Evaluation of β-D-Galactosidase from Escherichia Coli and Horseradish Peroxidase as Labels by Sandwich Enzyme Immunoassay Technique

Abstract

SUMMARY.~-D-galactosidase from Escherichia coli and horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human a-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The non-specific binding of Fab'-peroxidase conjugates to IgG-coated polystyrene halls was less than that of Fah'-[1-D-galactosidase conjugates. and the affinity-purified Fab'-peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding f1-D-galactosidase conjugates. However. a large quantity of Fah'-peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than thẽ -[)-galactosidase assay. Other advantages and disadvantages of the two enzymes are discussed.We developed a method for conjugating Fab' with~-D-ga\actosidase from Escherichia coli using thiol groups in the hinge of Fab' by the reaction between thiol and maleimide groups (the hinge methods};': 2 This enabled us to measure 1 amol of ornithine o-aminotransferase from rat liver. I 2 amol of human IgE, 0·05 amol of human ferritin and 2 amol of human thyroidstimulating hormone." We also developed a method for preparing a monomeric conjugate of Fab' and horseradish peroxidase using thiol groups in the hinge of Fab' by the reaction between thiol and maleimide groups" 5 or by the thiol-disulphide exchange reaction (the hinge method). The Fah-peroxidase conjugate thus prepared was shown to be superior to those prepared using amino groups of Fab' by the glutaraldehyde and periodate methods (the non-hinge methods) in both sandwich enzyme immunoassay for rnacromolecules": 5 and immunohistochemical staining of tissue sections. Furthermore. the use of affinity-purified antihuman growth hormone Fah'-peroxidase conjugate prepared by the hinge method enabled us to measure 3 amol of human growth hormone." Thus, both enzymes appear to provide a sandwich enzyme immunoassay at an 310 attomole level, but have not been critically evaluated.This paper describes a critical evaluation of -D-galactosidase from Escherichia coli and horseradish peroxidase as labels of Fab ' by the sandwich enzyme immunoassay technique. Materials and methods BUFFERThe most frequently used buffer was (l-Il! mol/l sodium phosphate buffer. pH 7·{), containing 0·\ molll NaCI and I gil bovine serum albumin (fraction V. Armour Pharmaceutical Co.. Kankakee, Illinois), and was abbreviated as buffer A. Calculation of the amount of enzymesThe amount of~-[)-galactosidase in mol was

Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays

“…The incubations were performed at room temperature without shaking throughout. After removal of the colored polystyrene beads with tweezers, the white polystyrene beads were washed, and bound ␤-Dgalactosidase activity was assayed by fluorometry using 4-methylumbelliferyl-␤-D-galactoside as substrate (19) at 30°C for 2.5 h. The fluorescence intensity was measured with a spectrofluorophotometer (F-3010; Hitachi, Ltd., Tokyo, Japan) using 360 nm for excitation and 450 nm for emission analysis. The fluorescence intensity of 10 Ϫ8 M 4-methylumbelliferone in 0.1 M glycine-NaOH buffer (pH 10.3) was adjusted to 100.…”

Recombinant p51 as Antigen in an Immune Complex Transfer Enzyme Immunoassay of Immunoglobulin G Antibody to Human Immunodeficiency Virus Type 1

“…The incubations with polystyrene beads were performed with shaking at 180 rpm at room temperature throughout. After washing as described above, ␤-D-galactosidase activity bound to the white polystyrene beads was assayed at 30°C for 1 h by fluorometry using 4-methylumbelliferyl-␤-D-galactoside as substrate (16). The fluorescence intensity was measured relative to 10 Ϫ8 M 4-methylumbelliferone in 0.1 M glycine-NaOH, pH 10.3, using 360 nm for excitation and 450 nm for emission analysis with a spectrofluorophotometer (F-3010; Hitachi, Tokyo, Japan).…”

Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV-1

“…After removing the colored polystyrene beads, the white polystyrene beads were washed, and bound β-D-galactosidase activity was assayed by fluorometry using 4-methylumbelliferyl-β-D-galactoside as substrate at 30°C for 2.5 hr. The fluorescence intensity was measured relative to 1 x 10 -8 mol/L 4-methylumbelliferone (15).…”