Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase.

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase.

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A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed.Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column.Routinely, Immulon 2 Removawell strips new balance 990 v3 red were coated with the purified antibody overnight at 4 degrees C.After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.

85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C.The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less.The pretreatment with SDS was necessary to achieve maximal immunoreactivity.

The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate.Rat H-TGL was detected by the animed aniflex complete color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.

1 M citrate phosphate, pH 5.0.A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard.Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.

05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions.Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.

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