Enhanced ELISA sensitivity using TCA for efficient coating of biologically active lipopolysaccharides or lipid A to the solid phase

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Enhanced ELISA sensitivity using TCA for efficient coating of biologically active lipopolysaccharides or lipid A to the solid phase. / Hardy, Eugenio; Ohlin, Mats; Llano, Manuel.

In: Journal of Immunological Methods, Vol. 176, No. 1, 10.11.1994, p. 111-116.

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T1 - Enhanced ELISA sensitivity using TCA for efficient coating of biologically active lipopolysaccharides or lipid A to the solid phase

AU - Hardy, Eugenio

AU - Ohlin, Mats

AU - Llano, Manuel

PY - 1994/11/10

Y1 - 1994/11/10

N2 - 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 (24). 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°C, when incubated for only 30 min. In the other procedure, LOS in 0.2% TCA was coated at 37°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.

AB - 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 (24). 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°C, when incubated for only 30 min. In the other procedure, LOS in 0.2% TCA was coated at 37°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.

KW - Coating efficiency

KW - ELISA

KW - Lipid A

KW - Lipopolysaccharide

KW - Trichloroacetic acid ([76-03-9])

UR - http://www.scopus.com/inward/record.url?scp=0028143672&partnerID=8YFLogxK

U2 - 10.1016/0022-1759(94)90355-7

DO - 10.1016/0022-1759(94)90355-7

M3 - Article

C2 - 7963589

AN - SCOPUS:0028143672

VL - 176

SP - 111

EP - 116

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 1872-7905

IS - 1

ER -