Jacalin, the human IgA1 and IgD precipitating lectin, also binds IgA2 of both allotypes.

The lectin jacalin from jackfruit seeds shows a human IgA-subclass specificity by gel precipitation and Western blotting. However, its reactivity with IgA2 is a matter of controversy. We further studied the immunoglobulin isotype specificity of jacalin by affinity chromatography with myeloma sera and by inhibition of jacalin binding to solid-phase IgA1 by purified monoclonal immunoglobulins. The lectin proved to bind IgA2 of both allotypes with a lower apparent affinity than for IgA1 and IgD.

The use of cellulose carbonate-based immunoadsorbents in the isolation of minor allotypic components of rabbit immunoglobulin populations.

Cellulose trans-2,3-carbonate has been used as a new insoluble matrix for the simple coupling of a1- and b4-positive rabbit immunoglobulin to make immunoadsorbents capable of purifying from serum, with great efficiency, alloantibodies to these allotypic determinants. The antibodies have themselves been conjugated to prepare specific antibody immunoadsorbents of high binding activity for their allotypic target molecules. With these anti-allotypic solid-phase reagents it has been possible to affinity purify a1- and b4-positive immunoglobulin molecules and to deplete serum immunoglobulin of these molecules to leave in the eluates only the allotypically uncontaminated minor immunoglobulin components which are a-negative or b-negative (lambda chain-bearing) molecules. lambda chain molecules were also purified in very small quantities by affinity chromatography on a sheep anti-rabbit lambda chain column. This method of purifying minor populations of rabbit immunoglobulin from normal serum by special immunoadsorbent applications offers new opportunities to study the products of rarely expressed immunoglobulin genes in normal rabbits.

Graft-versus-host reaction after allogeneic hemopoietic tissue transplantation in rabbits conditioned with cyclophosphamide.

Graft-versus-Host Reaction (GvHR) was investigated in rabbits conditioned with a cyclophosphamide, which was given as a single injection of 110 mg/kg of body weight or as 4 injections of 50 mg/kg given on each of four successive days, before allogeneic bone marrow or spleen cells transplantation. The GvHR was of a chronic type. Splenomegaly was significant in the animals receiving spleen cell grafts, but not in the recipients of bone marrow transplants. In each of experimental groups delayed body weight gain and autoallergic phenomena were observed. Autoantibodies against erythrocytes, tested by direct Coombs test, were found in almost all graft recipients. This was a long lasting phenomenon since sensitized erythrocytes were found even after six months after transplantation. Cytotoxic autoantibodies against lymphocytes were present in the sera of about 20% of the recipients, and not longer than 30 days after transplantation. They proved not to be anti-RLA antibodies.

Fab2 fragments from HLA xenoantisera specifically block cytolysis mediated by HLA-AB alloantisera.

Fab2 fragments from antisera raised in rabbits with partially purified cellular and serum HLA antigens were tested for their ability to block the cytolytic activity of operationally specific HLA-A,B alloantisera. One Fab2 fragment preparation blocked the cytolytic activity of all the HLA-A,B alloantisera tested; the remaining nine inhibited the lytic activity of alloantisera to certain HLA-A,B allospecificities, suggesting that these xenoantisera contain antibody to certain HLA-A,B allotype determinants or to closely associated structures. In contrast to previous reports in the literature none of the xenoantisera contained significant amounts of antibodies to human beta 2-microglobulin.

Preparation of IgG subclass allotypes from polyclonal IgG.

Two allotypes have been identified for each of the IgG subclasses IgG1, IgG2 and IgG3. These allotypes are referred to as G1m(a) and G1m(f), G2m(n) and G2m(-n), and G3m(g) and G3m(b). Using a pool of normal human serum and a combination of preparative electrophoresis, DEAE ion-exchange and protein A-Sepharose chromatography, it was possible to separate G1m(f) from G1m(a), G2m(-n) from G2m(n) and G3m(g) from G3m(b). Purification of G2m(-n) molecules is of special interest as no genetic marker has been found to identify this allotype.

The purification and properties of some less common allotypes of the fourth component of human complement.

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4-A1, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4-A and C4-B proteins, respectively, apart from the switch in their antigenic properties.

Structural polymorphism of murine complement factor H controlled by a locus located between the Hc and the beta 2M locus on the second chromosome of the mouse.

Structural polymorphism of murine factor H protein was demonstrated by using three different methods. 1) By prolonged agarose electrophoresis and immunofixation, factor H protein was visualized in the beta region as a single, distinct protein band in freshly bled EDTA-plasmas from many laboratory and wild mice. Two variants were detected among a large number of tested strains; one, referred to as H.1, moved faster to the anodal region (type strain, BALB/c), and the other, referred to as H.2, moved more slowly to the anodal region (type strain, STR). The F1 hybrid between BALB/c and STR exhibited a combining type of factor H protein, which was observed in each parent. 2) Two-dimensional peptide mapping analysis was carried out with tryptic peptides of these two factor H allotypes. Almost all of the spots in the maps of tryptic peptides were common to both allotypes. However, three distinct spots among the 57 spots detected in the map of tryptic peptides of the H.1 allotypes were not detected in that of H.2 allotype, whereas two spots among the 56 spots in the map of H.2 allotype were unique for this allotype. The F1 hybrid between BALB/c and STR showed a combining type of the map of parent. 3) Alloantisera against each of H allotypes were successfully produced in BALB/c or BALB/c-H.2 (a congenic strain with H.2 allotype) by repeated injection of each purified factor H protein either from the BALB/c or the STR strain. These findings indicated that the observed variants of factor H represent antigenically and structurally distinguishable allotypes. The allotypes of murine factor H protein are controlled by a single codominant locus located between the Hc locus and the beta 2M locus on the second chromosome of the mouse. This was shown by phenotyping the Hc locus and H locus with backcross progenies between A/J (one of strain with H.1) and MoA (one of strain with H.2). The recombination frequency between these two loci was 0.17 +/- 0.046.

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice.

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.

Three closely linked latent rabbit IgG allotype gene products: a1, d12, e14.

Rabbit latent allotypes in the serum are non-allelic immunoglobulin markers that are transiently expressed in low concentration. In this report we describe the isolation and characterization of a linked series of latent allotypes on the gamma-chain which were found in two rabbits. This latent allogroup contains the determinants a1, d12, e14, which corresponds to the known nominal haplotype I. The latent allogroup is only periodically expressed in the serum and transmitted in a non-Mendelian fashion. However, although only intermittently expressed, both a1 and e14 were found to be displayed in a polyclonal manner by isoelectric focusing. The a1 was associated with the Fab region, the d12 was associated with the hinge region, and the e14 was associated with the Fc region when the peptides were assayed after enzymatic cleavage. The non-allelic demonstration of these genetic markers indicates that each of the rabbits studied carries all the alternative genes for the major three allotypic loci in the gamma-chain. It is possible that other rabbits bear them as well, but they remain "silent" at any given time.

Coexistence of multiple monoclonal immunoglobulins with identical constant regions in one patient.

Two populations of IgG molecules, designated F-IgG3(chi) and S-IgG3(chi), were isolated from a patient with multiple myeloma. The constant regions of the two molecules appear to be identical, since the heavy chains of both belong to the gamma 3 subclass and both molecules are positive for G3m(5), G3m(10), and G3m(11) but negative for G3m(21), Km(1) and Km(2) allotypes. In contrast, the two molecules have different variable regions, since (a) they do not share idiotypic determinant(s); (b) their light chains have different NH2 terminal amino acid sequences, and (c) both their heavy and light chains have different peptide maps and amino acid compositions. SDS-polyacrylamide gel electrophoresis showed that the F-IgG3(chi) molecules have two kinds of light chains. One, designated, chi n, has a normal molecular weight (approximately 23,000); the other, designated chi h, has a molecular weight of approximately 28,000.

The four subclasses of IgG can be isolated from mouse serum by using Protein A-Sepharose.

We confirmed the findings of Ey and colleagues that mouse IgG is absorbed by protein A-Sepharose at pH 8.0. Also confirmed was their finding that IgG1 mainly elutes from such a column by means of a buffer with pH 6.0 and that the corresponding pH values for IgG2a and IgG2b were 4.5 and 3.5. We made the new finding that the bulk of IgG2a bearing allotypes a or j eluted already at pH 5, in contrast to IgG2a bearing allotype b. Another new finding was that IgG3 was mainly eluted at pH 4.5 regardless of the allotype. All four subclasses of IgG could thus be physically separated if the allotype was a or j (the only known exception is allotype b). Separation of IgG2a and IgG3 was achieved even when the allotype was b by using a pH gradient for elution. IgG2a came out at a slightly higher pH than IgG3. Mouse IgG antibodies against group A streptococcal polysaccharide belonged mostly to IgG3 and, to a lesser extent, to IgG2a and IgG2b.

Detection of IgG-associated determinants in reduced and alkylated preparations of human IgG3 by monoclonal antibodies.

Using classical typing antisera, previous experiments have failed to demonstrate IgG3 in partially reduced and alkylated preparations of human IgG intended for intravenous application (IGIV). To establish that IgG3 is actually present in such preparations, we designed an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies as solid-phase reagents and protein A-purified IgG3 as antigen. Three different samples of reduced and alkylated antigen were used: (1) IgG3 isolated from a ready-for-infusion IGIV; (2) IgG3 which was purified from an intramuscular (Cohn fraction II) IgG solution before being subjected to a mild reduction and alkylation procedure, and (3) completely reduced and alkylated IgG3. The reduction and alkylation procedure did not affect the solubility of IgG3, indicating that IGIV prepared in this manner should contain normal quantities of IgG3. In the ELISA, solid-phase monoclonals which were cross-reactive with multiple IgG subclasses clearly reacted with reduced and alkylated IgG3. Furthermore, there was no substantial difference between the quantities of modified and native antigen required for 50% maximal ELISA signal. In contrast, solid-phase monoclonals with IgG3-restricted specificity did not recognize reduced and alkylated material. These results indicate that IGIV prepared by reduction and alkylation has a normal IgG3 content and confirm that some IgG3-specific determinants are altered by the modification procedure.

Comparison of latent and nominal rabbit Ig VHa1 allotype cDNA sequences.

The genetic basis for the expression of a latent VH allotype in the rabbit was investigated. VH region cDNA libraries were produced from spleen mRNA derived from a homozygous a2a2 rabbit expressing an induced latent VHa1 allotype and, for comparison, from a normal homozygus a1a1 rabbit expressing nominal VHa1 allotype. The deduced amino acid sequences of the nominal VHa1 cDNA were concordant with previously published VHa1 protein sequences. A comparison of two complete VH-DH-JH and six partial VHa1 sequences reveals highly conserved sequence within VH framework regions (FR) and considerable diversity in complementarity-determining regions and D region sequences. Two functional JH genes or alleles are evident. Amino acid sequencing of the N-terminal 15 residues of pooled affinity-purified latent VHa1 H chain showed complete sequence identity with the nominal VHa1 sequences. Possible latent VHa1-encoding cDNA clones, derived from the a2a2 rabbit, were selected by hybridization with oligonucleotide probes corresponding to the VHa1 allotype-associated segments of the first and third framework regions (FR1 and FR3). cDNA sequence analysis reveals that the 5' untranslated regions of nominal and latent VHa1 cDNA were virtually identical to each other and to previously reported sequences associated with VHa2 and VHa-negative genes. Moreover, some latent VHa1 genes encode FR1 segments that are essentially homologous to the corresponding segment of a nominal VHa1 allotype. In contrast, other putative latent genes display blocks of VHa1 sequence in either FR1 or FR3 that are flanked by blocks of sequence identical to other rabbit VH genes (i.e., VHa2 or VHa-negative). These composite sequences may be directly encoded by composite germ-line VH genes or may be the products of somatically generated recombination or gene conversion between genes encoding latent and nominal allotypes. The data do not support the hypothesis that latent genes are the result of extensive modification by somatic point mutation.

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain.

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.

Myeloma class switch variant X63.2aRI-16 secretes IgG2a and IgG1 with identical VDJ sequence.

The myeloma line X63.2aRI-16 isolated in vitro as spontaneous tertiary class switch variant (gamma 1 leads to gamma 2b leads to gamma 2a,gamma 1) of X63 (IgG1, chi) by fluorescence-activated cell sorting using class-specific antisera expresses two heavy chains. X63.2aRI-16 secretes IgG2a,chi as does the parental cell line X63.2a-25, a hybrid molecule containing gamma 2a and gamma 1 heavy chains and IgG1,chi. The chain of the latter protein is the product of a reverse class switch in regard to the embryonic order of the CH genes. We purified the immunoglobulins of X63.2aRI-16, isolated the CNBr fragments of both heavy chains and determined the complete amino acid sequence of the VH domains and of the CH domains up to the first subclass-specific residues. The remaining CNBr fragments of the CH domains were characterized by amino acid analyses. It was found that both heavy chains of the double producer possess identical VH domains and CH domains characteristic for the subclasses gamma 2a and gamma 1, respectively. The identities of the two VDJ sequences expressed in X63.2aRI-16 cells suggest that the reverse class switch event to gamma 1 cannot simply be explained on the basis of a deletion model involving only a single CH locus.

IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs.

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies.

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.

Novel human light chain V kappa segment: serologic and structural analyses of the kappa III-like Bence Jones protein and IgG kappa light chain REE.

Immunochemical and sequence analyses of kappa light chain REE (Bence Jones protein REE and the light chain isolated from IgG kappa myeloma protein REE) revealed antigenic and structural features not previously described for human kappa-chains. Although closely related to proteins of the V kappa III subgroup, light chain REE is readily distinguished from light chains classified serologically as members of the kappa IIIa or kappa IIIb sub-subgroups. Light chains REE (Bence Jones protein REE and light chain REE) are identical in sequence and differ from kappa III proteins by at least 10 uncommon amino acid substitutions in the first three framework regions. Further, kappa-chain REE is unique by virtue of a four-residue deletion in the third complementarity-determining region. The deletion encompasses the three carboxyl-terminal residues in the V kappa-encoded segment and the first residue at the site of V-J recombination. Urine specimens from patient REE also contained a light chain fragment that lacked the first (amino-terminal) 85 residues of the native light chain but otherwise was identical in sequence to the light chain REE. The extensive amino acid differences and unique length of the V kappa segment in light chain REE indicate that this kappa-chain is the product of an unusual V kappa III gene or, alternatively, represents a rarely expressed and novel human V kappa gene.