Monoclonal antibodies against rat immunoglobulin kappa chains

Monoclonal antibodies directed against rat kappa light chains have been generated by immunizing SJL/J mice with soluble rat immunoglobulin, followed by fusion of immune spleen cells with the P3-X63-Ag8 myeloma cell line. Monoclonal antibodies from three of these hybridoma cell lines, MAR 18.5, 80.2, and 103.6, have been extensively characterized. MAR 18.5, 80.2, and 103.6 antibodies are of the gamma 2a kappa isotype, and bind strongly to protein A, allowing easy purification. Monoclonal antibody from clone 18.5 binds equally well to Ig of both RI-1a and RI-1b allotypes, whereas 80.2 and 103.6 antibodies selectively bind to RI-1b. These monoclonal antibodies can be FITC conjugated for use as a second antibody in indirect immunofluorescence assays, or radiolabeled for use in radio immunoassays requiring a specific antirat kappa antibody. The antiallotype specific monoclonal antibodies also may be of use in the study of rat immunoglobulin genetics.     

Basilea kappa light chains of rabbit immunoglobulins

Immunogenetics of the Basilea allotype were investigated. It was shown that the Basilea gene is controlled at a locus identical with, or closely linked to, the Ab kappa allotypic locus. Basilea-positive molecules have been physically separated by immunoabsorption from molecules carrying the lambda chain markers, c7 and c21. Injection of newborn Basilea homozygotes with anti-Basilea immune serum results in suppression of the synthesis of Basilea-positive molecules, but does not affect the synthesis of the lambda chain, or of the heavy chain allotypic markers. Allotype-suppressed Basilea homozygotes do not recognize either the Basilea allotype or the kappa isolate as 'self' and make anti-Basilea and anti-kappa antibody upon immunization with Basilea antigen. The specificity of this antibody was identical with that of a heterologous anti-kappa immune serum raised in a goat and made specific by absorption with purified lambda material. These results prove that Basilea gene products are of kappa isotype and are controlled at the Ab allotypic locus.     

Partial sequence of the variable region of IgG light chains of b5 allotype from A B4B5 rabbit.

The partial sequence (positions 29–71) of the variable region of light chains of predominately b5 allotype from the IgG of a single allotype-suppressed rabbit was obtained by traditional sequencing methods on isolated tryptic and chymotryptic peptides. The peptides from this region were isolated in relatively high yields and probably represent a dominant sequence. The framework sequence between positions 35 and 49 (FR 2) is identical to an FR 2 sequence commonly found in light chains from antibodies produced by b4 rabbits as well as murine and human myeloma light chains, with the exception of an interchange of threonine for proline at position 43 or 44. This may be b5 allotype-related since, to date, all the b4 light chains have had proline, and a b9 light chain was found with arginine at position 43. The fact that a dominant sequence could also be found for positions corresponding to the second complementarity determining region (CDR 2, 50–56) in other species, confirms previous observations that this portion of the light chain is not extremely variable in the rabbit.     

Serologic distinction between the rabbit kappa L chain allotype b4 and an allele b4.

Rabbit kappa light (L) chain allotypes are controlled by the allelic genes b4, b5, b6, b9 and a recently described allele b4v. The product of the b4v gene differs from that of b4 by at least two amino acid substitutions in the constant region. A serologic distinction between the b4 and b4v allotyes has now been detected by a radioimmune assay using one of six anti-b4 sera tested. The distinction was enhanced by fractionation of this antiserum on an immunoadsorbent containing cottontail rabbit IgG. IgG with L chains of the b4v type are deficient in their ability to inhibit binding of b4 IgG to the antibody preparation. L chains isolated from b4 and b4v IgG samples could not be distinguished by this assay indicating that interaction with an H chain is required for expression of the L chain determinants that differentiate b4 from b4v.     

Monoclonal antibodies specific for variable and constant domains of murine lambda chains

Rat monoclonal antibodies directed against the BALB/c myeloma protein M315 (alpha,lambda 2) are described. 9A8 (IgG1) binds the V domain of lambda 2 and cross-reacts with lambda 1 and lambda 3 chains. 2B6 (IgG2a) is directed to the C domain of lambda 2 and cross-reacts with C lambda 3. The antibodies bind isolated chains as well as complete immunoglobulins. The monoclonals detect soluble immunoglobulin (radioimmunoassay), immunoglobulin immobilized on polystyrene (enzyme-linked immunosorbent assay), immunoglobulin bound to nitrocellulose (immunoblotting), and surface immunoglobulin intercalated in cell membranes (immunofluorescence). The antibodies are easily purified on protein G immunosorbents and may be biotinylated or conjugated with fluorescein isothiocyanate without loss of capacity to bind. In addition to the anti-lambda antibodies, a C alpha 2/C alpha 3-specific monoclonal antibody, 8D2 (IgG2a) is described.     

Partial primary structure of the immunoglobulin light chain constant region of a single rabbit of b5 allotype

The partial amino acid sequence of the constant region of the b5 light chain from the normal IgG of a single rabbit is reported.

For structure determination, the IgG light chain was fully reduced and carboxymethylated, then digested with chymotrypsin or trypsin. All chymotryptic peptides covering the constant region from positions 116–210 (117–214 in the standardized numbering system of Kabat et al.^*) were isolated and purified by column and paper chromatography. Sequences were then determined using traditional sequencing methods. Overlapping was obtained by use of large tryptic peptides, covering positions 138–210 (139–214^*). The chymotryptic peptide ending with Leu 115(116 ^*) could not be obtained in a pure state owing to insolubility and perhaps heterogeneity. When the sequence of this light chain is compared to those of light chains of b4 and b9 allotypes, about 77% homology is found with b4 and 62% with b9. This confirms serological data which would indicate that b4 and b5 allotypes are more similar to each other in structure than they are to b9. The allotypic differences are distributed throughout the whole constant portion of the light chain.     

Escape from b locus allotype suppression in the rabbit is mediated by IgM molecules bearing an allotope-restricted variant of kappa light chain

Rabbits homozygous for b6 at the kappa light chain b locus were suppressed for the expression of the b6 allotype and then induced to produce auto anti-b6 antibody. Rabbits which subsequently escaped suppression produced auto antibody with restricted allotype specificity. Escape from allotype suppression was mediated by IgM bearing a kappa chain variant with a restricted number of b6 allotopes and having a diminished interaction with auto anti-b6 antibodies from the same and other rabbits escaping b6 suppression. This suggested that there were allelic variants or subpopulations of the b6 light chain which were under independent regulation of expression, clearly influenced by the specificity of auto anti-allotype antibody. Since escape from suppression was mediated by IgM it is proposed that a normal pathway of B cell differentiation occurs during recovery from suppression.     

The structural correlates of the rabbit light chain b allotypes: Sequence studies of b5 and b6 chains

Rabbit kappa light chains of allotype b5 and b6 were prepared from antibodies of restricted heterogeneity made by animals hyperimmunized with, respectively, strain III and strain II pneumococcal vaccines. The amino-acid sequence of several tryptic peptides were determined. The variable region fragments of the b5 and b6 chains appear to be quite similar to the corresponding fragments of b4 and b9 chains, albeit some residues seem to be allotype associated. In contrast the chains of different allotypes vary right from the start of the constant region in a number of positions, suggesting that b allotypes correlate with amino-acid substitutions in this region. The number of substitutions between the b5 and b6 and the previously determined b4 and b9 constant regions sequences ranges from 20 to 35%. Serological studies suggest that Leporidae b allotypes diverged no more than 2 × 10⁶ years ago. By this time only 1% of the substitutions could be generated by ‘conventional evolution’. Duplication and mutation of the individual C_K genes could account for the high level of divergence observed. The data reported here support the notion that the structural genes encoding the light chain constant regions of the various b allotypes coexist on the same chromosome and that the allelism is controlled by a regulatory mechanism.     

Monoclonal antibodies against heavy and light chains of domestic mink IgG.

A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).     

Monoclonal antibodies specific for kappa chain, lambda chain, and IgG1 of human gammaglobulin

Hybrid cell lines secreting antibodies specific for human gammaglobulin (HGG) were prepared by cell fusion and cloning techniques. The monoclonal antibodies were tested for their antibody reacts with a different antigenic determinant of HGG. One reacts with isolated kappa (kappa) light chains, one with isolated lambda (lambda) light chains, and one with the Fc fragment of IgG1 molecules. The reactivity patterns of two additional monoclonal antibodies are more complex. One reacts with a determinant present on the Fc of all IgG subclasses and the other binds to a determinant on the Fab of IgG molecules. The two monoclonal antibodies reactive with light chains also bind to surface components of human B cells. The murine immunoglobulin (Ig) class of each clone product was identified.     

Anti-immunoglobulin antibodies IV. Cross-reaction of anti-idiotypic antibodies specific for rabbit and murine anti-a1 allotype antibodies with Fc fragment of human immunoglobulins

Anti-idiotype (Id) antibodies against anti-a₁ rabbit allotype antibodies were produced in rabbits and mice by immunization with polyclonal rabbit anti-a₁ allotype or with a monoclonal murine anti-a₁ allotype antibody, respectively. These antibodies recognize an interspecies cross-reactive idiotope on anti-a₁ allotype antibodies. However, these anti-Id antibodies also react with various subclasses of human IgG. In multiple systems, it was shown that these anti-Id antibodies interact with the Fc fragment of human IgG and, therefore, exhibit a rheumatoid factor-like activity. These results support the hypothesis that γ-globulin may not be the only stimulus to clones producing rheumatoid factors and that perhaps other antibodies, especially anti-Id antibodies, also bind γ-globulin secondarily.     

Monoclonal antibody against sheep kappa light chain

The production of a monoclonal antibody specific for sheep kappa light chain protein is described. The monoclonal antibody was designated McM11 and its specificity was verified using western blots of sheep IgG and slides of efferent lymph cells. The specificity of McM11 was confirmed by specific recognition of fusion proteins expressed by recombinant phage containing sheep kappa cDNAs. N terminal sequence of the light chain recognized by McM11 showed homology to kappa type light chains. McM11, together with McM6, a lambda specific monoclonal antibody, was shown by two color FACS analysis of sheep blood lymphocytes to recognize all sheep light chains.     

Monoclonal antibodies to axolotl immunoglobulins specific for different heavy chains isotypes expressed by independent lymphocyte subpopulations

An immunoblotting analysis of purified axolotl immunoglobulins (Ig) separated by SDS-PAGE reveals two heavy (H) chains isotypes: a 76 kDA chain recognized by the monoclonal antibody (mAb) 33.45.1 and a 66-68 kDa doublet recognized by the mAb 33.39.2. The 76 kDa chain is associated to high molecular weight (HMW) Ig molecules and the 66-68 kDa H chains are associated to low molecular weight (LMW) Ig of 172 kDa. Both H chains isotypes are linked to identical light (L) chains, labelled in immunoblotting by the mAb 33.101.2. Two different axolotl lymphocyte subpopulations are characterized by these two distinct H chains isotypes. One population of splenic lymphocytes (approximately 40%) is labeled by indirect immunofluorescence with mAb 33.45.1, specific for the 76 kDa H chain isotype. Another population (approximately 20%) is labeled by mAb 33.39.2 specific for the 66-68 kDa H chain isotype. Both populations of splenic lymphocytes are stained by mAb 33.101.2 specific for the axolotl L chains. Therefore, the presence of at least two independent Ig classes is now confirmed in a urodele amphibian species at the humoral and cellular levels.     

Rat kappa chain allotypes. IV. Monoclonal antibodies to distinct RI-1b specificities

Four hybridomas are described which produce antibodies to the RI-1b rat kappa chain allotype. Three are rat alloantibodies of KG2b isotype, one is a mouse heteroantibody of KG2a isotype. These antibodies were labeled by incorporation of 75Se-selenomethionine and their specificity determined by a plate-binding assay. Inhibition studies using partially cross-reactive sera from Asian and Australian Rattus show that these four hybridomas detect four distinct RI-1b specificities. Differences in the slopes of the inhibition curves indicate that each of the specificities may exist in more than one form. In addition, in the form of 125I-labeled antibodies, two of these hybridomas show a high degree of specificity in binding to Ig-bearing lymphocytes from spleen and lymph nodes. These hybridomas will therefore be useful not only in studies on the phylogenetic distribution of RI-1 specificities but also in replacing conventional antisera for immunochemical and cellular immunological studies.     

Monoclonal antibodies for specific detection of Encephalitozoon cuniculi

Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized. The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E. cuniculi originating from clinical samples. They did not cross-react with other Encephalitozoon species (E. intestinalis and E. hellem) or with a collection of gram-negative bacteria, yeast, and other parasites. The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E. cuniculi. These epitopes were demonstrated to be E. cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We developed MAbs to strains of E. cuniculi that can be used successfully to distinguish E. cuniculi from other microsporidial species in cultures established from clinical specimens. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia.     

Demonstration of the clonality and specificity of an auto-antibody response to a suppressed immunoglobulin allotype of the rabbit kappa chain b locus

A regulatory idiotypic network is proposed to control allotype expression in normal rabbits. We have used suppression of the kappa chain b6 allotype in an attempt to restrict the number of regulatory idiotypes involved in an induced auto anti-allotype response. These auto anti-b6 antibodies were examined by an agarose imprint immuno-fixation IEF technique using iodinated allotype-bearing IgG. All totally b6-suppressed rabbits produced a clonally complex response which was generally spectrotypically unique - thus contradicting previous claims of a dominant common idiotypic pattern. The compensating light chains thus have available as many regulatory V genes as does the kappa 1 light chain. However, the b6/b6 homozygotes breaking b6 suppression produce an auto anti-b6 antibody which does not interact with their "escaping" molecules and which is clonally restricted. We propose a regulatory mechanism limiting the V genes utilised to produce the autoantibody, the latter then allowing only molecules bearing non-interactive allotopes to "escape" suppression.