A monoclonal antibody which can distinguish between the two isotypes of human C4.

A monoclonal antibody to Human C4 (L003) has been shown to bind to the polymorphic C4d fragment of the alpha-chain of C4. Another monoclonal antibody (L001) was shown to bind to the beta-chain. L003 reacts differently with the two isotypes of C4, C4-A and C4-B. The equilibrium constant of L003 for C4-B is approx. 7-fold higher than that for C4-A, while L001 has similar affinity for both C4 types. Also, L003 binding to C4-A is very much more pH dependent than is its binding to C4-B. These observations explain the very useful property of L003 in being able to separate the two isotypes by affinity chromatography.

A monoclonal antibody to C1q which appears to interact with C1r2C1s2-binding site.

A monoclonal antibody (SB-4) to human C1q was prepared. The equilibrium constant of the antibody for C1q was found to be greater than 10(10) M-1. It has been shown that the antibody binds to the A-B chain dimer, probably via the B chain of C1q. Pepsin digestion of C1q at pH 4.5, which fragments the globular regions but leaves the collagenous region intact, allowed the demonstration that the antigenic site is located in the collagenous region of the molecule. The effect of the antibody on haemolytic activity has shown that it is capable of inhibiting the formation of EAC1 cells from EAC1q cells plus C1r and C1s but is incapable of inhibiting the C1 activity of performed EAC1 cells. This indicates that the binding of the antibody to the collagenous portion of the B chain of C1q probably prevents interaction between C1q and the C1r2-C1s2 complex.

Phenotype of lymphocytes mediating copolymer-specific humoral immunity in ethanol-consuming C57BL/6 mice.

Little is known about the mechanisms of impaired immune function in alcoholic patients. We have previously shown that ethanol consumption by mice alters copolymer-specific humoral and cellular immune responses. Does ethanol consumption eliminate suppressor T cells, allowing nonresponder mice to make humoral immune responses to poly(Glu50Tyr50) (GT)? Female C57BL/6 mice were fed a nutritionally complete liquid diet containing 35% ethanol-derived calories for up to 33 days. Control mice were fed an isocaloric control liquid diet or remained on a solid diet and water. Mice fed the ethanol-containing diet made GT-specific plaque-forming cell (PFC) responses, whereas mice fed liquid control or solid diets did not. Lymphocytes from ethanol liquid diet-consuming mice helped splenocytes from either solid or liquid control mice to make a GT-specific PFC response. The cells mediating help were nylon wool nonadherent, CD4-bearing T cells. These findings suggest that ethanol does not eliminate copolymer-specific suppressor cells, but instead alters the functional capability of helper T cells for humoral immune responses.

Flow cytometric analysis of lymphocyte subsets of mice maintained on an ethanol-containing liquid diet.

Alcoholic patients often have impaired immune function, yet little is known about the precise mechanism(s) of this impairment. We have previously shown that ethanol consumption by mice alters copolymer-specific humoral and cellular immune responses. In this study, we asked whether alcohol consumption by mice would phenotypically alter lymphocyte populations. Female C57BL/6 mice were fed a nutritionally complete liquid diet containing 35% ethanol-derived calories for up to 8 days. As controls, mice either were fed a liquid control diet that isocalorically substitutes sucrose for ethanol or remained on a standard solid diet and water ad libitum. Although mice fed ethanol-containing liquid or pair-fed control liquid diets have decreased numbers of spleen cells compared with solid diet controls, only the ethanol-containing diet allowed normally nonresponder C57BL/6 spleen cells to make antibody responses to the poly(Glu50Tyr50) synthetic copolymer antigen. Flow cytometric analysis of splenic lymphocyte populations of mice on the ethanol-containing diet shows an increase in the relative proportion of T-lymphocytes as compared with mice on either solid or liquid control diets. No such change is seen for either B-cell or natural killer cell populations in these same mice. Both liquid control and liquid ethanol diets caused a slight decrease in the CD4:CD8 ratios of splenic T-lymphocytes. We see the relative percentage of T-cells bearing the alpha beta T-cell receptor (TcR) increases in the spleens of liquid ethanol diet mice; a smaller increase TcR alpha beta usage is seen in the spleens of liquid control mice, compared with solid diet mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration of copolymer-specific humoral and cell-mediated immune responses by ethanol.

Excessive alcohol consumption represents a major human health threat. The frequency and severity of infections in alcoholics is often pronounced, suggesting impaired immune function in these patients. The precise effect of ethanol on cells of the immune system is poorly understood. We have previously shown that synthetic copolymers of L-amino acids, GT and GAT, are powerful tools for clarifying the role of regulatory T-cells in both cell-mediated and humoral immunity in inbred mouse strains. We asked whether these same antigens would have application to a murine model of ethanol consumption. In this study, female mice were placed on a nutritionally complete liquid diet containing 35% ethanol-derived calories. As control, mice either were placed on a liquid control diet that isocalorically substitutes sucrose for ethanol or remained on a solid diet consisting of standard laboratory chow and water ad libitum. Our data show that the liquid ethanol diet severely inhibits two measures of cell-mediated immunity, the ability of responder B6 mice to make an anti-GAT delayed hypersensitivity and GAT-specific T-cell proliferative responses as compared with pair-fed liquid control diet or solid diet controls. On the contrary, this liquid ethanol diet does not significantly impair humoral immunity; it allows nonresponder C57BL/6 or C3H/HeN mice to respond in vivo to GT immunization. These findings suggested to us that the effect of ethanol may occur prior to antigenic stimulation, and this was confirmed by in vitro immunization.

CNBr cleavage of the light chain of human complement factor I and alignment of the fragments.

The light chain and heavy chain of reduced and alkylated human complement Factor I were purified by high-pressure gel-permeation chromatography. CNBr cleavage of Factor I light chain yielded four major fragments, which were purified by gel filtration. N-Terminal sequence analysis of the CNBr-cleavage fragments allowed identification of 200 of the approx. 240 amino acid residues of the light chain. An alignment is proposed, based on sequence analysis of peptides obtained after cleavage at arginine residues of the light chain and on homology of the sequence determined with that of other serine proteinases. The sequence around the active-site serine residue was established and three potential attachment sites for carbohydrate moieties were identified.

Purification of human C3b inactivator by monoclonal-antibody affinity chromatography.

Monoclonal antibody has been obtained to the human complement control protein C3b inactivator after immunization of mice with the enzyme prepared by conventional methods. Antibody from ascitic fluid was purified and coupled to Sepharose-CL-4B to give a specific affinity column, which was used to isolate C3b inactivator from human serum in 70% yield. The product was characterized by size, chain structure, amino acid analysis and proteolytic activity.