Subtilopeptidase A isoenzyme system. Interaction with serum components and its importance for quantitative immunoelectrophoresis

Abstract

A method was developed which involved electroimmunoassay and crossed immunoelectrophoresis of subtilopeptidase A (EC 3.4.21.14). Initial trials with unfractionated antiserum were not successful and interaction of the enzyme with non-immunoglobulin serum components were shown to be the cause of the failures. Quantitative immunoelectrophoresis was possible when purified immunoglobulins were used. A pH of6.5 (lower than the usual pH8.6) was necessary to obtain a proper baseline definition. Subtilopeptidase A was confirmed as a multiple isoenzyme system. Qualitative inter-batch variations were detected. Di-isopropyl phosphorofluoridate inhibition altered the electrophoretic pattern, but no loss of antigenic determinants was observed.

Antigenic and Allergenic Characterization of the Enzymes Alcalase and Savinase by Crossed Immunoelectrophoresis and Crossed Radioimmunoelectrophoresis

“…Our finding that the alcalase material contained two clearly distinct, antigenically different compo nents was in contrast to a previous report [9] showing that alcalase (subtilopeptidase A) was a multiple iso enzyme system in which different forms of the antigen exhibited partial antigenic identity. In that previous report, CIE analysis of subtilopeptidase A showed that it contained one major and one minor electropos itive antigen, each consisting of three and two iso zymes, respectively.…”

“…Some of the differences between the results of our analysis and those of previous studies may be attri buted to interbatch heterogeneity in the enzyme that had been previously reported [9], use of different buf fers for the CIE that gave different antigen mobility, or the fact that the current bacterial strains produce a more homogeneous product. We have confirmed the heterogeneity in other batches of alcalase we have an alyzed by CIE which contained three distinct antigens in contrast to the two for the batch used for this study.…”

“…As a result, stained gels exhibited a diffuse antigen-antibody precipitate on the anodic side of the gel opposite the large, well-defined cathodic antigenantibody precipitin peak. Similar retrograde mobility was previously reported for components of subtilopeptidase A, the major component of alcalase [9]. Had a charge change not occurred, the antigen would have migrated in the opposite direction during lst-dimension electrophoresis.…”

Efficacy of Oral Antigen Specific Immunotherapy on Desensitization of Some Autoimmune Diseases Associated with Milk Allergy

“…Serum total IgE: Total IgE concentration was determined by using microplate enzyme immunoassay (Biocheck, Inc. 323 Vintage Park Dr. Foster City, CA 94404, USA) according to the method of [18]. Serum IgG: It was measured using immunodiffusion (Bioscientifica S.A Company) according to the method of [19]. Serum RF: it was done according to the methods of [20] using Human Gesellschaft fur, Biochemica und Diagnostica kit (mbH, D-65205 Wiesbaden-Germany).…”