Analysis of human tumor cells for Ia-like antigens with monoclonal antibodies.

Cultured and noncultured human solid tumors were analyzed for expression of Ia-like antigens with the use of two monoclonal antibodies and a rabbit antiserum against human Ia-like antigens. Of 27 tumor cells tested, 3 melanomas bound antibodies [e.g., 21,563 cpm 131I-labeled staphylococcal protein A ([131I]SpA) with monoclonal antibody Q5/6], but 24 others (1 melanoma, 9 neuroblastomas, 1 medulloblastoma, 3 gliomas, 4 sarcomas, 2 colon carcinomas, 2 transitional cell carcinomas of the bladder, 1 teratoma, and 1 squamous cell carcinoma of the lung) did so minimally or not at all (0-427 cpm [131I]SpA with antibody Q5/6. Monoclonal antibody Q5/6 was quantitatively absorbed with homogenates of 32 noncultured tumors to determine if Ia-like antigens were expressed by neoplastic cells in vivo. Ten milligrams (wet wt) each of 5 of 7 noncultured melanomas removed more than 83% (median, 85%) of the antibody. In contrast, 10 mg each of 10 neuroblastomas, 7 carcinomas, 4 sarcomas, and 4 Wilms' tumors removed less than 47% (median, 19%) of the antibody; even 100 mg of these tumors removed less than 68% (median, 44%) of the antibody.

Rosetting of antibody-sensitized tumor cells with anti-immunoglobulin antibody-coated erythrocytes by a new method for detecting antigens on cells.

Tumor cells were treated with rabbit antibody to tumor-associated cell surface antigens and tested with erythrocytes coated with antibody specific for the sensitizing rabbit immunoglobulin. The sensitized tumor cells formed rosettes with the indicator cells. By this method, we confirmed that line 1 and line 10 hepatoma cells (from two tumors independently induced by diethylnitrosamine in strain 2 guinea pigs) bear antigens not present on normal liver cells. We also confirmed that line 1 and line 10 cells bear antigenically different tumor-associated cell surface antigens. This method appears simpler than other serological methods for detecting tumor-associated cell surface antigens on tumor cells. Also, this method may be a general one for detecting and enumerating cells bearing surface antigens.

One antigen may form two precipitin lines and two spurs when tested with two monoclonal antibodies by gel diffusion assays.

A monoclonal antibody reactive with human immunoglobulin (Ig)G4 and a monoclonal antibody reactive with IgG1, IgG2 and IgG4 were tested with an IgG4 myeloma protein by double diffusion in a polyethylene glycol-containing gel. In a three-well Ouchterlony pattern, the IgG4 myeloma protein formed lines of double partial identity (double spur) with the two monoclonal antibodies. The two spurs lengthened and thickened with decreasing concentrations of polyethylene glycol indicating that soluble immune complexes diffused past the precipitin lines and formed the spurs. In a two-well pattern, the myeloma protein formed two lines with mixtures of the two monoclonal antibodies indicating that the immune complexes formed by the two antibodies distributed bimodally in the gel as if two types of complexes were formed. These unpredicted findings indicate that the process of antigen-antibody precipitation in gels needs to be analyzed further by using monoclonal antibodies.

Two monoclonal antibodies to two different epitopes of human growth hormone form a precipitin line when counterdiffused as soluble immune complexes.

We have tested whether soluble immune complexes obtained by mixing human growth hormone (hGH) with one anti-hGH monoclonal antibody (MAb) can form a precipitin line when diffused against another MAb in a polyethylene glycol containing gel. By testing seven anti-hGH MAbs one against the other in this assay, we have found that 10 pairs of MAbs out of the 21 possible combinations formed a line. Apparently, the first MAb formed soluble hGH dimers that were linked by the second MAb into precipitating linear complexes. Since each precipitin line was formed by the cooperative reaction of two MAbs, this sequential reaction of MAbs may be used in methods for the positive selection of MAbs that are suitable for two-site immunoassays.

A monoclonal antibody may show cross-reactivities in Ouchterlony assays but not in other assays.

A monoclonal antibody to human IgG was tested with myeloma proteins of the four IgG subclasses. When tested by immunofluorometric assay, enzyme-linked immunosorbent assay, hemagglutination and hemagglutination inhibition assays, the antibody reacted with IgG3 but not with the other three IgG subclasses. When tested by Ouchterlony assays in the presence of polyethylene glycol, the antibody formed lines with all four IgG proteins. The line with IgG3 was sharp and stable, but the lines with the other three IgG subclasses tended to blur with time and with the lower PEG concentrations. These findings show that Ouchterlony assays can reveal cross-reactions of a monoclonal antibody that can be missed by more sensitive assays.

Identifying immunoglobulin isotypes, allotypes and idiotypes by a hemolytic assay in gel.

We developed a hemolytic radial immunodiffusion assay for identifying immunoglobulin (Ig) isotypes, allotypes and idiotypes by using gels containing erythrocytes coated with anti-Ig antibody or erythrocytes coated with Staphylococcal protein A. These indicator cells lysed specifically when treated sequentially with Ig antigen, the appropriate anti-Ig antiserum (developer) and complement. To identify these Ig subpopulations, we used monospecific indicator cells, e.g. erythrocytes coated with antibody specific for an Ig isotype, and developers with broader specificities ('multispecific'), e.g. antiserum to Fab. Alternatively, we used 'multispecific' indicator cells, e.g. erythrocytes coated with antibody to Fab and monospecific developers, e.g. antiserum to Ig idiotype. To identify Ig subpopulations specifically, either the indicator cells or the developer need to be monospecific. When both the indicator cells and the developer were monospecific, e.g. to allotype and to isotype, the specificity was determined by both reagents and ultimately restricted by the reagent with the narrower specificity, that is, reacting with the smallest Ig subpopulation. This sensitive hemolytic assay may be used to quantitate subpopulations of Ig molecules and may be modified into a reverse plaque forming cell assay to count lymphocytes secreting a given Ig class, type, allotype and idiotype.

Rosetting of human T lymphocytes with goat red blood cells: effect of treatment with 2-aminoethylisothiouronium bromide (AET) and comparison with AET treated sheep red blood cells.

In vitro treatment of goat red blood cells (GRBCs) with the sulphydryl compound 2-aminoethylisothiouronium bromide (AET) increases their specific reactivity with human T lymphocytes without affecting the specificity of the reaction. AET-GRBCs bind to only part of T lymphocytes rosetting with AET-sheep red blood cells (SRBCs): the receptors for both types of RBCs are very simular if not identical, but display higher affinity for AET-SRBCs than for AET-GRBCs. Rosetting of T lymphocytes with AET-GRBCs may be useful to enumerate T lymphocyte subsets in patients with abnormality of the immune system and to fractionate T lymphocyte subpopulations.

Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens.

Serological and immunochemical studies showed that monoclonal antibody Q2/70 (MoAb Q2/70), produced by the hybridoma technique, is specific for human Ia-like antigens. This antibody recognizes an antigenic determinant which is different from those defining the serologic polymorphism of Ia-like antigens, and is expressed on subsets of human Ia-like molecules and on lymphoid cells from other species. MoAb Q2/70 inhibits unidirectional MLRs* between allogenic human lymphocytes, but not between murine and human lymphocytes. In ADCC* assays. MoAb Q2/70 mediates lysis of cultured human B lymphoid cells RPMI 4098, effected by murine splenocytes. The antibody is suitable to isolate immunologically functional B lymphocytes from human peripheral blood.

Early ontogeny of rheumatoid factor antibodies. Characterization of a murine neonatal hybridoma autoantibody to IgGl in BALB/c mice.

The ontogeny and the function of rheumatoid factors (RF) are poorly understood. Here we describe a stable neonatal hybridoma cell line of BALB/c origin that secretes a RF autoantibody of the IgM class. By a series of in vitro assays we determined that this RF reacts specifically with the murine IgGl heavy chain. It also binds IgG of several mammalian species. By using mutant IgGl molecules, the reactive epitope was mapped to the CH3 domain of the constant region of IgGl. These findings indicate that clones with reactivity to autologous IgGl exist in normal mice at birth.