ABTS diammonium salt is a substrate for horseradish peroxidase (HRP) conjugate.

A micro-technique of enzyme-linked immunosorbent assay (ELISA) using ABTS, as a substrate for HRP conjugate is studied. In a comparative study among 4 substrates, namely; 5-aminosalicylic acid (5AS), O-phenylenediamine (OPD), O-tolidine (OT) and ABTS, for HRP in terms of sensitivity, ABTS is the most sensitive, stable and the best in visuality by its bluish-green color^[1]. ABTS is a typical peroxidase substrate. For purification and characterization peroxidase positive transformants are cultivated in large scale (XL) under conditions that yield active protein in the culture supernatant. After 160 h cultivation an activity of 55,000 U/L in relation to the substrate ABTS is achieved and the supernatant containing the peroxidase is harvested. With ABTS as substrate the peroxidase activity falls significantly when the H₂O₂ concentration rose above 0.125 mM, indicating that the enzyme is inhibited by H₂O₂. Maximum reaction rates depending upon substrate tested reache values between 31.2 and 125 µM^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

548.68

C₁₈H₂₄N₆O₆S₄

30931-67-0

Room temperature in continental US; may vary elsewhere.

4deg;C, sealed storage, away from moisture

*In solvent : -80deg;C, 6 months; -20deg;C, 1 month (sealed storage, away from moisture)

In Vitro:;

H₂O : ≥ 50 mg/mL (91.13 mM)

DMSO : 20.83 mg/mL (37.96 mM; Need ultrasonic)

* “≥” means soluble, but saturation unknown.

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂：;10% DMSO ;; 40% PEG300 ;; 5% Tween-80 ;; 45% saline

  Solubility: ≥ 2.08 mg/mL (3.79 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (3.79 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂：;10% DMSO ;; 90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.08 mg/mL (3.79 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (3.79 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂：;10% DMSO ;; 90% corn oil

  Solubility: ≥ 2.08 mg/mL (3.79 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (3.79 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Matsuda H, et al. Evaluation of ELISA with ABTS, 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate of peroxidase and its application to the diagnosis of schistosomiasis. Jpn J Exp Med. 1984 Jun;54(3):131-8.

  [2]. Lauber C, et al. Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus. AMB Express. 2017 Aug 23;7(1):164.

Enzyme activity is determined photometrically using a temperature controlled multi-mode plate reader or alternatively in a UV/Vis spectrophotometer. Reactions are initiated by addition of the enzyme. Enzyme activity is measured over a period of 10 min at 25°C at the appropriate wavelength for the substrate. One unit (1 U) is defined as the amount of enzyme that converts 1 µmol substrate per minute. Various H₂O₂ concentrations (0-1250 µM, enzyme concentrations (0.27-54 nM) and substrate concentrations are used to determine the enzyme activity. The activity of rPsaDyP vs ABTS is determined in 100 mM sodium acetate buffer at pH 3.8 and a final H₂O₂ concentration of 125 µM. Production of the ABTS cation radical is studied at 420 nm (ε₄₂₀ 36,000 L mol^-1 cm^-1)^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Matsuda H, et al. Evaluation of ELISA with ABTS, 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate of peroxidase and its application to the diagnosis of schistosomiasis. Jpn J Exp Med. 1984 Jun;54(3):131-8.

  [2]. Lauber C, et al. Identification, heterologous expression and characterization of a dye-decolorizing peroxidase of Pleurotus sapidus. AMB Express. 2017 Aug 23;7(1):164.