Study of heart-reactive antibody in antisera and hybridoma culture fluids against group A streptococci

Abstract

Heart-reactive antibody is known to be produced in animals immunized with group A streptococci. However, the detection and quantitation of these antibodies and their respective antigens have been limited to immunofluorescence or immunoprecipitation tests. In this study a more sensitive technique was used to detect heart-reactive antibody, i.e., the enzyme-linked immunosorbent assay (ELISA). Countercurrent immunoelectrophoresis and agar gel immunodiffusion were also used to confirm the reactivity of rabbit antisera to group A streptococci and human heart tissue. Human heart tissue antigen was prepared by Triton X-100 extraction, and checkerboard titrations of heart tissue antigen versus streptococcal antisera revealed optimal concentrations of each for the ELISA. When immune rabbit serum was reacted with heart tissue antigen, a 5to 10-fold increase was observed over the reactions of preimmune sera controls. Streptococcal antiserum was found to have a sixto eightfold greater reactivity with heart tissue antigen than with similar concentrations of kidney and skeletal muscle antigens. Heart tissue antigen was partially purified by DEAE-cellulose chromatography, and quantities of >10 to 100 ng (dry weight) were shown to react with streptococcal antisera in the ELISA. Heart, kidney, and skeletal muscle antigens were subjected to electrophoresis in polyacrylamide gel slabs and blotted onto a nitrocellulose filter. These filter blots reacted with streptococcal antisera and confirmed the tissue antigen specificity observed with the ELISA. In addition, the ELISA was found to be an effective method for the detection of heart-reactive antibodies produced by murine hybridomas that were producing antibody to group A streptococci.

Human rheumatoid factors with restrictive specificity for rabbit immunoglobulin G: auto- and multi-reactivity, diverse VH gene segment usage and preferential usage of V lambda IIIb.

“…The heavy chain V region (VH) and Vx sequences of nine of these IgMX tLF were determined at the cDNA level. Five VH genes in three V. families were used by these antibodies (Ab), including V.I (dp21/1-4b and dpl0 [51pl]/ hv1051), V.3 (dp38/3-15 and dp77/ [13][14][15][16][17][18][19][20][21], and V.4 (dp70/4-4b). The deduced V gene-encoded amino acid sequences of the X chains of these IgMX tLF confirmed their serological classification as xlII, and they were further classified as members of the relatively uncommon VxlII subgroup, designated VxlIIb.…”

“…The Vx-subgroup nature of the h-type IgM anti-rabbit IgG RF was determined by ELISA using murine mAbs specific for the VxI, VxII, VxIV, and VxVI subgroups and the VxIIIa, VxIIIb, and VxIIIc sub-subgroups (19). The ELISA for KF-binding to streptococcal extract was kindly performed by Dr. Madeleine W. Cunningham (University of Oklahoma, Oklahoma City, OK) (20). The affinity constants of R.F to rabbit IgG and myosin were measured by ELISA as described by Friguet et al (21).…”

Autoantibodies in the sera of patients with rheumatic heart disease: characterization of myocardial antigens by two-dimensional immunoblotting and N-terminal sequence analysis

“…Nine proteins could be characterized either by amino acid sequencing (spot nos. 1,7,8,9,10,11) or by MoAbs (spot nos. 2, 3, 5) ( Table 1).…”

“…Other cross-reactive proteins of the myocardial tissue have not yet been identified by their protein sequence but only by their molecular weight. Cunnigham & Russel [11] demonstrated that autoreactive antibodies from sera of patients with RHD bind, in one-dimensional Western blot analysis, to heart tissue proteins with molecular masses 40, 67 and 200 kD. In subsequent studies, myosin has been identified as the relevant 200-kD protein immune target in myocardial tissue [12].…”

Polyspecificity of antistreptococcal murine monoclonal antibodies and their implications in autoimmunity.

“…Monoclonal Antibodies. Murine hybridoma cell lines were selected, cloned, stored and maintained as previously described (20). mAbs used in this study included 36.2.2, 49 .8 .9, and 54 .2 .8, which were stored as culture fluids at 4°C .…”