Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays

The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP2.Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG1 and IgG2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and application of monoclonal antibodies directed to separate epitopes of glutathione-insulin transhydrogenase

Five monoclonal antibodies specific for glutathione-insulin transhydrogenase were characterized. None of the monoclonal antibodies cross-reacted with another insulin-degrading enzyme, neutral thiopeptidase. The isotype of four antibodies was IgG1 and of the fifth IgG2b. Affinity studies, competitive binding studies and immunoblot analysis of CNBr and trypsin cleavage products of glutathione-insulin transhydrogenase demonstrated that the four IgG1 antibodies were directed to an epitope of the enzyme which was distinct from the epitope recognized by the IgG2b antibody. Inhibition studies indicated that each monoclonal antibody, when added singly to glutathione-insulin transhydrogenase, was unable to inhibit the insulin-degrading activity of the enzyme. However, when monoclonal antibodies directed against separate epitopes of glutathione-insulin transhydrogenase were presented together (i.e., the IgG2b with any one of the four IgG1 antibodies), a loss in enzymatic activity was noted. Immunoblot analysis of rat organ extracts with the IgG1 antibodies demonstrated one immunoreactive protein band of Mr 56,000 in all tissues examined (liver, fat, pancreas and kidney) except the spleen, which demonstrated two immunoreactive protein bands of Mr 56,000 and 51,000. The same immunoblots, when probed with the IgG2b antibody, demonstrated the same immunoreactive protein banding pattern as above plus an additional immunoreactive protein band of Mr 67,000 in all tissues. Studies with spleen extracts from steptozotocin-induced diabetic rats demonstrated that there was a loss of the 51,000 immunoreactive band in diabetes. This 51,000 protein was restored upon insulin treatment of the diabetic rats and nullified upon concomitant administration of cycloheximide or actinomycin D with insulin. Immunoblots of human liver, adipose and skeletal muscle extracts indicated that each monoclonal antibody cross-reacted with the human form of the enzyme which had a molecular weight of Mr 63,000; a second minor immunoreactive band of 67,000 was detected with the IgG2b antibody. The physiological significance of additional molecular forms of the enzyme (i.e., 67,000 and 51,000) remains to be determined.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Mouse × pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodies

The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse × pig anti‐PRRSV antibodies. We cloned the constant regions of gamma‐1 and gamma‐2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western‐blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Production of a human monoclonal antibody recognising a determinant on mouse IgG2b from a patient receiving mouse monoclonal antibody for diagnostic imaging

Summary The development of human antibodies recognising mouse immunoglobulins represents an obstacle to effective antibody therapy. This study shows that patients produce modest titres of antibodies (predominantly antimouse rather than anti-idiotypic) after a single low-dose injection for immunoscintigraphy, suggesting that repeated imaging with the same or a different antibody could be a problem. Fusion of the lymphocytes from a patient who had been imaged twice previously resulted in a monoclonal antibody that specifically binds to an IgG2b isotypic determinant. Anti-IgG2b antibodies predominated in this patient's serum. Production of human monoclonal antibodies from patients given mouse monoclonal antibodies not only allows a finer dissection of the immune repertoire but also provides possible reagents for controlling the human anti-(mouse Ig) response, for selection of class-switch variants of mouse monoclonal antibodies and enhancing tumour imaging.     

Rat monoclonal antibody specific for prostaglandin E structure

Six different monoclonal antibodies directed against prostaglandin E2 were obtained from hybrid myelomas, following fusion of mouse NS-1 myeloma cells with spleen cells from a rat immunized with bovine serum albumin conjugates of prostaglandin E2. Four of them were of the IgG2a subclass and the other two were an IgG2b and an IgG2c. Affinities of antibodies for prostaglandin E2 were in the range 5.8 X 10(6)-6.7 X 10(8) M-1. Cross-reactivity experiments showed that one monoclonal antibody was directed almost exclusively against the prostaglandin E structure. The specific monoclonal antibody purified from ascites fluid was used for enzyme immunoassay, and as little as 30 pg of prostaglandin E1 and 100 pg of prostaglandin E2 were detected, which values are comparable to those obtained by radioimmunoassay. These results reveal that the hybridization technique is a reliable way to obtain prostaglandin E-specific antibody and that monoclonal antibodies can be valuable reagents for immunoassays.     

Normal human sera contain antibodies directed at Fab of IgA

Serum samples from 26 normal volunteers were evaluated by isotype-specific ELISA for the presence of IgG and IgM antibodies directed at IgA. Although there were wide variations in antibody levels, anti-IgA antibodies of both isotypes were found in all individuals tested. The anti-IgA activity was detected against a variety of polymeric and monomeric IgA1 and IgA2 myeloma proteins containing both kappa and lambda light chains. By using Fab and Fc fragments generated by incubation of an IgA1 myeloma protein with IgA1 protease, it was shown that the anti-IgA activity was specific for the Fab portion of the IgA molecule. It was also demonstrated that the serum of two individuals contained both IgG and IgM activity directed at autologous affinity-purified IgA. IgM antibody levels against both whole IgA and Fab of IgA were significantly higher than IgG antibody levels. Cells producing anti-IgA antibodies of both isotypes were detected in lipopolysaccharide-stimulated human spleen.     

Fast protein sequencing of monoclonal antibody by real-time digestion on emitter during nanoelectrospray

Growth in the pharmaceutical industry has led to an increasing demand for rapid characterization of therapeutic monoclonal antibodies. The current methods for antibody sequence confirmation (e.g., N-terminal Edman sequencing and traditional peptide mapping methods) are not sufficient; thus, we developed a fast method for sequencing recombinant monoclonal antibodies using a novel digestion-on-emitter technology. Using this method, a monoclonal antibody can be denatured, reduced, digested, and sequenced in less than an hour. High throughput and satisfactory protein sequence coverage were achieved by using a non-specific protease from Aspergillus saitoi, protease XIII, to digest the denatured and reduced monoclonal antibody on an electrospray emitter, while electrospray high voltage was applied to the digestion mixture through the emitter. Tandem mass spectrometry data was acquired over the course of enzyme digestion, generating similar information compared to standard peptide mapping experiments in much less time. We demonstrated that this fast protein sequencing method provided sufficient sequence information for bovine serum albumin and two commercially available monoclonal antibodies, mouse IgG1 MOPC21 and humanized IgG1 NISTmAb. For two monoclonal antibodies, we obtained sequence coverage of 90.5–95.1% for the heavy chains and 98.6–99.1% for the light chains. We found that on-emitter digestion by protease XIII generated peptides of various lengths during the digestion process, which was critical for achieving sufficient sequence coverage. Moreover, we discovered that the enzyme-to-substrate ratio was an important parameter that affects protein sequence coverage. Due to its highly automatable and efficient design, our method offers a major advantage over N-terminal Edman sequencing and traditional peptide mapping methods in the identification of protein sequence, and is capable of meeting an ever-increasing demand for monoclonal antibody sequence confirmation in the biopharmaceutical industry.     

Selective immunoprecipitate identification using monoclonal anti-rabbit IgG

A monoclonal mouse antibody directed against rabbit IgG has been conjugated with horseradish peroxidase and used to identify immunoprecipitates which contain rabbit antibodies. By combining a specific rabbit antisera with a general antiserum from another species (e.g., goat antiserum against human serum), immunoprecipitates containing the antigen(s) recognized by the rabbit antibodies have been selectively identified by colorimetric development of peroxidase activity. Since the monoclonal antibody is specific for rabbit IgG and nonprecipitating, the peroxidase conjugate can be included in the agarose with the primary antisera.