Pak C. Kwong

Miniaturization makes mixed hemadsorption assays more sensitive, reliable and economic

Abstract The mixed hemadsorption assay is a sensitive method for the detection of antibodies to cell surface antigens as well as to soluble antigens. Miniaturization of this assay through the use of microplates with 12 μl rather than 400 μl wells allows a 5-fold reduction in volume of test serum and equivalent reduction in target antigen and other reagents used in the indicator system. Using 5 different antigen systems, melanoma-associated antigens, carcinoembryonic antigen, HLA antigen, β-2 microglobulin and fetal calf serum, a 4–8-fold increase in sensitivity is realized. The geometry of the 12 μl wells results in no significant detachment of target cells which may account for the improved sensitivity and quality of the titration curves.

Difference in cell binding patterns of two monoclonal antibodies recognizing distinct epitopes on a human melanoma-associated oncofetal antigen

Two monoclonal antibodies (MAbs), 140.240 and 96.5, generated independently in different laboratories, have been shown to detect the target structures of 87,000 (gp87) and 97,000 (p97) glycoproteins, respectively, both strongly expressed by melanoma cells and fetal small intestine. To determine whether MAb 140.240 and MAb 696.5 recognized a same target structure, they were tested in immunoprecipitation/SDS-PAGE using NP-40 lysates of melanoma cells labelled with [35S]methionine for 18 hr. Both antibodies precipitated a single band with Mr = 87,000. Reciprocal immunodepletion studies showed that neither of the two antibodies detected the 87,000 band in the lysate immuno depleted by either antibody, suggesting that these two antibodies recognize the same or extremely similar molecules. Two-dimensional tryptic peptide mapping analysis showed that the two identified molecules shared the same finger-printing pattern. A 40,000 fragment of the 87,000 molecule produced by protease digestion was precipitated by MAb 96.5 but not MAb 140.240, indicating that the epitopes recognized by the two antibodies are localized at discrete sites on the molecule. Serological studies on these two antibodies revealed slightly different binding patterns in the MAb 140.240 exhibited a more melanoma-restricted specificity, while MAb 96.5 had a specificity to melanoma and to some other cell types. The observed difference in epitope specificity may be important in the clinical applications of these antibodies.

A simple microassay for detection of antibodies to fetal calf serum and related antigens and its application to the serological definition of human tumor antigens.

Abstract The use of cultured cells for the production and testing of anti-tumor antisera is complicated by the fact that cultured cells can adsorb macromolecules derived from the serum supplement used in the culture medium onto their surface membrane. The need to monitor antisera for the presence of this unwanted reactivity has led us to adapt the mixed hemadsorption assay for the detection of antibodies to antigens present in fetal calf serum (FCS), the most widely used serum supplement. Our improved microassay involves the fixation of FCS to the bottom of plastic microplates with glutaraldehyde for the production of an antigen substrate which is stable for at least 16 weeks. The microassay is simple, reproducible, and capable of detecting nanogram quantities of FCS antigens. Anti-FCS reactivity is detectable with this microassay in approximately 90% of sera from both normal individuals and melanoma patients. Similarly, anti-tumor antisera raised in monkeys, rabbits and patients with melanoma against cultured human melanoma material contain high titers of anti-FCS reactivity. But this reactivity can be completely removed by absorption with insolubilized FCS. Xenoantisera raised against melanoma cells grown in human adult serum still show strong reactivity against FSC. This is due to immunologic cross-reactivity between FCS and normal human serum antigens.

Selective extraction by 1-butanol of surface glycoprotein antigens from human melanoma cells

SummaryThe butanol extraction method has previously been used to achieve selective release of tumor-specific transplantation antigens from mouse sarcoma cells. In this study we investigated the feasibility of this method for extracting four surface glycoprotein antigens (87K, 95–150K, HLA-DR, and HLA-A,B,C) from cultured human melanoma cells. Of the four antigens examined, only 95–150K and HLA-DR antigens could readily be detected in material extracted by 2%, 3%, or 5% butanol. The 3% butanol was found to be most effective in releasing these two antigens. Treatment of melanoma cells with ≤3% butanol did not decrease the viability of extracted cells as judged by either Trypan Blue dye exclusion or plating efficiency. Thus the noncytolytic butanol extraction method offers a promising approach to the isolation of certain glycoproteins such as 95–150K and HLA-DR from viable human melanoma cells for further purification and structural analysis.

Identification of Immunoglobulin Class and Subclass of House Monoclonal Antibodies to Human Cell Surface Antigens by Mixed Hemadsorption Assay

A microassay is described to determine immunoglobulin Ig class or IgG subclass of mouse monoclonal antibodies by mixed hemadsorption assay. Monoclonal antibody bound to adherent target cells is reacted with serial dilutions of a panel of class or subclass specific rabbit anti-mouse Ig antisera and binding of the latter is traced by anti-rabbit globulin-coated indicator erythrocytes. The class or subclass of the bound monoclonal antibody is revealed by preferential binding of the corresponding rabbit antibody. Unlike gel immunodiffusion analysis, the mixed hemadsorption assay may be performed with unconcentrated hybridoma culture supernatants.

Effect of Glutaraldehyde Treatment of Human Melanoma Cells on the Expression of Melanoma-associated Antigens

SummaryHuman melanoma cells were treated with different concentrations of glutaraldehyde, and retention of serological reactivity with antisera against melanoma-associated antigens, HLA antigen, and β2-microglobulin was assessed by quantitative absorption analysis in mixed hemadsorption microassays. Glutaraldehyde concentrations of 0.025% or greater significantly impaired binding to melanoma cells of antibody against melanoma-associated antigens. At a concentration of 0.0025% antibody binding was not decreased although plating efficiency was reduced to less than 1%. Glutaraldehyde concentrations of 0.25% or greater significantly reduced binding to the same melanoma cells of antisera against HLA antigen and β2-microglobulin. Glutaraldehyde treatment (up to 2.5%) of HT-29 colon carcinoma cells failed to reduce reactivity of antisera against CEA and blood group A isoantigen, which are present on these cells. These studies indicate that the effect of glutaraldehyde treatment of cells on retention of surface antigens is critically dependent on the concentration of glutaraldehyde used and the type of antigens involved.