PubMed · 6357874
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J H Newman. 1983. Circulating immune complexes: a review.. https://pubmed.ncbi.nlm.nih.gov/6357874/
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We investigated the interaction of immune complexes (IC) isolated from hepatitis C virus (HCV)-infected individuals with several cell lines that differentially express Fc receptors, and analyzed viral infection by the presence of HCV RNA sequences. Monocytic (U937 and Monomac-6) and lymphocytic (MOLT-4 and Jurkat) cell lines were incubated with interferon- plus phorbol myristate acetate to stimulate the expression of Fc receptors before addition of IC. Cell interaction with IC was monitored by flow cytometry. Positive cell fluorescence was detected in U937 and Monomac-6 cells [mean fluorescence intensity (MFI) 10.56+/-0.8 and 11.60+/-0.8, respectively]. Incubation of cells with monoclonal antibodies against Fc receptors for IgG before addition of IC decreased MFI in both cell lines (U937 2.1+/-0.5, Monomac-6 4.4+/-0.8, P<0.001), indicating that cell-IC interaction through these receptors was inhibited. In particular, the blockage of FcgammaRII was responsible for this effect. No binding of IC with either MOLT-4 or Jurkat cell lines was detected, which correlated with a very low Fc receptor expression. HCV RNA sequences were identified in the cells up to 120 h of post incubation with IC. These results suggest that IC can mediate entry of HCV to both U-937 and Monomac-6 cell lines mainly through the FcgammaRII.
Bacterial proteins A and G (SpA and SpG) are immunoglobulin receptors that can be used as probes for monitoring change in the conformation of heavy chain constant (C(H)) domains. Interaction of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody (Ab) with SpA and SpG were measured by isothermal titration calorimetry and surface plasmon resonance in order to address the question of whether hapten-binding induces a conformational change in the C(H) domain. The interactions of IgG2a or its enzymatic fragments with SpA were measured in the presence or absence of the hapten. Although binding of Fab and F(ab')2 fragments were not observed to free SpA, they did bind to immobilized SpA. In addition, the association constant (K(a)) for interaction of IgG2a with immobilized SpA was approximately 20-fold higher than that with free SpA. This was explained in terms of high avidity resulting from multivalent interaction between IgG2a and immobilized SpA on the chip. Interestingly, the hapten-binding weakened the interaction between the F(ab')2 fragment and SpA. Furthermore, approximately half of the IgG2a was incapable of binding to immobilized SpA in the presence of hapten. These results were explained using a model which assumed the formation of two kinds of SpA/IgG complexes; one through sites on F(ab')2 arms and the other through sites on the Fc region. The former type dissociated as a result of hapten-binding, as did the F(ab')2 fragment and suggested that a conformational change had occurred around the Fab arms, while the latter type did not dissociate because of the higher avidity of the Fc region. However, using a mutant SpA with a lower K(a) value for the interaction with IgG2a, it was shown that hapten-binding induced long range conformational changes in the Fc region of IgG2a. Similar evidence of conformational change upon hapten-binding was also obtained using SpG as a probe.
Reliable analysis using an immunosensor strongly depends on the specificity, activity, and sensitivity of the antibody. Immobilization of antibody on the solid matrix enables its repeated use, for which it is required to dissociate the antigens and antigen-enzyme conjugate from the immobilized antibody matrix after each use and while doing so, a maximum retention of activity and specificity are crucial requirements. In the present investigation, on the development of an immunosensor for the organophosphorus pesticide ethyl parathion (EP) using EP antibodies, different dissociating agents such as organic solvents, detergents and acidic buffers, that is, dimethyl sulphoxide (DMSO), Tween-20, cetyl trimethylammonium bromide (CTAB), methanol, chloroform, guanidium chloride (GdmCl), glycine-HCl (Gly-HCl) buffer in the pH range of 1.5-3.0, pierce buffer and combination of DMSO and methanol in phosphate buffer and Gly-HCl buffer and salts like NaCl and MgCl2 were used. Generally about 50-60% dissociation was obtained with some degree of denaturation of the antibody immobilized on the sepharose matrix. However, 1% DMSO in combination with 0.2 M Gly-HCl buffer at a pH of 2.3 showed 97% dissociation and the immobilized antibody retained sufficient activity to carry out 14 reproducible assays for EP.