Simple methods for preparing EAC1,4b,2a,3b and EAC4b,3b with human or guinea pig complement components using an anticomplementary agent, K-76 monocarboxylic acid.

Abstract

Abstract Simple methods were developed for preparing EAC1–3b, EAC4b,3b, EAC–3b,P, and EAC4b,3b,P with either human or guinea pig complement (C) components. The methods are based on the inactivation of both C5 and C3b inactivator (C3blNA) in serum or serum deficient in properdin (RP) with the anticomplementary compound K-76 monocarboxylic acid (K-76COOH). EAC1–3b,P is readily obtained by incubating sensitized erythrocytes (EA) with whole C serum pretreated with K-76COOH. EAC1-3b is obtained similarly using RP instead of whole serum. The numbers of C3b sites per cell are inversely proportional to the cell concentration used during the preparation. EAC1–3b,P cells possessed only C3b molecules with no other derivatives of C3. These C3b molecules were fully sensitive to the action of C3blNA and β1H. EAC4b,3b,P and EAC4b,3b are obtained by removing C1 with ethylenediaminetetraacetate (EDTA) and incubating the mixture at 37°C to allow decay of C2a. These intermediate cells can initiate the alternative pathway of C activation in the presence of Mg ion and ethyleneglycolbis(aminoethyl) tetraacetate (EGTA). When tested by agglutination assay with antiserum to factor B, properdin, or βH, factor B is detected only on EAC1–3b,P; a small amount of properdin is found even on EAC1–3b and EAC4b,3b when they have been prepared at lower cell concentration, and β1H H globulin is detected on all these intermediates. Guinea pig C5 is more effectively titrated with the intermediate cells EAC1–3b or EAC1–3b,P containing guinea pig C components than with those containing human components; human C5 is more effectively titrated with intermediate cells containing human components. Factor B can be titrated hemolytically with EAC4b,3b or EAC4b,3b,P cells followed by treatment with rat or guinea pig serum in EDTA buffer (C-EDTA). None of the intermediate cells prepared by these methods react with bovine conglutinin. Treatment of the cells with C3blNA and β1H renders the cells able to show conglutination. The intermediate cells prepared by these methods form rosettes with Raji cells but not with Daudi cells. Treatment of the cells with C3blNA and β1H enhances rosette formation with Raji cells, and enables the cells to form rosettes with Daudi cells.