Comparison of Chromogens for the Determination of Horseradish Peroxidase as a Marker in Enzyme Immunoassay

Abstract

o-Phenylenediamine, 2,2'-azino-di(3-ethylbenzthiazoline sulphonic acid-6) (ABTS), odianisidine and 4-aminoantipyrine were compared as chromogens for the determination of horseradish peroxidase. Highest sensitivity in the determination of horseradish peroxidase-IgG conjugates in dissolved form was obtained with o-phenylenediamine. When these conjugates were used in a two-site binding enzyme immunoassay for hepatitis B surface antigen (HBsAg), the steepest calibration curve and the lowest detection limit were obtained when ABTS was used to determine the immune complexes bound to the solid phase. Non-ionic detergents, such as polyoxyethylene^sorbitol ester, retarded horseradish peroxidase inactivation, resulting in a chromogen-dependent activity rise of horseradish peroxidase. An optimised determination of horseradish peroxidase is reported, in which the sensitivity of the solid phase enzyme immunoassay is doubled by the use of 0-dianisidine. Vergleich von zur Aktivitätsbestimmung von Meerrettich-Peroxidase als Marker im Enzymimmunoassay verwendeter ChromogeneZusammenfassung: Die Empfindlichkeit der Aktivitätsbestimmung von Meerrettich-Peroxidase wurde mit den Chromogenen 0-Phenylendiamin, 2,2'-Azino,di(3-ethylbenzthiazolin-sulfonat-6) (ABTS), o-Dianisidin und 4-Aminoantipyrin verglichen. An IgG gekoppelte Meerrettich-Peroxidase wird in gelöster Form am empfindlichsten mit ö-Phenylendiarnin bestimmt. Nach Einsatz der Konjugate in einem zwei-Seiten Enzymimmunoassay zur Bestimmung des Hepatitis B-Oberflächenantigens (HBsAg) und quantitativer Bestimmung der an die feste Phase gebundenen Immunkomplexe werden die steilste Ständardkurve und geringste untere Nächweisgrenze mit ABTS erhalten. Nichtionische Detergenzien wie Polyoxyethylen*orbitester verzögern die Inaktivierung von Meerrettich-Peroxidase während der Reaktion und fuhren zu einer chromogenabhängigen Aktivitätssteigerung der Meerettich-Peroxidase. Es wird eine .optimierte Bestimmung von Meerrettich-Peroxidase mit <7,Dianisidin vorgestellt, wodurch die Empfindlichkeit des Festphase-OEnzymimmunoassays verdoppelt werden kann.

Astrocyte Overgrowth in the Brain Stem and Spinal Cord of Mice Affected by Spinal Atrophy, Wobbler

“…After blocking for 1 h at 37°C with 1% (v/v) bovine serum albumin in PBS, plates were incubated with polyclonal anti-GFAP (dilution 1:20, 2 h, 37°C), washed, and then incubated with HRP-conjugated sheep anti-rabbit y-globulin (dilution 1:200, 1 h, 37°C). The plates were developed using 2,2'-azino-di-3-ethylbenzyithiazolinsulfonate as substrate for HRP [Porstmann et al, 1981]. The absorbance was measured at 410 nm after 30 min at 37°C.…”

Sucrase-alpha-dextrinase in the rat. Postinsertional conversion to inactive molecular species by a carbohydrate-free diet.

“…Residual binding sites on the nitrocellulose were then blocked by exposure to 2% FCS for 1 h. The sheet was washed three times for 15 min on a rocking platform with 200 ml of 0.15 M NaCl, and 0.05 M Tris, pH 7.0 (Tris-saline), picked up with Teflon-coated tweezers, and submerged sequentially (four times) into plastic dishes containing 200 ml of Tris-saline, and then incubated with 100 ml of a 1: 1,000 dilution of monospecific, polyclonal anti-S-D in 2% FCS for 3 h. The nitrocellulose was then washed three times with Tris-saline and exposed to goat anti-rabbit IgG bound to horseradish peroxidase at 1:3,000 dilution for 2 h at 220C. After three washes with Tris-saline, 1 X 1-cm squares were cut and transferred to 12 X 75-mm test tubes and the colorimetric reaction was performed with ABTS as the peroxidase substrate as described by Porstmann et al (18). The reaction was stopped with azide, and, after removal of the nitrocellulose square, the absorbance of the substrate solution was measured at 418 nm.…”

Evaluation of Peroxidase Activity in Selected Vegetables from Hyderabad, Telangana, India

“…Enzyme activity was determined in triplicates and results are represented as Mean ± SD. The substrate used was hydrogen peroxide (H 2 O 2 )/phenol/4-aminoantipyrine solution (Porstmann, 1981). H 2 O 2 rapidly reacts with 4-aminoantipyrine-phenol solution in the presence of peroxide to produce a quinoneimine chromogen (http://www.amanoenzyme.co.jp) which shows intense pink colour with a maximum absorbance at 510 nm.…”