A haemagglutination method for detection of rheumatoid factor using preserved erythrocytes covalently coated with human IgG.

The preparation and use of a new haemagglutination assay for rheumatoid factor using preserved erythrocytes covalently coated with human IgG is described. Test serum heat inactivation or adsorption with uncoated erythrocytes is not required. This human IgG-coated erythrocyte agglutination test (H/CEAT) appears stable and specific, is suitable for routine determinations of serum IgM-rheumatoid factor, is simple to perform and compares favourably in these properties with either the Rose-Waaler or latex agglutination assays for rheumatoid factor.

The ultrastructure of C1t, a subcomponent of the first component of complement: an E.M. and ultracentrifuge study.

C1t is a 9.5 S alpha1 glycoprotein that has been shown to be a fourth subcomponent of the first component of complement. The m.w. of C1t was found to be 233,000 by sedimentation equilibrium in the ultracentrifuge. A subunit m.w. of 23,000 was obtained by sedimentation equilibrium in 5.95 M guanidinium chloride. No change in either m.w. was produced by prior reduction and alkylation. In the electron microscope characteristic pentagonal figures of 85 A diameter were observed together with rod-like figures which appear to be stacked assemblies of the pentagonal figures. These observations lead us to propose that C1t is a noncovalent, decameric protein with the subunits disposed at the vertices of two regular pentagons joined at one of their faces. A possible relationship between C1t and the P-component of amyloid is discussed.

Clq inhibition of the interaction of collagen with human platelets.

Human Clq, isolated in pure state after affinity chromatography on IgG-Sepharose, inhibited collagen-induced aggregation and release of 14C-Serotonin from prelabeled human platelets. Platelet aggregation induced by ADP or thrombin was not inhibited by Clq. Also, the adherence of platelets to glass surfaces was significantly diminished by Clq. In contrast, aggregated Clq mimicked the effect of collagen in causing platelet aggregation and release of serotonin. It appears that monomeric Clq, which has structural similarities to collagen competes with collagen for specific sites on the platelet surface.

The identification of a previously unrecognized subcomponent of the first component of complement.

The use of an affinity chromatography method designed to isolate C1 from serum has led to the discovery of a novel plasma protein, II-P2, associated with C1. The persistent Ca++-dependent association of II-P2 with C1 subcomponents following euglobulin precipitation, affinity chromatography on Sepharose-IgG, and density gradient ultracentrifugation indicates that II-P2 might be a C1 subcomponent. Using purified preparations of II-P2 it was found that a) II-P2 was retained on Sepharose-IgG through a Ca++-dependent link with C1q,b) II-P2 enhanced the C1 activity of mixtures of C1s and C1q in a dose-dependent fashion, c) II-P2 bound firmly to EAC1q4 cells and enhanced their C1s-binding ability. Fractionation of C1 by DEAE-Cellulose chromatography under the conditions that led to the original identification of C1q, C1r, and C1s resulted in recovery of II-P2 in the fractions containing C1r. The evidence presented confirms that II-P2 is a C1 subcomponent (C1t).

The macromolecular structure of the first component of complement.

The binding of C1 to IgG and the interactions between C1 subcomponents have been studied by affinity chromatography of serum C1 and purified C1 proteins on Sepharose-IgG. Affinity chromatography of serum on Sepharose-IgG resulted in the binding of C1; subsequent washing with EDTA removed only C1s and C1t. Affinity chromatography of serum-EDTA on Sepharose-IgG resulted in binding of only C1q and C1r. Affinity chromatography of serum on Sepharose-tryptophan-modified-IgG resulted in the binding of only C1r and C1s. By the use of purified C1 proteins and Sepharose-IgG in binding studies it was confirmed that both C1q and C1r bind independently to sites on IgG and hold C1s and C1t by Ca++-dependent bonds. Measurements of the hemolytic activity of various combinations of C1 subcomponents confirmed the data obtained by the affinity binding studies. Both C1t and C1r independently enhanced the C1 activity of C1q-C1s mixtures; maximal activity required all four subcomponents. Sucrose gradient ultracentrifugation of mixtures of C1 proteins showed formation of the following complexes: C1qs (12S), C1qrs (15S), C1qst (18-23S), and C1qrst (19S). The evidence suggests that the spatial sequence of the components of the Sepharose-IgG-Serum C1 complex is: Sepharose-IgG: C1q: C1t: C1s: C1r: IgG-Sepharose. The probable physiologic significance of this model is discussed.