Immunoglobulin VH determinants defined by monoclonal antibodies.

Abstract

The heavy and light chains of immunoglobulins (Ig) consist of two distinct regions: variable and constant. Variable regions, which determine the antibody specificity and idiotypic determinants, are further divided into three hypervariable regions and four relatively invariant, so-called framework, regions (1, 2). The variable regions of both heavy chains (VH) 1 and light chains (VL) have been divided into subgroups based upon similarities between amino acid sequences in their framework regions. For human Ig, three major VH subgroups have been characterized on the basis of amino acid sequences of the aminoterminal 20 residues within the first framework region (2). One set of goat antibodies specific for each human VH subgroup has been prepared by selective adsorption and was found to be useful in typing secreted and membranebound Ig on B cells (3).Evidence from studies (4-8) using anti-idiotype antibodies has suggested that T cells and their antigen-specific, soluble factors might possess Ig-like idiotypic determinants. VH allotypic determinants have also been demonstrable on rabbit T cells by several groups (9, 10) and not by others (11-15). Antibodies against VH framework regions of the mouse myeloma protein MOPC-315, which cross-react with other mouse Ig regardless of their antibody specificity, class, subclass, subgroup, or allotype (16), may inhibit T cell function (17-19) and cross-react with soluble factors ofT cells (20,21). The molecular nature of antigen receptors on T lymphocytes, however, is still controversial. Recent studies (22, 23) using recombinant DNA techniques have suggested that antigen-specific helper or killer T cell clones may not use the Ig-joining and constant gene segments.We prepared monoclonal hybridoma antibodies against VH fragments isolated from human IgM myeloma proteins with the hope of using these to analyze the products of normal or abnormal B and T cells. This paper describes the preparation and characterization of four such monoclonal anti-human VH antibodies. Our studies *

A Mouse Monoclonal Antibody Reactive Preferentially with Human IgM λ

“…9) , and lymph node cells from immunized mice were fused with a non-Ig-producing myeloma cell line P3-X63-Ag.8.653 (10). Hybridoma screening, cloning and ascites production were basically the same as described previously (3,7,10). LP4 mAb (mouse Ig isotype: y2bx) was selected based upon its reactivity with subpopulations of the IgM paraproteins, and purified from its ascites fluid by absorption to Sepharose 4B coupled with IgMX.…”

“…The following mouse yl* monoclonal antibodies (mAb) specific for human Ig isotype were used in this study: SA-DA4-4 anti-//, SA-BA1-19 anti-K, and 1-155-2 anti-X (4, 6, 7). These mAbs were purified from ascites fluid by affinity chromatography on Sepharose 4B columns coupled with the appropriate human Ig (2,3) . A rat mAb (187.1) specific for mouse k chain, a generous gift of Dr. M. D. Scharff, Albert Einstein Medical School, NY (8), was purified from its culture supernatant by affinity columns coupled with mouse IgMK.…”

“…To determine the specificity of LP4 mAb, the previously described ELISA (3,4,7) was conducted. Briefly, polyvinyl microtiter wells (Dynatech Laboratories, Inc., Alexandria, VA) precoated with purified paraproteins or normal IgG (5 ug/ml) were incubated with 100 |il of purified LP4 mAb (5 ug/ml) for 6 hrs at 4°C.…”

“…Twenty three monoclonal paraproteins (13 IgM, 2 IgD, 5 IgA, 3 IgG) listed in Table 1 were isolated from patients' sera by euglobulin or salt fractionation, DE-52 ion exchange cellulose-and Sepharose 6B gel filtration-column chromatography as described elsewhere (2)(3)(4). Polyclonal IgG was similarly isolated from normal pooled human sera.…”

“…Affinity-purified goat antibodies with specificity to mouse Ig or human u chain determinants and without cross-species reactivity (Southern Biotechnology Associate, Birmingham, AL) were labeled with alkaline phosphatase (AP), fluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (RITC) as described (2)(3)(4)(5). The following mouse yl* monoclonal antibodies (mAb) specific for human Ig isotype were used in this study: SA-DA4-4 anti-//, SA-BA1-19 anti-K, and 1-155-2 anti-X (4, 6, 7).…”