Increased sensitivity in detecting tumor-associated antigens in sera of patients with colorectal carcinoma

A monoclonal antibody defining the Lewis blood group determinant was used to immobilize antigen in sera of patients with adenocarcinoma of the gastrointestinal tract, and a second radiolabeled antibody, which defines a gastrointestinal cancer-associated antigen (GICA), was used to detect the immobilized antigen. With this approach, elevated antigen levels were found in 34 of 49 (69%) of sera from patients with advanced colorectal carcinoma as compared with 9 of 292 (3%) of sera from patients with non-malignant gastrointestinal diseases and of healthy donors. For early primary colorectal carcinoma, the combination of anti-Lewis and anti-GICA monoclonal antibodies was more sensitive in detecting GICA than using anti-GICA antibody alone. Double determinant radioimmunoassay revealed the glycolipid determinant lacto-N-fucopentaose (LNF) III circulating in colorectal carcinoma patients' sera. 53% of patients older than 65 years had elevated levels of the LNF III determinant compared to none of age-matched, apparently healthy donors or patients with benign gastrointestinal tract lesions, and 18% of patients with inflammatory gastrointestinal tract diseases. In younger patients, the differences were less marked. Our results suggest the potential usefulness of Lewis and LNF III determinants as markers for the detection of gastrointestinal tract malignancies.     

Anti-HLA-B7,B27,Bw42,Bw54,Bw55,Bw56, Bw67,Bw73 monoclonal antibodies: specificity, idiotypes, and application for a double determinant immunoassay

The monoclonal antibodies (MoAbs) KS3 and KS4 are secreted by hybridomas constructed with splenocytes from a BALB/c mouse sequentially immunized with the cultured lymphoid cells JKu and LG-2 which share only the HLA-B27 specificity. Serologic and immunochemical assays have shown that the two MoAbs recognize the same (or spatially close) determinant expressed by HLA-B7,B27,Bw42,Bw54,Bw55,Bw56,Bw67, and Bw73 alloantigens. This determinant is spatially close but distinct from those defined by the anti HLA-B27 monoclonal antibodies described in the literature. The syngeneic antiidiotypic MoAb T12-105 and T12-211 elicited with MoAb KS4 were shown to recognize idiotopes within the antigen combining site of MoAb KS3 and KS4. Neither idiotope was detected on the anti HLA class I and anti HLA class II monoclonal antibodies tested. The MoAb KS4 in combination with the anti human beta 2-microglobulin MoAb NAMB-1 was utilized to develop a double determinant immunoassay (DDIA). The latter represents a sensitive method to detect and quantitate HLA-B27 antigens in spent culture medium of lymphoid cell lines and in serum. Typing for HLA-B27 antigens with the DDIA of sera from HLA typed donors yielded results highly correlated with those of the conventional lymphocytotoxicity assay.     

Another case of a lymphocytotoxic antibody with blood group A1, Leb and A Led associated specificity

A lymphocytotoxic antibody with blood group A1 Leb, and A (A1 + A2) Led, associated specificity was found together with an anti-HLA-DR2 in the serum of a multiparous woman. The A1 Leb and A Led-antibodies could be absorbed with erythrocytes from persons with blood group A1 or A2, irrespective of their Lewis antigens, even if their lymphocytes reacted negatively in this antibody, leaving anti-HLA-DR2 in the serum. Lymphocytes of blood group A1 were able to absorb the anti-A1 Leb and -A Led, whereas those of A2 could only absorb the anti-A Led. Saliva from persons with blood groups A1 Le (a - b +), A2 Le (a- b +), A1 Le (a - b -), secretor, and A2 Le (a - b -), secretor, inhibited the anti-A1 Leb and -A Led when tested in the serum/saliva ratio 50:1, which was not the case with other ABO-Lewis combinations. The woman who produced this antibody has blood group O Le (a - b +) and secretes H, Lea and Leb substances in about the same amount as do other individuals with blood group O Le (a - b +) used as controls. The anti-A in her serum has equal titers against A1 Le (a + b -) and A1 Le (a - b +) red cells.     

Effect of Lewis blood group antigen expression on bacterial adherence to COS-1 cells.

Epithelial cells from secretor individuals demonstrate decreased bacterial adherence compared with cells from nonsecretors. Lewis blood group antigen expression is one component of the secretor/nonsecretor phenotype and several epidemiologic studies have suggested a link between Lewis blood group antigen phenotype and susceptibility to urinary tract infections. In this study, we examined the possibility that the expression of the difucosylated Lewis blood group determinants, Leb and Ley (associated with the secretor phenotype), made cells less susceptible to Escherichia coli adherence by masking receptors for pili. COS-1 cells, which do not produce Lewis (Lea, Leb, Le(x), and Ley) blood group antigens, were used as target cells for bacterial adherence. The surface blood group antigen expression pattern of the cells was then modified by cotransfection with plasmids containing DNA inserts encoding alpha (1,2)-fucosyltransferase and alpha (1,3)- and alpha (1,4)-fucosyltransferases, resulting in the expression of Leb and Ley. E. coli HB101 expressing various adhesins (type 1, PapJ96, PapIA2, PapAD110, Prs, and S) from recombinant plasmids bound equally well to untransfected cells and transfected cells expressing Lea and Le(x) (nonsecretor phenotype) and Leb and Ley (secretor phenotype) antigens. We conclude that the presence of Leb and Ley antigens on cells from secretors does not alone mask receptors for E. coli pili or hinder bacterial adherence.     

Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor

The hyperthyroidism of Graves' disease (GD) is caused by TSH-receptor (TSH-R) stimulating autoantibodies (TSAb), leading to overproduction of thyroid hormones. We present evidence for TSAb interaction with three distinct regions of the TSH-R, one in immediate vicinity of the carboxy terminal serpentine. Three murine monoclonal antibodies (MoAbs 28.1, A9 and 31.7) directed to amino acids 36-40, 147-228 and 382-415 were labelled and tested for their binding to human recombinant TSH-R on solid phase. All MoAbs bound to TSH-R with a K(d) of 8-12 nm and showed no competition among themselves. We tested 114 sera from euthyroid controls, 118 TBII positive sera from patients with GD (containing TSAb confirmed by bioassays), 16 TBII positive sera from patients with autoimmune thyroid disease (AIT), who were hypothyroid and had TSH blocking antibodies (TBAb), and 20 patients with AIT, who were hypothyroid but negative for all TRAb. Mid-regional MoAb A9 tracer achieved the highest sensitivity in the GD group (72.0%), whereas C-terminal MoAb 31.7 found most sera positive in the AIT group (87.5%). Surprisingly, the N-terminal MoAb 28.1 had the lowest sensitivity in the GD (10.4%) and AIT group (43.8%). Using a mixture of all three tracer MoAbs did not increase the sensitivity in the GD or AIT group, compared to the best single MoAb alone. Median inhibition of MoAb A9 was significantly (P < 0.001) higher than inhibition of MoAbs 28.1 or 31.7 in the group of GD patients but not in other groups. Almost all patient sera with positive reactivity in the MoAb tracer assays had TBII values in the higher range. However, there were many highly TBII positive sera, which did not show a displacement of the MoAb tracers. We conclude that, contrary to some reports, the binding of TSAb and TBAb to the TSH-R is not restricted to distinct and distant epitopes. The middle part of the TSH-R seems to be more relevant for TSAb binding than the N-terminal part, while a proportion of TSAb autoantibodies also binds to a C-terminal epitope of the TSH-R. The method described here is a TSH independent competitive assay for the detection of TSH-R autoantibodies.     

The 40-kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies

To characterize the 40-kilodalton (kD) major allergen of Candida albicans (C. albicans), six monoclonal antibodies (MoAbs) against this allergen were generated. In SDS-polyacrylamide gel electrophoresis and immunoblot analysis, these MoAbs showed four different reaction patterns to antigens of six different Candida species. With the exception of one MoAb, other MoAbs were resistant to periodate treatment indicating non-carbohydrate epitopes were probably being recognized by these MoAbs. These MoAbs were used in the molecular cloning and immunological analysis of the gene coding for the 40-kD allergen. Nucleotide sequence determination of the two lambda gt11 cDNA clones obtained showed that the 40-kD allergen is an alcohol dehydrogenase (ADH) which shares a 70% amino acid sequence homology with the ADH isozyme I of Saccharomyces cerevisiae. This finding was confirmed by positive immunological response of the lysates of the clones obtained and a preparation of ADH of Saccharomyces cerevisiae to various MoAbs and to IgE antibodies in sera of allergic patients.     

Significance of erb B-2 Gene Product as a Target Molecule for Cancer Therapy

The new monoclonal antibodies (MoAbs) E401, E811, E907 and E919 were prepared and characterized. These recognized an extracellular domain (amino acids No. 292–370) on the human c-erbB-2 gene product. Utilizing MoAb E811 and MoAb E919, a double determinant immunoassay (DDIA) was established to detect the soluble and the shed forms of the c-erbB-2 molecule. The levels of circulating erbB-2 antigen in the sera of patients with benign diseases and healthy controls were very low. The incidence of positivity for shed c-erbB-2 antigen in gastric cancer, eolonic cancer, gall-bladder caneer, pancreatic eancer and other cancers were 7.4%, 4.2%, 0%, 6.7% and 0%, respectively. Four of 54 patients with gastric carcinoma showed high levels of serum c-erbB-2 antigen. They belonged to clinical stage IV and their histological types were all well differentiated adenoeareinomas (two papillary and two tubular adenoeareinomas). Furthermore, the incidence of positive staining in gastric cancer was 34.6%; higher than that for shedding erbB-2 antigen. Most of the cases v-hich showed erbB-2 expression on cells were well-differentiated adenoeareinomas. Meanwhile, the distribution of erbB-2 antigen was limited in normal tissues. The results suggest that the expression of erbB-2 antigen is largely restricted to adenocarcinoma cells. It may not shed easily from these cells, and therefore It may be a very useful target molecule for passive immunotherapy.     

Lewis antigens in normal and neoplastic urothelium.

The Lewis (Lea and Leb) antigens are closely related to the A, B, H blood group antigens and have been demonstrated in several secretory epithelia, but their expression in nonsecretory cells has not been studied systematically. This report provides detailed data on the expression of Lea and Leb in normal and neoplastic urothelium. The authors have examined multiple biopsy specimens of normal bladder mucosa and transitional cell carcinomas (TCCs) from 74 patients whose red blood cells (RBCs) were also typed for A, B, H, Lea, and Leb antigens and have correlated tissue antigen detectability with the RBC phenotype and the cytologic grade of malignancy. Antisera of human and animal sources were used in a modified red cell adherence test (RCA), and multiple controls were employed for determination of the specificity of the reactions. Both fresh-frozen and paraffin-processed tissues were examined from each patient. Paraffin processing as well as treatment with ethanol significantly suppressed the tissue reactions. Ninety-four percent of normal mucosa specimens and 73% of TCCs gave positive reactions with both anti-Lea and anti-Leb sera. Abnormal patterns of Lewis reactivity were observed in 43% of Grade III or IV and in 14% of Grade I or II TCCs. Although there was no direct correlation between A, B, H reactivity and Lewis reactivity, all TCCs which had abnormally low reactivity for both the expected Le and A, B, or H antigens were of high grade and invasive.     

Analysis of the expression of H, Lewis, X, Y and precursor blood group determinants in saliva and red cells using a panel of mouse monoclonal antibodies

Salivary glycoproteins from 33 normal individuals were analyzed with a panel of mouse monoclonal antibodies to H-1, H-2, Lea, Leb, X, Y and precursor blood group determinants. Samples from 19/33 individuals co-expressed Leb and Y-determinants (secretors) and 6/33 co-expressed Lea and X-determinants (non-secretors). Erythrocytes of these individuals were typed Le (a-b+) and Le (a + b-), respectively. In seven other salivas, only one specificity, either Lea, Leb, X or Y, was expressed and in one sample none of these determinants could be detected. Only one saliva sample expressed H-1 specificity and none expressed H-2 or type 1 precursor determinants. The absence of H-1 and H-2 structures in secretors and the resulting expression of difucosylated Leb and Y-structures is probably a tissue-specific trait of salivary gland secretions. The strict co-expression of Leb with Y and Lea with X supports the conclusion that only one 2-O-fucosyl-galactose transferase, which can fucosylate both type 1 and type 2 chains, exists in salivary glands. The finding that a number of individuals expressed neither X- nor Y-specificities was unexpected in view of previous work showing that the 3-O-fucosyl N-acetylglucosamine transferase involved in forming this structure is a ubiquitous enzyme. The individualistic expression of blood group phenotypes in tissues should be considered when the altered expression of blood groups in malignancy and other diseases is studied.     

A unique human IgE-binding epitope on the Bermuda grass pollen recognized by mouse lambda-type monoclonal antibodies

A group of six mouse monoclonal antibodies (MoAbs) with the unusual lambda-type light chain were generated by fusion of NS-1 cells with splenic cells derived from BALB/c mice immunized with crude extracts of Bermuda grass pollen (BGP). Four of them were IgG1, one was IgG2b, and one was IgG3. Binding inhibition assay showed that they recognized the same (or very similar) epitope. Using sera from BGP-allergic patients, it was found that the specific binding between the IgE antibodies and the MoAb 26-11-fixed antigen could be blocked by MoAb 26-11 itself and another MoAb 9-13 in a dose-dependent manner. It appears that the epitope recognized by the lambda-type MoAbs is a human IgE-binding antigenic determinant. Further physico-chemical analyses showed that this epitope was stable under heat but sensitive to treatments of sodium periodate and proteinase K. Results from these studies indicate that this unique epitope which leads to the generation of lambda-type MoAbs is part of a glycoprotein.     

Analysis of the repertoire of anti-HLA antibodies with anti-idiotypes to a murine anti-HLA-A2,A28 monoclonal antibody

Xenoantibodies to idiotypes of the anti-HLA-A2,A29 MoAb CR11–351 were isolated from an antiserum raised in rabbit #81 by immunizations with purified MoAb CR11–351. The purification procedure involved absorption with insolubilized mouse immunoglobulis and monoclonal antibodies and affinity chromatography on insolubilized MoAb CR11–351. Two antibody populations were identified in the xenoantibody preparation #81: one recognizes a recurrent idiotope expressed by the MoAb CR10–215, CR10–402, Q1/28, Q6/64, and 6/31 to monomorphic determinants of HLA-A,B antigens and by the MoAB CR10–343, CR11–462, and Q516 to human la antigens. The other antibody population recognizes a private idiotope. Neither idiotope was detected on the anti-HLA-A2, A28 variant (A28¹) MoAbs BB7.2, MA2.2, and PA2.1, on the anti-HLA-A2,B17 MoAb MA2.1 and on antibody populations in conventional anti-HLA-A2,A28 antisera. The idiotopes were also not detected on the anti-HLA-A2,A28 MoAb A2,A28 M1 which recognizes a determinant spatially close to that identified by the MoAb CR11–351. The idiotope(s) recognized by the xenoantibodies #81 may be located in the combining site of the MoAb CR11–351, since its incubation with the anti-idiotype antibodies specifically blocks the reactivity with lymphoid cells with the appropriate HLA phenotype.     

Detection of urothelial Lewis antigens with monoclonal antibodies.

The detectability of Lewis a and b antigens (Lea, Leb) was examined in the normal urothelium of 28 human subjects whose red blood cells (RBCs) were also tested for Lea and Leb. Mouse monoclonal antibodies as well as goat and human antisera were used on paraffin-processed and fresh-frozen tissues. Multiple biopsy specimens from the same individual were studied in order to evaluate temporal as well as topographic consistency of the results. In addition, the expression of the Lewis antigens in the appendiceal mucosa was investigated in 9 of these patients. The antibodies against Lea reacted with the urothelium of all patients with either Lea+b- or Lea-b+ RBC phenotype. None of the 5 patients with Lea-b- RBCs had Lea urothelial reactivity. The antibodies against Leb reacted with the urothelium of all patients with Lea+b- or Lea-b+ RBCs and with 2 of the 5 patients with Lea-b- RBCs. The mucin in the goblet cells of the appendiceal mucosa was positive only for the Lewis antigen that was also detectable of the individual's RBCs. These findings indicate that the expression of Lewis antigens in nonsecretory epithelia may not follow the same principles as in the blood and secretions.     

Human Polyreactive IgM Monoclonal Antibodies with Blocking Activity Against Self-Reactive IgG

Natural IgM antibodies have been found to be involved in the control of IgG reactivity in normal serum. The authors investigated the blocking activity of four human IgM monoclonal antibodies (BY-2, BY-7, BY-10 and IRM-7) derived from B-cells from blood samples of three renal dialysis patients, which had shown multispecific properties similar to those observed for natural polyreactive autoantibodies. To achieve this, competitive inhibition assays were performed with these MoAbs on the binding of IgG purified from a healthy control, three patients with SLE, and two patients with autoimmune thyroiditis, to histone, dsDNA, RNP and thyroglobulin. MoAbs inhibited binding of self-reactive IgG to histone and dsDNA, but not to thyroglobulin or RNP, of natural and active or inactive phase disease-associated autoreactive IgG. The inhibitory effect of the MoAbs was mediated by V-region dependent interactions with autoreactive IgG, as shown by the ability of these MoAbs to block the binding of F(ab')₂ fragments of autoreactive IgG to antigens (histone and dsDNA). The blocking of autoantibody activity was dose-dependent with maximal inhibition occurring at a specific molar ratio between the patient's IgG and a given MoAb. In contrast, MoAbs did not inhibit binding of IgG alloantibodies present in the sera of four polytransfused renal dialysis patients to target antigens on the surface of different cells. These results support the concept of a functional idiotypic network regulating autoimmune responses, and suggest that the IgM MoAbs under study may be natural polyreactive antibodies belonging to the physiological network of autoantibodies with highly connected V-regions, capable of binding and functionally neutralizing V-regions of natural and pathologic autoantibodies.     

Detection of circulating intercellular adhesion molecule-1 antigen in malignant diseases.

A new monoclonal antibody (MoAb) HA58 (IgG1) was prepared, which recognizes the binding site on the intercellular adhesion molecule-1 (ICAM-1) antigen to the lymphocyte function-associated antigen-1 (LFA-1). The double-determinant immunoassay (DDIA) was established with use of MoAb HA58 and another anti-ICAM-1, MoAb CL207, to detect the soluble, shedding ICAM-1 antigen. Human recombinant interferon-gamma (IFN-gamma) induced not only the expression of cell surface ICAM-1, but also the shedding ICAM-1 antigen in an IFN-gamma concentration-dependent and incubation-time-dependent manner. DDIA was applied to detect the shedding ICAM-1 antigen in the sera of patients with malignant or benign diseases. The incidence of positivity for ICAM-1 antigen in malignant diseases was higher than that in benign diseases or in healthy controls. Furthermore, the sera of cancer patients with liver metastasis showed higher levels of the shedding ICAM-1 antigen. These findings suggest that serum ICAM-1 antigen may be a useful marker to monitor tumor burden in cancer patients.     

Antisera Recognising HLA-A, -B and DRW Antigens Raised in Rhesus Monkeys

Rhesus monkeys were immunized with partially purified HLA-A, -B, -C and DR antigens. The resulting sera were shown to have activity against species-specific determinants on both HLA-A, -B, -C chains and beta 2 microglobulin by the use of somatic cell hybrids. When this was removed by absorption, the sera showed activity against three of the four HLA-A and -B antigens in the immunogen when tested on a panel of peripheral blood lymphocytes and T cells. Antibodies recognizing HLA-DR antigens were detected by testing platelet absorbed sera on a panel of typed lymphoblastoid cell lines. After absorption to remove activity against species-specific determinants on the HLA-DR antigens, two cross reacting specificities were defined. One consisted of a determinant in common between HLA-DRw1, 2 and 6 and the other a putative determinant in common between HLA-DRw4, and 5. The nature and significance of these cross-reacting groups of HLA-DR antigens is discussed in the light of current HLA-DR serology and the nature of HLA antigens in general.     

Detection of carcinoembryonic antigen and related antigens in sera of patients with gastrointestinal tumors using monoclonal antibodies in double-determinant radioimmunoassays

Of 14 monoclonal antibodies produced in six different laboratories, 13 bound to purified preparations of carcinoembryonic antigen (CEA). All antibodies reacted to spent medium of colorectal carcinoma cell lines. Competitive binding studies indicated that 12 different antigenic determinants representing six different groups were detected on the CEA molecule(s). Six antibodies were used in double determinant radioimmunoassays (RIA) to detect CEA and CEA-related antigens in sera of 311 patients with various gastrointestinal diseases and of normal donors. None of up to 115 sera of healthy donors had elevated antigen levels with four out of the six monoclonal antibodies tested, whereas up to 9% of sera showed elevated antigen levels when tested with two antibodies. Between 1.4% and 4.4% of sera from patients with inflammatory and benign neoplastic diseases of the gastrointestinal tract were positive. Antigen levels were elevated in 56 to 75% (depending on antibody used) of sera from patients with advanced gastrointestinal tumors. These preliminary results indicate that double-determinant immunoassays with a panel of monoclonal antibodies might improve conventional CEA assays by reducing the number of false positive sera detected by polyclonal sera in patients with benign inflammatory bowel diseases.     

Detection of blood group A and H-related antigens in normal and neoplastic bladder epithelium: a comparative study using monoclonal antibodies with defined fine specificities.

Three monoclonal anti-blood group H and four monoclonal anti-blood group A antibodies directed at the blood group antigens on Type 1 or Type 2 backbone structures were evaluated as immunohistochemical reagents by indirect immunofluorescence of normal and neoplastic bladder epithelia. The results were compared with fluorescence using polyclonal rabbit anti-A or H sera or Ulex europaeus lectin. The monoclonal antibodies gave less intense or more restricted immunofluorescence than the conventional reagents but showed considerable variation in the extent of their reactivities with urothelial samples from different individuals. In some cases they failed to give immunofluorescence with tissue samples known to contain the immunodominant blood group structures they recognize. In addition, hitherto unsuspected heterogeneity was revealed in the expression of the Type 2-based blood group H and A-structures in the endothelia of neighbouring small blood vessels.     

Hyperplastic polyps of the colon and rectum. An immunohistochemical study with monoclonal antibodies against blood groups antigens (sialosyl-Lea, Leb, Lex, Ley, A, B, H)

We studied 40 hyperplastic polyps (HP) immunohistochemically with monoclonal antibodies against 8 different blood group antigens (BGA) comparing their reactivity with normal control colon and colorectal adenocarcinomas. The 8 BGA studied were: Sialosyl-Lea, Lea, Leb, Lex, Ley, A,B, and H. sialosyl-Lea, Lea, Lex, and Ley can be though of as differentiation antigens. The former 2 BGA are expressed on mature (differentiated) epithelium while the latter 2 BGA are expressed by undifferentiated epithelium of the crypt base. A, B, H and Leb are not expressed in the normal distal colon, however, they are extensively expressed on distal colorectal cancers and adenomas and can be considered oncofetal BGA. HP expressed Lea, Lex, Ley in the same compartment of the crypt as normal colon and extensively expressed Sialosyl-Lea throughout the entire length of the crypt. This latter finding indicated maturation at a lower point in the crypt than in normals. All HP failed to express B and H BGA, while 6 of 40 expressed Leb and 5 of 40 HP expressed A BGA. Of the 6 HP expressing Leb BGA, 3 were from patients with synchronous or metachronous cancers and 2 from patients with mixed hyperplastic polyp-adenomas (HP/AD). Two of the HP expressing A BGA were from patients with HP/AD. The expression and distribution of these BGA in HP, especially the extensive expression of sialosyl-Lea correlates with the known cell kinetics of HP. While nonneoplastic in nature, HP may occasionally express true oncofetal BGA. Similarly, the HP component of HP/AD may also express true oncofetal BGA. These data suggest that the lesions classified morphologically as HP may be antigenically heterogenous.     

Specificity of monoclonal antibodies directed against human and murine class II histocompatibility antigens as analyzed by binding to HLA-deletion mutant cell lines

The specificity of 70 monoclonal anti-Ia monoclonal antibodies (MoAbs) (18 mouse allo-induced and 52 rodent anti-human) was studied with a panel of 17 HLA-deletion mutants that were derived from a single parent line and vary in expression of Ia antigens due to deletion of different subregions of HLA. MoAb binding was analyzed both by ELISA and flow microfluorometry. Characterization of the MoAbs with respect to specificity for products of subregions of a DR1-DC1-SB2 haplotype revealed great complexity. Many antibodies were quite specific for DR-linked determinants (26 MoAbs), DC-linked determinants (5 MoAbs), or determinants indistinguishable from SB (4 MoAbs). However, many MoAbs bound to products of more than one subregion: DR + SB (± weak DC) (22 MoAbs); Dr + DC (3 MoAbs); or DR + DC + SB (1 MoAb). Furthermore, a number of the MoAbs bound unequally to products of the two HLA haplotypes analyzed, particularly among those recognizing DC1-linked determinants and the murine alloinduced MoAbs. Finally, despite strong structural homologies of murine I-A to human DC and murine I-E to human DR, the intraspecies cross-reactions of MoAbs do not closely follow that pattern. These data: (1) illustrate the usefulness of HLA-deletion mutant cell lines for analysis of the specificity of MoAbs and for delineation of HLA subregions; (2) demonstrate the great diversity of MoAbs specific for class II molecules and the high frequency of MoAbs that bind to products of more than one Ia subregion, particularly DR and SB. In view of such complexity, many (perhaps most) MoAbs cannot be relied on to unambiguously identify products of a particular Ia subregion, without extensive characterization.     

Identification of allergen components of the opportunistic yeast Pityrosporum orbiculare by monoclonal antibodies

The yeast Pityrosporum orbiculare (P. orbiculare) is a member of the normal human cutaneous flora, but it is also associated with several clinical manifestations of the skin. We have previously observed IgE-binding components in P. orbiculare extracts, using sera from patients with atopic dermatitis. In the present study, we raised several monoclonal antibodies (MoAbs) against P. orbiculare to characterize some of its antigens, and used Candida albicans (C. albicans) as a control. We obtained several IgGI MoAbs which specifically recognized P. orbiculare in ELISA. Two of these were selected for immunoblotting studies on P. orbiculare and two patterns of reactivity emerged. Firstly, one MoAb showed a distinct band at a molecular mass of 67 kDa. In the second pattern, a sharp band at about 37 kDa appeared. In contrast, the IgM antibodies raised reacted with a 14-kDa component; but they reacted with C. albicans in addition to P. orbiculare The IgGI antibodies seemed to react with proteins, as their ability to react in ELISA with extract pretreated with protease was greatly reduced. In contrast, IgM MoAbs were much less affected, suggesting that they recognized nonprotein components. To determine whether these MoAbs-binding components were also recognized by human IgE, we adopted a radioimmunoassay (RIA) using the MoAbs as catcher antibodies. Both the 67-kDa and the 37-kDa components were IgE-binding proteins. P. orbicular RAST positive sera were scored as positive in the RIA, whereas the control serum was not.