Abstract
A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.
Highlights
A panel of mouse monoclonal anti-CD4 antibodies The functional duality of the variable (V)’ region of the was characterized in termsof idiotypic expression by immunoglobulin molecule results in the ability to recognize using specific anti-idiotypicantibody(anti-Id)rethe antigen and toexpress antigenic determinants which can agentsgeneratedinrabbits immunized with anti- be recognized by specific antibodies
LeuBa, a monoclonal anti-CD4 which inhibits the hu- nants expressed on the V region of the immunoglobulin molman immunodeficiency virus (HIV) gp120 binding to ecules are termed idiotypes (Id) and the specific antibodies
Id may be associated with the antigen-combining antibodies able torecognize CD4 epitopes overlapping site of the antibody molecule [3, 4] or can be localized to the epitope recognized by anti-Leu3aexpressedan regions not associated with the antigen-combining site [5].In antigen-combining site-related cross-reactive idiotype addition, Id can be unique to a particular antibody molecule (IdX)
Summary
(Calbiochem).The injections were administered at monthly intervals. For the initial injection, synthetic peptides were emulsified in Production of MouseMonoclonal Anti-CD4 Antibodies-Mouse Freund’s complete adjuvant, and for subsequent immunizations in monoclonal anti-CD4 antibodies were obtained by fusing spleen cells Freund’s incomplete adjuvant.Rabbits received a total of seven from BALB/c mice immunized with the CD4-expressing human T injections. 200 ng of the various monoclonal anti-CD4 antibodies was diluted in 50 pl of BBS and coated onto individual microtiter wells overnight at 4 "C.Nonspecific sites were blocked by adding 10%. A dilution in 10% NGS of rabbit anti-Id antiserum corresponding to the linear part of the Id-anti-Idbinding curve was mixed with either different concentrations of soluble recombinant CD4 (sCD4) (kindly provided by Dr George Cianciolo,Genentech, Inc., So. San Francisco, CA) or an irrelevant inhibitor (bovine serum albumin), added to triplicate wells in 50-pl aliquots and allowed to incubate overnight at. 10% NGS of rabbit antisera corresponding to the linear part of the anti-Leu3a binding curve was mixedwith different concentrations of the free peptides, added to triplicate wells in 50-pl aliquots and allowed to incubate overnight at 4 "C. The paper was incubated with ['251]proteinA, dried, and exposed to x-ray film
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