Factors affecting the fine specificity and sensitivity of serum antiganglioside antibodies in ELISA.
The major problem associated with ELISA of serum antiganglioside antibodies is the high background values (absorbancy of sera added to wells without ganglioside), which interfere with the accurate assessment of the fine specificity and sensitivity of these antibodies. This investigation identifies factors elevating the background values and/or decreasing the fine specificity, and describes strategies to minimize their influence. Using sera of neuropathy and melanoma patients, we found that highest background values were observed with the polystyrene 'tissue culture' microtiter plates; of the various 'non-tissue culture' microtiter plates tested, the lowest background values (> 0.060) were observed with Costar-3590 (H), Immunolon-3, Immunolon-1, Falcon-3915 (in increasing order). Background artifact of polystyrene microtest plates was significantly reduced by gamma irradiation (at 40 kRad) and/or use of detergent Tween-20 (0.1%) in the washing step. Even after controlling the background values, the fine specificity, namely, the ability of the antibody to distinguish between the target epitope of an antigen and epitopes of related antigens (when moles of antigen/well is constant) varied with different microtiter plates. Using sera with high affinity and specificity for GM2, GD3 or GM3, we observed that Immunolon-1, Immunolon-3 and particularly Falcon-3915 were superior for assessing the abilities of the antibodies to distinguish closely related epitopes found on other gangliosides. The reactivity of antiganglioside antibodies was more consistent after detergent treatment. The reactivity of antibodies to GD3 is significantly enhanced after treatment with Tween-20, but that of antibodies reacting to GM1 and GM2 is reduced. Fine specificity of the antiglycolipid antibodies was resolved better by coating glycolipids in mol/well rather than by weight/well. Based on these results, a protocol for a sensitive and reproducible ELISA for serum antiganglioside antibodies is recommended. The protocol takes into consideration the suitability of polystyrene plates, coating based on the number of molecules, pertinency of the solvent for coating, use of human serum albumin for blocking, dilution and washing steps and use of 0.1% Tween-20 to further minimize the background absorbancy.
Ravindranath
M H
MH
Laboratory of Glycolipid Immunotherapy, John Wayne Cancer Institute, Santa Monica, CA 90404.
Ravindranath
R M
RM
Morton
D L
DL
Graves
M C
MC
D001323:Autoantibodies / Q000032:analysis*; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D005732:Gangliosides / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D007158:Immunologic Techniques / Q000295:instrumentation; D008545:Melanoma / Q000276:immunology; D008969:Molecular Sequence Data; D009422:Nervous System Diseases / Q000276:immunology; D011136:Polysorbates; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D000939:Epitopes; D005732:Gangliosides; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011136:Polysorbates