Human immunoglobulin G subclass immune response to outer membrane antigens in meningococcal group B vaccine

The immunoglobulin G (IgG) subclass distribution of antibodies against the major outer membrane proteins from serotype 2a Neisseria meningitidis in human vaccinees was studied by immunoblotting. The volunteers received two doses of a noncovalent complex of group B polysaccharide and outer membrane material from the same meningococcal strain. Six weeks after the first vaccination the antibodies mounted against the class 1 and 5 proteins belonged mainly to the IgG1 and IgG3 subclasses. However, the binding of IgG3 to the class 5 proteins decreased markedly in serum samples taken after 25 weeks. Antibody binding to the serotype-specific class 2 protein was dependent on renaturation of the antigen by a dipolar ionic detergent (R. E. Mandrell and W. D. Zollinger, J. Immunol. Methods 67:1-11, 1984). The immune response against this protein showed more individual variation and consisted of IgG1 or IgG3 or both, often combined with IgG4.     

Human immune response to meningococcal outer membrane protein epitopes after natural infection or vaccination

Antibody levels in 41 sets of human acute- and convalescent-phase meningococcal sera were compared with those in 23 sets of human prevaccination and 2-week postvaccination sera. We used a modification of a solid-phase radioimmunoassay (SPRIA) technique to test each of the human serum samples as inhibitors of monoclonal antibodies (MAbs) that bind (HIMSPRIA) to the outer membrane complex from a 2a:P1.2:P5.1 strain. We used three murine MAbs specific for the 2a, P1.2, and P5.1 epitopes on meningococcal class 1, 2, and 5 proteins, respectively, to detect antibodies with similar specificities in human sera. Each of 40 available matching strains from patients were also screened with the three MAbs in a nitrocellulose spot blot assay. A total of 37 (92%) were positive for the 2a epitope, 36 (90%) were positive for the P1.2 epitope, and 16 (40%) were positive for the P5.1 epitope. Of 38 available convalescent-phase sera, 27 (71%) matched with these strains and had detectable inhibiting antibody for each of the MAb-defined protein epitopes of the infecting strain. Three convalescent-phase sera had no HIMSPRIA activity for MAb-defined epitopes that were present on the infecting strain; others had activity for one or two of the epitopes. The results were similar for pre- and postvaccination sera. The average level of HIMSPRIA activity for the P1.2 epitope was greater than fivefold higher in postvaccination sera compared with that in convalescent-phase sera. Sera with distinct patterns of HIMSPRIA activity also were tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis and showed a correlation between the HIMSPRIA activity for particular epitopes and the level of antibody binding to the immunoblotted proteins possessing those epitopes. A comparison of the HIMSPRIA and the bactericidal activity of selected postvaccination sera indicated a possible correlation between HIMSPRIA and bactericidal activity, but it also suggested the presence of bactericidal antibodies with specificities other than those defined by the MAbs.     

Human antibody response to outer membrane proteins of Campylobacter jejuni during infection.

Two techniques were used to isolate outer membrane proteins from Campylobacter jejuni, EDTA-lysozyme extraction and sodium-N-lauroylsarcosinate (Sarkosyl) solubilization. The protein profiles of the two preparations were similar, with a few additional bands in the EDTA-lysozyme preparations. The major outer membrane protein was 43,000 (43K) daltons, and there were 8 to 10 minor bands ranging from 92K to 14K daltons. There was no difference in the protein profile of a strain causing an infection (strain 17) and the resulting stool isolate (strain 17J). Sera collected before the infection and during the acute and convalescent stages were used with Western blotting and immunoautoradiographic techniques to determine the antigenicity of outer membrane proteins. A number of antigenic proteins were detected before the infection by their reaction with preinfection serum (61K, 51K, 43K, 40K, 34K, and 31K daltons), and three additional bands appeared during the infection when acute and convalescent sera were used (92K, 56K, and 19K daltons). Furthermore, an area of the gel at less than 14.4K daltons that did not stain with Coomassie brilliant blue became visible in the immune blots when the convalescent serum was used.     

Restoration of antibody binding to blotted meningococcal outer membrane proteins using various detergents

Restoration of IgG antibody binding to heat-denatured meningococcal outer membrane proteins has been studied on immunoblots with a series of 14 detergents. Nitrocellulose strips with the blotted proteins were incubated with the detergents and sera from human volunteers vaccinated with meningococcal membrane proteins. Zwitterionic and ionic detergents, containing substituted quarternary ammonium or amino groups with a minimum of 10 C atoms in the alkyl chain, restored the antigenicity of the serotype-specific class 2 porin protein. The concentrations of the Zwittergent detergents necessary for activation decreased with increasing alkyl chain length of the homologues. Only zwitterionic detergents renatured the class 1 protein. Both proteins were weakly antigenic in the presence of the nonionic detergents Triton X-100 and Tween 20. Meningococcal lipopolysaccharide restored antibody binding to the porin, but not to the class 1 protein. Similar concentrations of lipopolysaccharides from two other gram-negative bacteria had no effect.     

IgG subclass antibodies to serogroup B meningococcal outer membrane antigens following infection and vaccination

IgG and IgG subclass antibodies to the outer membrane antigens from Neisseria meningitidis (serogroup B, serotype 15:P1.16) were quantitated by an enzyme-linked immunosorbent assay (ELISA) in sera from 40 patients with group B:15:P1.16 meningococcal disease and 24 volunteers immunized with a serotype 15:P1.16 outer membrane vesicle vaccine. A second injection was given 6 weeks after the first immunization. Patient sera obtained two and six weeks after onset of the disease had significantly higher levels of total IgG, IgG1, IgG2, and IgG3 antibodies to the outer membrane antigens than acute sera, convalescent sera from patients with systemic non-meningococcal bacterial infections and sera from healthy controls. The levels of total IgG and IgG1 remained high one and three years later. Sera from the vaccinees showed high levels of total IgG and IgG1 6, 12 and 26 weeks after the first immunization and high levels of IgG3 6 weeks after the second immunization. No increase of IgG2 or IgG4 levels was observed in the postimmunization sera. Immunoblotting of three convalescent sera demonstrated individual patterns of IgG subclass binding to various outer membrane antigens with most distinct binding of IgG1 and IgG3 antibodies to the class I protein, the H.8 lipoprotein and the lipopolysaccharide. Since IgG1 and IgG3 are the most effective antibodies for complement activation and phagocytosis, group B meningococcal disease and immunization with the serotype 15:P1.16 outer membrane vesicle vaccine stimulate production of those IgG subclasses which have the strongest opsonic and bactericidal activity.     

Antibody responses to serogroup B meningococcal outer membrane antigens after vaccination and infection.

Antibody responses of adult volunteers given a vaccine containing meningococcal capsular polysaccharides (serogroups A, C, Y, and W-135) noncovalently complexed with serotype 2b:P1.2 and 15:P1.16 outer membrane proteins have been studied. Sera were analyzed by enzyme-linked immunosorbent assay methods for immunoglobulin G (IgG), IgM, and IgA antibodies and for bactericidal activities against the homologous strains. The vaccination was performed as a double-blind experiment with 47 volunteers, of whom 23 received the protein-polysaccharide vaccine and 24 received the control preparation containing the polysaccharides only. Ten additional persons volunteered for the protein-polysaccharide vaccine. Before vaccination, carriers of meningococci had significantly higher levels of specific IgG and IgA and also higher bactericidal activities than noncarriers. At 2 weeks postvaccination we found significant IgG and bactericidal antibody responses against both the 2b:P1.2 and 15:P1.16 strains in about 70% of the protein-polysaccharide vaccinees. The immune response induced by disease was compared with that induced by vaccination by analyzing paired sera from 13 survivors of serogroup B serotype 15 meningococcal disease. We found that the mean specific IgG level in acute-phase sera was lower than average in prevaccination sera from the vaccinees but similar to that of healthy noncarriers before vaccination. The convalescent-phase sera showed IgG responses similar to those of the vaccinees, but the IgM response to disease was significantly higher than after vaccination. The immune response to disease caused by serogroup B serotype 15 meningococci was found by enzyme-linked immunosorbent assay analysis to be about the same with outer-membrane antigens from a serotype 2b strain as it was with antigens from a serotype 15 strain.     

Quantification of specific antibody response to Cryptosporidium antigens by laser densitometry.

Cryptosporidium spp. is a protozoan parasite with worldwide distribution associated with diarrhea in immunocompromised patients (particularly those with acquired immunodeficiency syndrome [AIDS]) and in immunocompetent humans. Immunoglobulin M (IgM) and IgG antibody responses are readily detected by an enzyme-linked immunosorbent assay. To determine which Cryptosporidium antigens invoke antibody responses in humans, we performed polyacrylamide gel electrophoresis using purified oocysts, followed by Western blots with human sera from various populations. Of 40 sera from persons with cryptosporidiosis (24 AIDS and 16 non-AIDS patients), in 37 (93%) a 23,000-dalton antigen measured quantitatively by laser densitometry was recognized. Of 63 sera from IgM- or IgG-positive individuals, as determined by enzyme-linked immunosorbent assay, in 58 (92%) this same antigen was recognized. Up to three additional bands between 125,000 and 175,000 daltons were identified by some of these sera. These results suggest that most persons infected with Cryptosporidium spp. produce antibodies which recognize at least one common low-molecular-weight antigen. Isolation of this antigen will be useful in development of diagnostic tests and may be important in the study of immunity.     

Human bactericidal antibody response to outer membrane protein P2 of nontypeable Haemophilus influenzae.

The human bactericidal antibody response to the major outer membrane protein, P2, of nontypeable Haemophilus influenzae was studied. P2 was isolated from two strains of nontypeable H. influenzae and coupled to affinity columns. Pooled normal human serum was subjected to affinity chromatography with the P2 columns and a control column. Reducing the titer of antibody to P2 resulted in reduced bactericidal activity of that serum for the organism. Immunopurified antibody to P2 from human serum was bactericidal for the homologous strain. The extent to which these bactericidal determinants on P2 are conserved among strains was investigated. Immunopurified antibodies to P2 of two epidemiologically unrelated isolates were bactericidal for four of six strains tested. We conclude that P2 is a target for human bactericidal antibody and that some of these determinants that are recognized by human bactericidal antibody are conserved among strains of nontypeable H. influenzae.     

Human immune response to Vibrio cholerae O1 whole cells and isolated outer membrane antigens.

The serum immunoglobulin G (IgG) and mucosal secretory IgA (SIgA) response of human volunteers challenged with Vibrio cholerae O1 was analyzed for reactivity to V. cholerae O1 antigens by the immunoblot technique. Components of both in vitro- and in vivo (rabbit ligated ileal loop)-grown V. cholerae O1 were separated by sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis. Postchallenge serum IgG reacted uniquely with 15 antigens and with greater intensity than did prechallenge serum with at least 16 antigens. Serum IgG and SIgA reacted with antigens present in preparations from the homologous challenge strain of V. cholerae as well as antigens from strains of heterologous biotype or serotype. These heterologous antigens may represent antigens responsible for protection to rechallenge with a heterologous strain of V. cholerae. All the antigens detected by postchallenge jejunal fluid SIgA had an apparent molecular size of less than 25 kilodaltons. Serum IgG and jejunal fluid SIgA also reacted with antigens unique to in vivo-grown cells and several antigens in outer membrane preparations, suggesting that studies of protective immunity and V. cholerae O1 pathogenesis should include examination of both in vitro- and in vivo-grown V. cholerae O1 cellular antigens.     

Human antibody response to outer membrane protein G1a, a lipoprotein of Moraxella catarrhalis

Moraxella catarrhalis is an important cause of respiratory infections in adults with chronic obstructive pulmonary disease (COPD) and of otitis media in children. Outer membrane protein (OMP) G1a is an approximately 29-kDa surface lipoprotein and is a potential vaccine candidate. The gene that encodes OMP G1a was expressed and purified using a novel plasmid vector. [(3)H]palmitic acid labeling demonstrated that both native and recombinant OMP G1a contain covalently bound palmitic acid. To assess the expression of OMP G1a during human infection, paired sera and sputum supernatants from adults with COPD followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant lipidated OMP G1a to detect antibodies made specifically during carriage of M. catarrhalis. Overall, 23% of patients developed either a serum immunoglobulin G (IgG) response (9%) or sputum IgA response (21%) to OMP G1a, following 100 episodes of acquisition and clearance of M. catarrhalis. Patients developed antibody responses at similar rates following episodes of clinical exacerbation compared to asymptomatic colonization. Serum IgG antibodies following natural infection were directed predominantly at OMP G1a epitopes that are not exposed on the bacterial surface. These data show that OMP G1a is expressed during infection of the human respiratory tract and is a target of systemic and mucosal antibodies. These observations indicate that OMP G1a, a highly conserved surface protein, should be evaluated further as a vaccine candidate.     

Serum antibodies to cross-reactive Neisseria outer membrane antigens in healthy persons and patients with meningococcal disease

Outer membranes (OMs) were prepared from the Neisseria meningitidis (Nm) strain 44/76, N. gonorrhoeae (Ng) NRL 8658, and N. lactamica (N1) ATCC 23970. Paired serum samples from 16 patients with serogroup B Nm disease and single samples from 30 blood donors were tested for IgG antibody levels against the three OMs in indirect ELISA, before and after absorption of the sera with N1 OM. Immunoblot analysis was used to identify OM target antigens for cross-reacting and strain-specific antibodies. Most of the Nm- and NG-antibodies in sera from healthy adults were directed against OM antigens shared by the three Neisseria strains. Nm disease induced antibody formation against common Neisseria antigens, identified as the H.8 antigen and LPS determinants, against LPS determinants shared only by the Nm and Ng strains and against a variety of Nm-specific OM antigens. Very low levels of Nm-specific antibodies characterized the Nm patients in the acute phase. Also, the results indicate that the OM ELISA which has been used for the diagnosis of Nm disease, would be more useful if antibodies against common Neisseria antigens were removed from the sera before testing.     

Human antibody response to a group B serotype 2a meningococcal vaccine determined by immunoblotting

The antibody response of 30 volunteers vaccinated with a complex of group B polysaccharide and outer membrane vesicles (OMV) from serotype 2a Neisseria meningitidis and of 3 individuals who received a placebo vaccine was determined by immunoblotting. OMV were separated by sodium dodecyl sulfate-gel electrophoresis and electrotransferred to nitrocellulose filters. Binding of immunoglobulin G (IgG), IgA, and IgM antibodies in the human sera to OMV components was detected with class-specific peroxidase-conjugated antibodies. The immunoblotting results were also related to the bactericidal activity of the sera and the meningococcal carrier status of the volunteers. Before vaccination weakly reactive bands in the molecular weight range of 140,000 to 10,000 were observed on the blots. Sera from carriers showed more marked bands. Individual patterns of increased reactivity were seen 6 weeks after vaccination. The main immunoreactive components of OMV corresponded to a molecular weight of 43,000 (class 1 protein), 30,000 (class 5 proteins), and 22,000. IgG antibodies in postvaccination sera of high bactericidal titers showed distinct binding to the 43,000-molecular-weight antigen. Meningococcal carriers had antibodies against an antigen of 22,000 molecular weight; in polyacrylamide gels this component did not stain with Coomassie brilliant blue or silver. The marked binding of IgG antibodies to the class 5 proteins decreased considerably between weeks 6 and 25 after vaccination. Periodate oxidation of OMV abolished the binding of IgG antibodies to the class 5 proteins, whereas the antigenicity of the 43,000-molecular-weight (class 1 protein) and 22,000-molecular-weight antigens was unaffected.     

Confirmation of invasive meningococcal disease by single point estimation of IgM antibody to outer membrane protein of Neisseria meningitidis

OBJECTIVES: The sensitivity of laboratory confirmation of invasive meningococcal disease (IMD) by culture or PCR is affected by prior antibiotic treatment and decreasing use of early lumbar puncture. Serological diagnosis of IMD is not widely used because of reliance on paired serum samples. The application of single point estimations of IgM antibodies in the diagnosis of IMD was explored. DESIGN: Outer membrane proteins from a mix of commonly encountered meningococcal serotypes were partially purified and used as an antigen in an enzyme immunoassay for the detection of IgM antibody. The cut-off for the assay was derived using sera from blood bank donors and the accuracy then evaluated with sera from patients with culture-confirmed IMD, other bacterial infections and culture-proven nasopharyngeal colonisation with Neisseria meningitidis. RESULTS: The coefficient of variability of the assay was < 10% in negative, mid- and high-range positive sera and the specificity of the assay was at least 93%. In sera collected from 49 adult patients at various times after positive blood or CSF culture-confirmed IMD, the assay had a sensitivity of 100% in specimens collected between 5 and 18 days. At the time of isolation of meningococci from either blood or CSF, eight of 29 sera were IgM-positive, but beyond 70 days no positive results were detected. No differences were seen in the IgM responses in patients from whom different serogroups of N. meningitidis were recovered. CONCLUSIONS: Serological examination by single point IgM enzyme immunoassay (EIA) offers the possibility of an expanded laboratory confirmation of IMD in adults for samples taken between 5 and 18 days after onset.     

Human opsonins induced during meningococcal disease recognize outer membrane proteins PorA and PorB

Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class 3) proteins purified from mutants of the same strain (44/76; B:15:P1.7. 16) were adsorbed to fluorescent beads, opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs) demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians [ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immunosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an indication that both of these important steps in the in vitro phagocytic elimination of meningococci are initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in future vaccines.     

Human immune response to iron-repressible outer membrane proteins of Neisseria meningitidis.

Neisseria meningitidis grown under iron-limiting conditions in vitro expresses additional iron-repressible outer membrane proteins (FeRPs). To see which FeRPs were expressed and immunogenic in human infection, we examined purified membranes from four meningococcal disease isolates with Western blotting of patient sera. Convalescent serum from each patient contained immunoglobulin G (IgG) and IgM antibodies against the homologous 70-kilodalton (kDa) FeRP and IgG antibody to the homologous 94-kDa FeRPs. Three other immunoreactive FeRPs were identified in two or more strains. Neither acute-phase sera nor pooled normal human sera contained appreciable levels of these antibodies. Antigenic cross-reactivity among FeRPs was suggested by the observation that the convalescent sera of two patients contained IgG antibodies reactive with the 70- and 94-kDa FeRPs and IgM antibodies reactive with the 70-kDa FeRPs from all four strains. Additionally, rabbit antiserum against the 70-kDa FeRP from one of these disease isolates contained IgG and IgM antibodies that reacted in Western blots with the 70-kDa FeRPs in all four strains. These results demonstrate that meningococcal FeRPs are expressed and immunogenic in vivo and that certain of these proteins are immunologically cross-reactive.     

Human serum antibody response against Streptococcus mutans antigens.

Antigens from Streptococcus mutans were examined to identify specific polypeptides that may have stimulated antibody responses and possibly play some role in caries immunity. A group of 10 adult human subjects was screened for serum antibodies reactive with antigens from S. mutans. Extracellular and cellular protein preparations from S. mutans LM7 (Bratthall serotype e) and V403 (biotype c) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western electrophoretic transfer and immunoblotting analysis. Antibodies reactive with polypeptides ranging from 34 to 400 kilodaltons in apparent molecular mass were detected by these means. Radioimmunoassay competition experiments revealed that the cellular and extracellular antigens did not compete with each other for serum antibodies. Preabsorption of sera with extracellular proteins from other oral streptococcal species prior to immunoblotting indicated that the antigens unique to S. mutans have molecular masses greater than 100 kilodaltons, and each individual produced antibodies against different antigens of high molecular mass. Examination of sera from young children also indicated heterogeneous responses against S. mutans LM7 antigens.     

A serologic study of cultured breast cancer cell lines: lack of antibody response to tumour specific membrane antigens in patients.

Humoral antibodies to tumour associated membrane antigens of cultured human breast cancer cell lines were studied using the immune adherence (IA) test. Sera from 353 post-operative breast cancer patients and from twenty-five patients immunized by allogeneic breast cancer cells were tested against the MDA-MB-436 cell line. Fifty-five (15.6%) sera samples from the non-vaccinated group and 131 (77.3%) of 168 sera samples from the immunotherapy group were IA-positive to this cell line after absorption with bovine erythrocytes to exclude antibody to heterologous membrane antigens (HM Ag). Forty-five of the 55 positive-sera from the non-immunized group and 113 of the 131 positive sera from the immunized group became IA-negative after further absorption with lymphoblastoid cells autologous to MDA-MB-436. Subsequently, the twenty-eight positive sera remaining sere tested for oncofetal antigens (OFA). After absorption with OFA rich tissues (fetal brain and M14 melanoma cells), no reactivity remained in the sera samples. In order to identify antibodies specific to breast cancer antigens, the 129 sera samples from non-immunized patients were tested against four other breast cancer cell lines; MDA-MB-157, MDA-MB-231, MCF-7 and UCLASO-B1. Four sera which reacted to more than three of the cell lines were identified. The reactivity of three of the four was due to anti-OFA antibody. The last serum sample was reactive to anti-HLA antibodies. These results indicate that sera of patients with breast cancer contain antibodies to OFA, but do not detect breast histologic type specific antigens as tested by IA using five breast cancer cultured cell lines.     

Measurement of the human immune response to meningococcal lipooligosaccharide antigens by using serum to inhibit monoclonal antibody binding to purified lipooligosaccharide.

We developed a human inhibition monoclonal enzyme-linked immunosorbent assay (HIMELISA) to investigate the human immune response to the lipooligosaccharides (LOS) of Neisseria meningitidis. Monoclonal antibodies (MAb) were used to define seven epitopes on four LOS molecules of a meningococcal strain (126E) previously shown to express immunogenic LOS epitopes. The assay could distinguish epitope-specific antibody within whole sera. Neither the specificity nor the amount of the antibody measured by HIMELISA in sera of vaccinates changed during the immune response to meningococcal capsular polysaccharides, a chemically unrelated antigen. By using the HIMELISA, it was determined that sera from adults convalescing from meningococcal disease strongly inhibited MAb binding to two of the seven defined epitopes. The 3.6-kilodalton LOS of strain 126E expressed both of these epitopes. In addition, one of the inhibited epitopes was also expressed on the 4.0-kilodalton LOS of strain 126E. The convalescent-phase sera inhibited MAb binding to these two epitopes when they were expressed on LOS of diverse meningococcal strains. An acute-phase serum blocked MAb to the two epitopes to a lesser degree than did a convalescent-phase serum from the same patient. Immunoblotting the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated LOS with convalescent-phase sera confirmed the specificity of the human anti-LOS antibody identified by HIMELISA.     

In vitro neutralization of Chlamydia trachomatis by monovalent Fab antibody specific to the major outer membrane protein.

Monovalent Fab antibodies to serovar- and subspecies-specific epitopes of the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized infectivity for hamster kidney cells by preventing chlamydial attachment. These findings exclude the aggregation of chlamydiae as a mechanism of anti-MOMP neutralization and provide additional evidence in support of the MOMP as a chlamydial adhesin.     

Human melanoma associated antigens: A solid-phase assay for detection of specific antibody

A solid-phase radioimmunometric binding assay is described utilizing ¹²⁵I-labeled protein A for the detection of antibody to human melanoma associated antigens. The novel aspect of this assay is the use of chemically defined spent culture medium of melanoma cells as target antigens previously depleted of fibronectin by affinity chromatography. This makes it possible to screen for antibody in unabsorbed antiserum. Sensitivity, reproducibility and ease of performance of the assay are optimized by conjugating target antigens to a background of bovine serum albumin dried onto polyvinyl 96-well microtiter plates and cross-linked with glutaraldehyde. The use of an immobilized soluble antigen target derived from a large pool of spent culture medium facilitates direct interassay comparisons and permits extensive absorption analysis of antisera. The assay has considerable potential in screening for alloantibody and both poly- and monoclonal xenoantibody to human melanoma associated antigens.