Auto-antibody to an Ig VH region allotype: induction of anti-a1 antibody in an a1-suppressed a1a2 heterozygous rabbit.

Two a1a2 heterozygous sibling rabbits were first suppressed for the paternally inherited a1 VH region allotype and then immunized with a1 IgG. Anti-a1 antibody was detected in the serum of one of the rabbits. The anti-a1 auto-antibody reacted with the same amount of a1 IgG as did a conventional anti-a1 allo-antibody. Most of the IgG and IgM of this rabbit was of the a2 allotype and no significant amount of the a1 allotype was detected as would be expected for an a1 suppressed a1a2 heterozygous rabbit. However, allotype suppression in this rabbit is maintained by endogenous anti-allotype antibody. Rabbits with anti-allotype auto-antibody may be exploited to produce litters of heterozygous and homozygous rabbits efficiently suppressed for selected allotypes.     

Identification of rabbit genomic Ig-VH pseudogenes that could serve as donor sequences for latent allotype expression

Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.     

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.     

Nucleotide sequence of a rabbit IgG heavy chain from the recombinant F-I haplotype

We report the sequence of a cDNA encoding a rabbit immunoglobulin gamma heavy chain of d12 and e14 allotypes with high homology to partial cDNA sequences from rabbits of d11 and e15 allotypes. The encoded rabbit protein shows homologies with human (68-70%) and mouse (60-63%) gamma chains. The nucleotide sequence homologies of the CH domains range from 76-84% with human and 64-76% with mouse sequences. Comparison of the portion of VH encoding amino acid positions 34-112 with a previously determined VH sequence of the same allotype shows high conservation of sequences in the second and third framework segments but more marked differences both in length and encoded amino acids of the second and third complementarity-determining regions (CDRs). We also found a high degree of homology with a human genomic V-region, VH26 (77%) and a remarkable similarity between rabbit and human second CDR sequences and human genomic D minigenes. These results provide additional evidence that D minigene sequences share information with the CDR2 portion of VH regions.     

Rabbit latent group a allotypes: characterization and relationship to nominal group a allotypic specificities.

Latent group a allotypes were detected with a sensitive radioimmune inhibition assay. Sera, IgG preparations, and antibody fractions containing these allotypes inhibited the binding of insolubilized allotypic antisera to various radiolabeled antigens including IgG pools, homogeneous antibodies, and, in the case of a3, a VH fragment from a3/b4 IgG. Several different group a antiallotypic sera were used in the assays and all gave similar results. Comparison of inhibition curves for nominal and latent allotypes indicated that the full spectrum of allotypic subspecificities may be expressed in latent allotypes. Hemagglutination studies carried out with five sera containing high levels of latent allotypes confirmed the results obtained with the radioimmunoassay and indicated that inhibition values did not, at least in four of the five samples studied, reflect the presence of antiallotype antibodies.     

Germline VH genes in an a3 rabbit not typical of any one VHa allotype

We have undertaken investigations in the rabbit of VH genes that may be responsible for the observations of VHa allotypes unexpected from an animal's pedigree (latent allotypes). A short cDNA probe was prepared and shown to be specific for VHa2 mRNA. Southern analyses with short and large probes were unrevealing but screening of a lambda phage library from a VHa3-expressing animal identified a number of unusual genes. These VH genes are remarkable in that they are far closer to one another in the genome (in one case 3085 bps) than VH genes reported in mouse or man, they are highly homologous over long stretches of sequence, and they encode proteins not typical of any one VHa allotype. Proteins similar to the sort encoded by these genomic V-regions may explain some of the observations of latent allotype. Our data suggest that the allelic behavior of VHa allotypes is not due to allelism of a regulatory mechanism that acts upon identical VH genes in rabbits of different VHa phenotypes.     

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.     

Serologic and structural characterization of immunoglobulin chains secreted by rabbit-mouse hybridomas

Serologic and primary structural analyses of Ig chains secreted by several rabbit-mouse hybridomas have shown that these hybrid cells produce heavy (H) or light (L) chains identical to those isolated from rabbit sera. Two of the cell lines (7D2, 7D6) secreted rabbit H chains with a m.w. of 55,000 each of which expressed a full complement of variable and constant region allotypes (a3, d11, e15). These apparently normal rabbit H chains were secreted in a complex with a m.w. about 130,000, and serologic studies indicated that this complex contained a covalently linked mouse kappa L chain. Two other cell lines (4C1, 12F2) produced allotype b4 L chains with m.w. of 23,000 and 25,000, and a third (1D4P5) produced an allotype b5 L chain with a m.w. of 23,000. Serologic analyses indicated that the allotypes on these chains are equivalent to those expressed by normal rabbit Ig molecules. Partial amino acid sequence data obtained for the L chain products showed them to be typical of rabbit L chains, and to be significantly different from mouse L chains.     

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.     

VDJ genes in VHa2 allotype-suppressed rabbits. Limited germline VH gene usage and accumulation of somatic mutations in D regions

In this study we investigate the molecular genetic basis for VHa- Ig. Knowing that the expression of VHa allotype Ig is suppressed by neonatal injection of rabbits with anti-VHa allotype antibody, and that the decreased level of VHa allotype Ig, VHa+, in the suppressed rabbits is compensated for by an increase in VHa- Ig, we determined the nucleotide sequences of 41 VDJ genes from a2/a2 rabbits neonatally suppressed for the expression of a2 Ig. We compared these nucleotide sequences to each other and identified two groups of VH sequences. We predict that the molecules of each group are encoded by one germline VH gene. Inasmuch as VHa+ Ig is encoded predominantly by one germline VH gene, VH1, it appears that more than 95% of the VDJ repertoire of rabbits may be encoded by as few as three germline VH genes. A genomic VDJ gene whose VH sequence was similar to those of group I molecules was expressed in vitro and was shown by ELISA to encode molecules of the VHa- allotype, y33. Analysis of the D regions in the VDJ gene indicated that germline D2b and D3 gene segments were preferentially used in the VDJ gene rearrangement. A comparison of sequences of D regions of the 41 VDJ gene rearrangements in 3-, 6-, and 9-wk-old rabbits to sequences of germline D gene segments showed an accumulation of mutations in the D region. Inasmuch as we have previously shown that V regions of rabbit VDJ genes are diversified, in part, by somatic gene conversion, it appears now that rabbit VDJ genes diversify by a combination of somatic mutation and somatic gene conversion.     

Partial amino acid sequence and genetic control of latent a2 allotype induced in rabbits by immunization with anti-a2 antibody

We have shown that after immunization of homozygous a1 rabbits of the B immunoglobulin (Ig) heavy chain haplotype with anti-a2 antibody (Ab) a population of molecules appears that has all of the serologic characteristics of the a2 allotype. We have now isolated these putative latent a2 molecules, have separated the heavy chains, and after enzymatic deblocking, have determined the first 19 N-terminal amino acids. For all eight allotype-associated residues, these putative latent a2 molecules have the amino acid residues typical of a2 allotype. As expected, the preimmune IgG from this a1a1 rabbit has the amino acids typical of the a1 allotype. Thus by partial amino acid sequence analysis, we provide additional evidence that the latent a2 allotype can be induced in a1a1 rabbits of the B heavy chain haplotype by immunization with anti-a2 Ab. Rabbits of other heavy chain haplotypes were also immunized with anti-a2 Ab and were tested for their ability to synthesize latent a2 allotype. Thus far, a1a1 rabbits of the A, B, C, and I heavy chain haplotypes all synthesize latent a2 allotype. In contrast, a3a3 rabbits of the G and H heavy chain haplotypes did not synthesize latent a2 allotype.     

RNA conversion of lymphoid cells to synthesize allogeneic immunoglobulins in vivo

Ribonucleic acid extracts (“5 day immune” and “nonimmune”-RNA) obtained from lymph nodes and spleens of rabbits homozygous for the b⁴ or b⁵ allele of light chain immunoglobulin allotypes were injected iv into nonimmunized rabbits homozygous for the alternate allele. The recipient rabbits were then given multiple iv injections of sheep red blood cells (SRBC). The spleens were assayed 13, 21, and 37 days following the RNA injection for “direct” IgM and “indirect” IgG plaque forming cells (PFC) specific for SRBC. The b4 or b5 light chain allotype and the a1, a2, and a3 heavy chain allotype of the antibody in the plaques was identified by radioautography and by inhibition of plaque formation using anti-allotype antibodies. The b light chain allotype of the RNA donor was identified in 22–32% of the IgM plaques and in 25–42% of the IgG plaques. The allotype of the host rabbit b light chain allotype was identified in 56–67% of the IgM plaques and in 57–71% of the IgG plaques. Likewise the a heavy chain allotype of the RNA donor was identified in 10–19% of the IgM plaques and in 12–19% of the IgG plaques. The allotype of the host rabbit a heavy chain allotype was identified in 51–60% of the IgM plaques and in 55–63% of the IgG plaques. The concentrated lysates of spleen and lymph node cells were also analyzed for immunoglobulins of each light chain allotype by immunodiffusion with radiolabeled antibody. The allotype of both the RNA donor rabbit and host rabbit were found in most of the lysates of lymphoid tissues and in some of the IgG isolated from the serum and concentrated.     

Identification and genetic control of two rabbit high-density lipoprotein allotypes

Rabbits were immunized with high-density lipoprotein (HDL) isolated from the serum of other rabbits by ultracentrifugation and gel filtration. Two different precipitating antibodies were elicited which distinguished two antigenically different genetic variants, i.e., allotypes, of HDL. The allotypes were identified as HDL based on the observation that on immunoelectrophoresis the antigen-antibody precipitate formed by the reaction of each of the antiallotype antisera with electrophoresed rabbit serum appeared electrophoretically in the α₁ region and reacted with Sudan black and with β-naphthyl acetate. In addition, the precipitin bands formed by the reaction of each antiallotype antiserum with a normal rabbit serum coalesced with the precipitin band formed by the reaction of goat anti-HDL with the same normal rabbit serum. The inheritance of the two allotypes is controlled by a pair of allelic genes, as shown by genetic studies of 312 progeny from 83 rabbit families. This HDL locus, designatedLhj, was shown not to be linked to theLpq locus of low-density lipoprotein nor to any of five other loci controlling allotypic specificities of different rabbit serum proteins. The use of these allotypes for studying the structure and biosynthesis of HDL is discussed. This study was supported by Research Grant PHS AI-07043 (Dr. S. Dray) and PHS AI-09241 from the National Institutes of Allergy and Infectious Diseases. One of us (K. L. K.) is the recipient of National Institutes of Health Career Development Award (AI-28687).     

Immunoglobulin classes of anti-Sendai virus antibody detected by ELISA in infected nude mouse serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Rabbit collagenase. Immunological identity of the enzymes released from cells and tissues in normal and pathological conditions.

1. The immunological cross-reactivity between rabbit collagenases from a variety of normal and pathological sources was examined. The specific antibody raised against collagenase secreted from normal rabbit synovial fibroblasts gave reactions of complete identity with collagenases secreted from fibroblasts derived from rabbit skin, and from synovium from experimentally arthritic rabbits. 2. The rabbit fibroblast collagenase was immunologically identical with collagenases obtained from the organ culture medium of normal rabbit skin, synovium, ear fibrocartilage and subchondral bone. 3. Collagenases from the culture media of normal rabbit synovium and from hyperplastic synovium of rabbits made experimentally arthritic were identical. 4. The collagenase secreted from rabbit fibroblasts gave a reaction completely identical with that of a collagenase extracted directly from a rabbit carcinoma. 5. IgG (immunoglobulin G) from a specific antiserum to rabbit fibroblast collagenase was a potent inhibitor of the collagenases obtained from the culture media of the various rabbit cells and tissues. 6. Collagenases from human synovium and from mouse macrophages and bone were neither precipitated nor inhibited by antibodies to rabbit collagenase. 7. No immunoreactive material was found in lysates of rabbit polymorphonuclear leucocyte granules with the specific antisera to rabbit fibroblast collagenase. No evidence for inactive forms of rabbit collagenase in lysates of the rabbit synovial fibroblasts could be found, either by double immunodiffusion against the specific collagenase, or by displacement of active enzyme from inhibition by the IgG.     

Partial amino acid sequence in the N-terminal region of an anti-pneumococcal immunoglobulin heavy chain of allotype a2 (Short Communication)

The amino acid sequence of the N-terminal 48 residues of the heavy chain derived from a homogeneous rabbit antibody to type III pneumococci is described. This chain of allotype a(2) is compared with other rabbit heavy chains of allotypes a(1), a(2) and a(3). Within the N-terminal 25 positions, two chains which carry the same allotype a(2) possess identical amino acid sequences, but differ markedly from heavy chains of allotypes a(1) and a(3). Sequence variability is observed in residues 26-27 and 30-34, but not in residues 35-48.     

黑线毛足鼠和金仓鼠血清IgG的纯化及抗血清的制备

目的 纯化黑线毛足鼠和金仓鼠血清IgG,制备兔抗金仓鼠和黑线毛足鼠血清IgG的抗血清。方法 用Hitrap Protein G亲和层析纯化黑线毛足鼠和金仓鼠血清IgG,经SDS-PAGE电泳鉴定纯度,标准免疫方法免疫兔子制备抗血清。结果 黑线毛足鼠和金仓鼠血清IgG对protein G有很高的亲合性,用Hitrap Protein G亲和层析纯化,得到高纯度黑线毛足鼠和金仓鼠IgG,利用纯化的IgG作抗原制备了高效价的抗血清,免疫双扩散测定效价达1∶32和1∶16。结论 证实黑线毛足鼠和金仓鼠IgG和Protein G具有很高的亲和性,Protein G亲和层析是纯化黑线毛足鼠和金仓鼠IgG有效的方法之一,制备了黑线毛足鼠和金仓鼠IgG的抗血清。     

DNA polymorphism of human HLA-linked complement C4 allotypes, including C4 null alleles, in the Finnish population

Human HLA-linked complement C4 gene products, C4A and C4B, show extensive genetic polymorphism. In both loci, an allele without a gene product, C4 null, is also observed. We have performed a restriction enzyme analysis of genomic DNA samples from individuals having all common (frequency over 1%) C4 protein allotypes observed in the Finnish population. Only one allotype-specific RFLP marker was observed. With some enzymes a DNA polymorphism was observed, which was not detectable by C4 protein typing. Analysis of 10 different C4B null haplotypes and 4 C4A null haplotypes suggested that only one haplotype, HLA-B8 C4A0 B1, carried a C4A gene deletion. This was observed in all 4 unrelated individuals homozygous for this haplotype.