Introduction

The Triton-X (4-t-octylphenoxy polyethoxyethanols) is a nonionic detergent, which is often used in biochemical application to solubilize proteins. Their use in industrial and household cleaners and as emulsifiers for agricultural chemicals is among the many roles for these important chemicals. Direct human exposure to these and related surfactants also occurs through their use in the cosmetic and pharmaceutical industries. In addition, numerous biological and biochemical investigations depend upon the use of nonionic detergents, particularly in the field of membrane biochemistry. It is often difficult to remove detergents from the macromolecules, and analysis of bound detergent in biochemical sample becomes important. Quantification of these nonvolatile detergents is therefore a frequently encountered problem both for their residue analysis as well as for their presence in biochemical products. Most nonionic detergents present difficult analytical problems not only are they nonvolatile and lacking a chromophore with a high extinction coefficient, but most such detergents consists of a complex mixture of closely related, rather unreactive compounds. Due to their surfactant properties, extraction, workup, and concentration of these compounds are further complicated.

Principle of Triton-X Determination

The enzyme immunoassay for Triton-X is based on the competition between the Triton-X to be assayed and the Triton-X-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against Triton-X, coated onto microwells. The sample containing the Triton-X, and the Triton-X-Alkaline Phosphatase conjugate, when added to the microtiter wells, compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed in order to remove non-bound components. The bound enzymatic activity is then measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of Triton-X in the sample. The concentration is calculated on the basis of a standard curve.

Reagents

• 96-well microtiter plate. Twelve strips of 8 detachable wells, coated with rabbit anti-Triton-X antibody.
• One vial of 0.5 ml Negative Calibrator containing 0 ng/ml of Triton-X.
• One vial of Positive Calibrator containing 0.25 ml 1 µg/ml of Triton-X.
• One bottle containing 10.5 ml of Triton-X-Alkaline Phosphatase (TTX-ALP) conjugate.
• One bottle containing 10.5 ml of p-Nitrophenyl phosphate (pNPP) substrate.
• One bottle containing 15 ml of Wash Buffer (PBS-Tween).
• One bottle containing 5.5 ml of Stop Solution, 3N NaOH.

Assay Procedure

• Add 25 μl of standard or sample to the bottom of each well.
• Add 100 μl of TTX-ALP conjugate to each well. Incubate at room temperature for 40 minutes.
• Wash micrtiter wells 3 times
• Add 100 μl of pNPP substrate to each well and incubate at room temperature for 20 min.
• Add 50 μl of Stop Solution to each well.
• Read absorbance at 405 nm using an ELISA reader.

Triton-X-100 Inhibition Curve