Comparison of rat MHC class I antigens by peptide mapping

Monoclonal antibodies specific for the rat major histocompatibility complex (MHC) class I antigens RT1.Aⁿ, RT1.A^u, and RT1.E^u were used for immunoprecipitation of antigens biosynthetically radiolabeled with¹⁴C- or³H-labeled arginine, lysine, and tyrosine; with arginine or tyrosine alone; and with or without tunicamycin in the culture medium. Heavy chains of the glycosylated and unglycosylated antigens were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their tryptic and chymotryptic peptides were compared by high performance liquid chromatography. The antigens coded by the same locus in two different haplotypes (Aⁿ and A^u) differed by 30%, whereas the products of two different loci in the same haplotype (A^u and E^u) differed only by 1–3%. Comparative analysis of the data for samples labeled with single amino acids indicated that two amino acids in A^u have been substituted by an arginine and probably by a tyrosine residue, respectively, in E^u. The high degree of homology between the products of theA andE loci in the same haplotype accounts for the difficulty in detecting recombinational events within the MHC of the rat by classical serological approaches.We dedicate this publication to Professor Paul Doty on the occasion of his sixty-fifth birthday     

Development and validation of an enzyme-linked immunosorbent assay for the quantification of trastuzumab in human serum and plasma

Trastuzumab, a humanized monoclonal antibody, is used for the treatment of breast cancer patients who overexpress the HER2 receptor. To optimize therapy, pharmacokinetic studies are necessary. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for trastuzumab to support these pharmacokinetic studies. For this immunoassay, we raised anti-idiotype antibodies in rabbits. After purification of the rabbit material, the anti-idiotype antibodies are used as capturing antibodies on the ELISA plate. After trastuzumab has bound to the catcher antibody, a sandwich ELISA procedure is followed whereby biotinylated anti-idiotype antibodies can bind to trastuzumab. Detection is performed by streptavidin-polyHRP (poly-horseradish peroxidase) conjugate and (3,5,3′,5′)-tetramethylbenzidine (TMB) substrate. The reaction is stopped using sulfuric acid, and the absorbance is measured at 450 nm. The calibration range of the assay is 0.039 to 5 ng/ml in well. Because samples are analyzed in multiple dilutions, the validated range corresponds to 1.6 to 1600 ng/ml in undiluted serum. Samples above the upper limit of quantification (ULOQ) can be diluted before transfer to the assay plates. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum and plasma. The assay is now used to support pharmacokinetic studies with trastuzumab in human serum and plasma.     

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N<Superscript>pro</Superscript>) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N^pro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N^pro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N^pro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N^pro rabbit serum. When rabbits were immunized with the N^pro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N^pro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N^pro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N^pro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.     

Induction of multiple anti-c-erbB-2 specificities accompanies a classical idiotypic cascade following 2B1 bispecific monoclonal antibody treatment

 The bispecific monoclonal antibody (bsmAb) 2B1, targeting the extracellular domain of c-erbB-2, the protein product of the HER-2/neu proto-oncogene, and FcγRIII (CD16), expressed by human natural killer cells, neutrophils and differentiated monocytes, mediates the specific cytotoxic activity of these effector cells to tumor cells. A group of 24 patients with c-erbB-2-overexpressing tumors were treated with intravenously administered 2B1 in a phase I clinical trial and followed after treatment to evaluate the diversity and extent of the 2B1-induced humoral immune responses. As expected, 17 of 24 patients developed human anti-(murine Ig) antibodies (HAMA) to whole 2B1 IgG in a range from 100 ng/ml to more than 50 000 ng/ml; 10 of these patients (42%) had strong (at least 1000 ng/ml) HAMA responses, some of which were still detectable at day 191. These responses were usually associated with similar reactivity to the F(ab′)₂ fragments of the parental antibodies 520C9 (anti-c-erbB-2) and 3G8 (anti-CD16). We sought evidence of an idiotypic cascade induction, indicating a prolonged specific treatment-induced effect on at least one selected target of 2B1. Using competition-based enzyme-linked immunosorbent assays, specific anti-idiotypic antibodies (Ab2) were detectable against 520C9 in 11 patients and against 3G8 in 13 patients. Peak anti-idiotypic antibodies generally occurred 3–5 weeks from treatment initiation, with a downward trend thereafter. There was a statistically significant correlation among the induction of significant HAMA responses, anti-idiotypic antibody production and the development of antibodies to c-erbB-2. The anti-c-erbB-2 responses, which were distinct from anti-anti-idiotypic (Ab3) antibodies, were detected in the post-treatment sera of 6/16 patients examined. No obvious correlation could be made between the development of humoral immune responses, the dose received, and the clinical response. Future investigations involving 2B1 therapy will concentrate on investigating an association of these humoral responses to any c-erbB-2-specific cellular responses. Manipulations of 2B1 therapy effects that augment immunity to c-erbB-2 could provide additional avenues for immunotherapy with this and other bispecific antibodies. Received: 1 August 1996 / Accepted: 28 March 1997     

Class I MHC allochimeric presentation of composite immunogenic and self epitopes induces tolerance to genetically diverse rat strains

Functional topography of rat class I major histocompatibility complex (MHC) molecule was studied. The α₁-helical sequences that are shared by class I RT1.A^l and RT1.A^u were substituted in the RT1.A^a molecule to produce the composite -RT1.A^a MHC class I allochimeric molecule. Dominant immunogenic epitopes that induce accelerated rejection were identified within the hypervariable regions of the α₁ domain of RT1.A^a, RT1.A^l, and RT1.A^u. Peri-transplant portal venous delivery of MHC class I allochimeric proteins, that included composite α₁ helical immunodominant epitopes of RT1.A^u and RT1.A^l, induced donor-specific tolerance to RT1^u (Wistar Furth, WF) and RT1^l Lewis, LEW) disparate cardiac allografts in ACI (RT1^a) hosts. Allochimeric generated tolerance was characterized by absence of T cell deletion or anergy. Donor specific IgM allo-Abs was not detected, while IgG alloresponse was markedly attenuated in sera of tolerant hosts. Further, long-term allografts in allochimeric-conditioned hosts exhibited moderate B cell infiltration when compared to rejecting controls. Analysis of intragraft cytokines revealed selective upregulation of IL-10 and marked inhibition of IL-2, IFN-γ, and IL-4. Our findings indicate the emergence of a peripherally induced tolerant state, afforded by the novel approach of soluble class I allochimeric conditioning that presents donor immunogenic epitopes in the context of recipient class I determinants.     

Production of antibodies specific to human chorionic gonadotropin in mice immunized against its chemical analogs

Mice immunized against DS₅-hCG-Β and DS₆-hCG-Β, chemical analogs of Β-subunit of human choriogonadotropin (hCG-Β) in which 5 and 6 disulphide bonds respectively were reduced and alkylated, were found to produce antibodies specific to hCG without significant crossreactivity with human lutropin (hLH) as tested in a radioimmunoassay. In contrast, mice immunized against the native hCG-Β subunit produced hLH crossreacting antibodies. While the anti-DS5, DS6-hCG-Β serum was capable of selectively blocking the binding of [¹²⁵I]-hCG to rat testicular LH/hCG receptors, it failed to inhibit the binding of [₁₂₅I]-hLH to the same receptors. The radioimmunoassay for hCG using the mouse anti-DS₅, DS₆-hCG-Β serum was not as sensitive as that employing rabbit anti-DS₅, DS₆-hCG-Β serum. The minimal detection limit was 5 ng/ml for the mouse antibody as compared to 1 ng/ml for the rabbit antibody. Supported by U.S. Public Health Service Grant HD 08766 to OPB. An erratum to this article is available at .     

Preparation monoclonal β-type anti-idiotype antibody of zearalenone and development of green ELISA quantitative detecting technique

Abstract

Immunoassay has been widely used in the screening of mycotoxins, which may be hazardous to the operator or the environment. This study was to develop a green way to measure zearalenone (ZEN) with a monoclonal β-type anti-idiotype antibody (Ab2β) against ZEN in place of ZEN standard. Six monoclonal β-type anti-idiotype antibodies were prepared. The 50% inhibitory concentration (IC₅₀) value to ZEN of the six antibodies was between 34.45?±?1.12–182.12?±?15.40?nM. A green ELISA was then developed and validated. The quantitative conversion formula between ZEN and the monoclonal Ab2β against ZEN was y?=?0.092x^0.722, R² = 0.990. The working range was 2.63–100.64?ng ml^?1. The recovery rate in spiked feed samples was from 82.15% to 102.79%, and the within-assay and between-assay coefficient variation (CV) level were less than 10.00%. A good correlation was obtained by high-performance liquid chromatography method (HPLC) to validate the developed method.     

PD-1对伯氏疟原虫感染小鼠体液免疫应答的影响

利用伯氏疟原虫Plasmodium berghei ANKA(P.b ANKA)感染BALB/c小鼠,PD-1单抗阻断后,流式细胞术检测脾脏浆细胞、滤泡辅助性T细胞(Tfh)数量。qRT-PCR检测IL-21、IL-10和IL-6 mRNA水平,ELISA检测血清抗体,以探讨程序性死亡受体-1(programmed cell death-1, PD-1)在疟原虫初次感染中对体液免疫应答的影响。结果发现,PD-1单抗阻断加速了P.b ANKA感染小鼠的死亡。与对照组相比,PD-1阻断组感染后第12天短寿浆细胞(CD138~+CD44~+)数量明显降低(P0.05),长寿浆细胞(CD138~+CD44~-、CD138~-CD44~+)和Tfh(CD4~+CXCR5~+)细胞数量无差异性改变,脾细胞IL-21的mRNA水平明显下降(P0.05),血清抗裂殖子表面蛋白(merozoite surface protein, MSP)-1特异性IgG无明显改变。P.b ANKA感染中PD-1通路可能通过影响Tfh分泌IL-21进而干扰浆细胞数量影响体液免疫应答。     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997     

Genetic definition of a further gene region and identification of at least three different histocompatibility genes in the rat major histocompatibility system

Two new recombinant haplotypes of the rat major histocompatibility system,RT1, have been detected in [LEW.1A (RT1 ^a ) ×LEW.1W (RT1 ^u )] × LEW 1N(RT1 ⁿ ) segregating hybrids. Recombinantr3 carries theRTL1. A region (determining classical transplantation antigens) and theRT1.B region (determining strong mixed lymphocyte reactivity and genetic control of antipolypeptide immune responsiveness) of the RT1^a parent, bur rejects RT1^a skin grafts. Recombinantr4 carries theA andB regions of the RT1^u parent, but rejects RT1^u skin grafts. The two histocompatibility genes detected are allelic to each other. The relevant locus, designated asH-C, maps to theB-region side of theRT1 system and appears to mark a thirdRT1 gene region,RT1.C. Availability of haplotypes r3 andr4 allowed the definition of a histocompatibility locus in theB region,H-B. The products ofH-C, H-B and of the previously describedH-A gene vary in antigenic strength.     

Gene order in the major histocompatibility complex of the rat

The loci in the major histocompatibility complex (MHC) of the rat which code for class I and class II antigens—RT1.A and RT1.B, respectively — have previously been separated by laboratory-derived recombinants and by observations in inbred and wild rats. Closely linked to the MHC is the growth and reproduction complex (Grc) which contains genes influencing body size (dw-3) and fertility (ft). These phenotypic markers were used in this study to orient the A and B loci of the MHC. Two recombinants were used for mapping. The BIL(R1) animal is a recombinant between the MHC and Grc, and it carries the haplotype RT1.A ^lB^lGrc⁺. The r10 animal is an intra-MHC recombinant, and it has the haplotype RT1.A ⁿB¹ Grc. These recombinants were characterized serologically, by mixed lymphocyte reactivity, by immune responsiveness to poly (Glu⁵²Lys³³Tyr¹⁵) and by the presence of the dw-3 gene. The data demonstrate that the gene order of the loci is: dw-3-RT1.B-RT1.A.     

Induction of CD4(+) and CD8(+) Bordetella pertussis toxin subunit S1 specific T cells by immunization with synthetic peptides.

In this study two synthetic peptides from the Bordetella pertussis toxin subunit S1 were conjugated to human anti-idiotypic antibodies and used as an immunogen in cancer patients to induce immunity. The aims of the present report are to explain why no carrier or adjuvant effect of the conjugated pertussis peptides could be established regarding induction of responses against the anti-idiotype and to explore the type and quality of induced anti-pertussis immune responses. The lack of carrier and adjuvant effect of the peptides might be related to the fact that the anti-idiotypic antibodies by themselves include helper epitopes and that none of the patients had a detectable T cell response against any of the selected peptides before immunization, which might be a requirement for an adjuvant effect. However, three of four immunized patients mounted a humoral as well as cellular response against the pertussis peptides used. The induced T cell immunity was restricted to one of the two peptides in responding patients. Established T cell lines and MHC blocking studies indicated that the T cell epitopes of the two peptides had a different MHC restriction. The type of T cell response induced seemed to govern the humoral response. The only durable antibody response was accompanied by the presence of a CD4(+) T cell response against the same peptide. Immunization with an anti-idiotype conjugated to synthetic peptides might thus induce both a B and a T cell response against the peptides and the type of induced T cells (CD4 or CD8) governs the quality of the humoral response. Moreover, the possibility of boosting or inducing a response against the antigen from which the peptide sequences were deduced also seemed feasible.     

Soybean 11S globulin polypeptides have an antigenic homology with 11S globulins from various plants

Summary Rabbit serum antibodies (AB) against glycinin acidic polypeptides were separated by cross exhausting, and the antibody fractions for each of the two subfamilies of glycinin subunits (A₁ and A₃) were obtained. The antibodies were used in the immuno blot assay with seed protein of various plant classes. Polypeptides homologous to soybean glycinin were detected. Homology with A₁ polypeptide was revealed in more cases than with A₃. Total seed protein preparations were subjected to centrifugation in sucrose density gradient, and the polypeptides, imunochemically related to glycinin, occurred only in fractions with sedimentation constant about 11S. The nativity of conservative antigenic determinants of 11S globulins is discussed.     

B para-Bombay phenotype in China caused by homozygous mutation for site 328 G > A of FUT1 gene: a case report

The aim of this paper is to accurately identify a case of B para-Bombay and to analyze the genetic mutation. ABO and Lewis blood groups were identified by standard serological methods, and trace antigens on RBCs were detected by adsorption-elution test, while blood group substances in the saliva were detected by agglutination inhibition test. The ABO gene exons 6-7, FUT1 gene exon 4 and FUT2 gene exon 2 were directly sequenced. Serological results showed that there were B antigens on RBCs without H antigens, anti-A and anti-HI antibodies in serum, and B and H blood group substances in the saliva. The Lewis phenotype was Le (a-b+). According to gene sequencing analysis, ABO, FUT1 and FUT2 genotypes were B101/O02, h^328G/Ah3^28G/A and Se^357C/TSe^357C/T, respectively. This rare phenotype can be mislabeled as "O" if any of the detailed investigations are not performed. Therefore, in order to ensure the safety of blood transfusion, genetic and serological tests are necessary for the correct identification of difficult blood groups.     

Carbohydrate moieties of rat MHC class I antigens

The rat major histocompatibility complex class I antigens RT1.A^u and RT1.E^u from the u haplotype and RT1.Aⁿ from the n haplotype were labeled with ¹⁴C-asparagine or with ³H-fucose, mannose, galactose, and N-acetylglucosamine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed complete removal of radioactivity from the sugar-labeled antigen heavy chains by digestion with glycopeptidase F, an enzyme that removes N-linked glycans completely. High performance liquid chromatography analysis of the tryptic digests of the mixed sugar-labeled and asparagine-labeled antigens demonstrated that all the sugar-labeled peptides were coincident with asparagine-labeled peptides. The Aⁿ antigen showed three glycopeptides, each of which had different amounts of sugar radioactivity. The antigens A^u and E^u showed two glycopeptides with different amounts of radioactivity but at identical positions in the two antigens. Antigen E^u had an additional glycopeptide with a lower amount of radioactivity. The positions of the glycopeptides from the A^u and E^u antigens were different from those of the Aⁿ antigen. The peptide profiles of the ¹⁴C-asparagine-labeled A^u and E^u antigens demonstrated distinct differences between the molecules. The results of this study show that: (a) all the glycans on rat class I antigens are N-linked, as they are on H-2 and HLA class I antigens; (b) there are compositional differences among the glycans in each of the three antigens; (c) the glycosylation pattern of the rat class I antigens is similar to that of the mouse class I antigens, which contain two or three glycans, in contrast to that of the human class I antigens, which contain only one glycan; and (d) the antigens A^u and E^u from the same haplotype are more closely related to each other than they are to the Aⁿ antigen.     

B para-Bombay phenotype in China caused by homozygous mutation for site 328 G>A of FUT1 gene: a case report

The aim of this paper is to accurately identify a case of B para-Bombay and to analyze the genetic mutation. ABO and Lewis blood groups were identified by standard serological methods, and trace antigens on RBCs were detected by adsorption-elution test, while blood group substances in the saliva were detected by agglutination inhibition test. The ABO gene exons 6-7, FUT1 gene exon 4 and FUT2 gene exon 2 were directly sequenced. Serological results showed that there were B antigens on RBCs without H antigens, anti-A and anti-HI antibodies in serum, and B and H blood group substances in the saliva. The Lewis phenotype was Le (a-b+). According to gene sequencing analysis, ABO, FUT1 and FUT2 genotypes were B101/O02, h^328G/Ah^328G/A and Se^357C/TSe^357C/T, respectively. This rare phenotype can be mislabeled as "O" if any of the detailed investigations are not performed. Therefore, in order to ensure the safety of blood transfusion, genetic and serological tests are necessary for the correct identification of difficult blood groups.     

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.     

Linkage disequilibrium between two genetic loci of the major histocompatibility complex in rats

The major histocompatibility complex (MHC) in natural populations of rats is composed of genetic phenotypes that are similar, if not identical, to those seen in inbred laboratory strains. Examination of individual wild rats from a single location in the city of Pittsburgh, Pennsylvania, resulted in the identification of seven different RT1.A histocompatibility serotypes and three RT1.B mixed lymphocytes responses. In this population of animals there is a significant association (p < 0.005) between four RT1.A and RT1.B phenotypic pairs: RT1.A⁸B¹, RTl.A^kBⁿ, RTl.A^dB^a and RT1.A¹B^a. The observed values for linkage disequilibrium (0.211,0.076,0.070 and 0.085, respectively) are very high and are close to the maximum expected, given the individual allelic frequencies. Although the animals included in this study were obtained from one location, agreement with Hardy-Weinberg equilibrium is demonstrated for other loci in the same population. The demonstration of equilibrium suggests that significant inbreeding is not affecting this population of rats. Not enough is known about the allelic frequencies in surrounding rat populations to determine how important the effect of migration is on these disequilibrium values. The large linkage disequilibria may indicate that, in the rat, environmental selective forces are operating to ensure the nonrandom association of separate components of the MHC.     

High chemotactic response to platelet-derived growth factor of a teratocarcinoma differentiated mesodermal cell line

Summary Evidence is presented that a differentiated mesodermal line (MES-1) from P19 EC cells express a high chemotactic response to platelet-derived growth factor (PDGF) as assayed in a blind-well modified Boyden chamber. Compared to the NIH 3T3 fibroblasts the chemotactic response of MES-1 is increased by 10-fold at 0.3 ng/ml of PDGF, 4-fold at 1.25 ng/ml of PDGF, 2-fold at 2.5 ng/ml of PDGF. In contrast, PDGF induces the same increase in [³H]thymidine incorporation in both cell lines, made quiescent under reduced serum concentration. This high chemotactic response to PDGF seems specific for these mesodermal cells. Among the different teratocarcinoma cells tested, including stem cells (F9, PC 13, PCC4) and endodermal derivatives (PYS, F9 with retinoic acid, PSA 5E), only the visceral endodermlike cells (PSA5E) are slightly attracted by PDGF. This chemotactic response to PDGF is not related to the presence or characteristics of the type B PDGF receptors, which are less numerous in MES-1 cells (10⁵ receptors/cell, KDa 1,2 mM) compared to NIH 3T3 cells (64×10⁴ receptors per cell, KDa 1,8 nM). The MES-1 cell line might be of interest for studying the chemotactic effect of PDGF. These results also suggest a role for this soluble factor in cell migration during early embryogenesis. This investigation was supported by a grant of La Fondation pour la Recherche Médicale.     

Recognition of the blood group H type 2 trisaccharide epitope by 28 monoclonal antibodies and three lectins

The patterns of cross-reaction of 30 monoclonal antibodies and three lectins were determined by ELISA with 21 ABH, Ii or Lewis related synthetic oligosaccharides coupled to bovine serum albumin. At least seven main groups of cross-reactive patterns were identified among the antibodies, plus several intermediate patterns between two of the main antibody groups. The three lectins had different cross-reaction patterns,Galactia tenuiflora was different from all the antibodies,Ulex europaeus lectin 1 andLotus tetragonolobus were similar, but not identical to groups III and V of antibodies respectively. The anti-H antibodies cross-reacting with A type 2 gave similar agglutination scores with all the normal ABO erythrocytes, while the anti-H antibodies not cross-reacting with A type 2 reacted with different scores: O>A₂>A₂B>B>A₁>A₁B>O_h, suggesting that these antibodies react better with the free H epitopes and do not recognize the H in A or B epitopes. Based on the ELISA and agglutination results and the lowest energy conformations of each oligosaccharide obtained by computer modelling, the most probable oligosaccharide surface areas recognized by each antibody main group are illustrated.