Enhanced ELISA sensitivity using TCA for efficient coating of biologically active lipopolysaccharides or lipid A to the solid phase.
A new simple, reproducible and sensitive ELISA that uses trichloroacetic acid (TCA) for the coating of lipooligosaccharide (LOS), smooth lipopolysaccharide (LPS) or lipid A to the solid phase has been developed. The experimental parameters (temperature of coating, time of coating, antigen concentration and TCA concentration) were evaluated by a complete factorial design (2(4)). As a result of the evaluation, two main coating procedures were developed. In one, LOS was shown to coat efficiently in 0.2% TCA, at 37 degrees C, when incubated for only 30 min. In the other procedure, LOS in 0.2% TCA was coated at 37 degrees C for 16 h. The slower procedure proved, as expected, to be even more efficient than the former. The new ELISA was compared to three previously reported ELISAs, and showed the greatest sensitivity, probably, as a consequence of the higher coating efficiency of LOS to plates. The biologic activity of LOS was not modified by the low TCA concentration used, as proven by retention of its biological activity in the induction of procoagulant activity in blood mononuclear cells. We conclude that small amounts of biologically active LOS/LPS or lipid A can be coated on solid surfaces by this approach to achieve a rapid and economical assay procedure.
Center for Genetic Engineering and Biotechnology, Pilot Plant Division, Havana City, Cuba.
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Ohlin
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Llano
D000818:Animals; D000911:Antibodies, Monoclonal; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008050:Lipid A / Q000032:analysis*; D008070:Lipopolysaccharides / Q000032:analysis*; D051379:Mice; D009345:Neisseria meningitidis / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012706:Serratia marcescens / Q000737:chemistry*; D014238:Trichloroacetic Acid
D000911:Antibodies, Monoclonal; D008050:Lipid A; D008070:Lipopolysaccharides; D014238:Trichloroacetic Acid