients with colorectal carcinoma

The tumour-associated antigen (TAA) GA733-2 is overexpressed by >90% of human colorectal carcinomas (CRC). The antigen has previously been shown to be recognised by B and T cells. The aim of the present study was to define B cell epitopes of GA733-2. Fifteen percent of CRC patients with no previous immunotherapy have recently been shown to elicit an anti-GA733-2 IgG antibody response. Sera of these patients ( n=136) were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies against 23 partly overlapping synthetic peptides (18 amino acids: aa) derived from the extracellular domain of GA733-2. An 18-aa long sequence at the N-terminal region of the antigen (peptide 2) was found to be an immunodominant B cell epitope. Fifty percent of the patients had antibodies against peptide 2, while 8% to 9% had antibodies against peptides 1, 4, 7, 8 or 20. In healthy donors ( n=30) antibodies against peptides 2 and 8 were also detected in 13% and 3% of cases respectively, while no antibodies were found against the other peptides and the complete protein. Thirteen percent of CRC patients ( n=30) with no IgG antibodies against the GA733-2 antigen elicited antibodies against peptide 2. The specificity of peptide-reactive sera was verified by inhibition ELISA. The binding of sera to GA733-2 was significantly inhibited by peptides to which CRC sera bound, but not by control peptides. Binding to peptide 2 of sera showing both peptide 2 and GA733-2 reactivity was specifically inhibited by the complete GA733-2 antigen, while binding of peptide 2-reactive sera showing no GA733-2 reactivity was not inhibited. CRC sera interfered with the binding of monoclonal antibody (mAb) 17-1A and mAb C215 that recognise distinct epitopes of GA733-2. No significant correlation was found between the presence of anti-peptide antibodies in CRC patients and clinical stage or overall survival. The results provide additional evidence for immune recognition of CRC by the host.