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Production of monomeric antigen-enzyme conjugate to study requirements for follicular immune complex trapping

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Author: Laman, J.D. · Hart, H. ter · Boorsma, D.M. · Claassen, E. · Rooijen, N. van
Institution: Medisch Biologisch Laboratorium TNO
Source:Histochemistry, 2, 97, 189-194
Identifier: 231742
Keywords: Enzyme · Monoclonal antibody · Animal cell · Antigen antibody complex · Dendritic cell · Immunohistochemistry · Lymph follicle · Mouse · Nonhuman · Priority journal · Technique · Animal · Antibodies, Monoclonal · Antigen-Antibody Complex · Electrophoresis, Polyacrylamide Gel · Enzyme-Linked Immunosorbent Assay · Epitopes · Female · Horseradish Peroxidase · Mice · Molecular Weight · Penicillins · Serum Albumin · Spleen


Studies concerning the localization of immune complexes in lymphoid follicles and the involvement of these trapped immune complexes in the regulation of the immune response have thus far been performed with poorly defined complexes in terms of size and composition. For that reason, the minimum requirements for trapping in terms of number of antigen- and antibody molecules present in immune complexes could not be determined. We here describe the production and in vivo use of a monomeric HSA-HRP antigen-enzyme conjugate, readily demonstrable in cryostat sections and ELISA. This conjugate was obtained by combining the glutaraldehyde coupling-method with chromatography to fractionate monomeric and multimeric constituents. SDS-PAGE analysis showed that the conjugate consisted of a single molecular species of 109 kDa, whereas the often used periodate oxidation coupling method yielded a heterogeneous population of multimeric, oligomeric and monomeric molecules. We investigated the minimal size requirements for the composition of immune complexes to be trapped in murine spleen follicles using three different conjugates (monomeric HSA-HRP, multimeric HSA-HRP and multimeric HSA-HRP-Penicillin) and a panel of anti-HSA and anti-Penicillin monoclonal antibodies. We demonstrate that the smallest immune complexes, consisting of one antibody and two conjugate molecules, do not localize in splenic follicles. Immune complexes prepared with a single monoclonal antibody localize in follicles only if the epitope recognized occurs repeatedly on the antigen. The relevance of these results for physiological follicular trapping of protein antigens is discussed. The described method for the production of monomeric enzyme-labelled protein applicable in histochemistry and ELISA should prove useful to prepare other conjugates of defined size for studies of trapping and other applications.