1. Introduction

Imidazole is incorporated into many important biological molecules. The most obvious is the amino acid histidine, which has an Imidazole side chain. One of the applications of Imidazole is in the purification of His-tagged proteins in immobilized metal affinity chromatography (IMAC). Imidazole is used to elute tagged proteins bound to Ni ions attached to the surface of beads in the chromatography column. An excess of Imidazole is passed through the column, which displaces the His-tag from nickel co-ordination, freeing the His-tagged proteins. The Imidazole EIA kit can be used to determine quantitatively the concentration of Imidazole in the aqueous sample.

2. Principle of Imidazole Determination

The enzyme immunoassay for Imidazole is based on the competition between the Imidazole to be assayed and the Imidazole-alkaline phosphatase conjugate, for binding to antibody directed against Imidazole, coated onto microtiter wells. The sample containing the Imidazole, and the Imidazole-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 then is measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of Imidazole in the sample. The concentration is estimated by comparison with standard Imidazole solution.

3. Reagents

Reagents are for in vitro research use only. All reagents of the kit are stable, if stored at 2-8 °C, until the expiration date stated on the kit.

1. 96-well microtiter plate (#S). Twelve strips of 8 detachable wells, coated with Anti-Imidazole antibody.
2. One vial of 1.2 ml Negative Calibrator containing 0 μg/ml of imidazole.
3. Three vials of Positive Calibrator (0.9 ml) containing 1.0, 3.0 and 9.0 μg/ml of imidazole.
4. One bottle (#3) containing 9.5 ml of Imidazole-Alkaline Phosphatase conjugate (IDZ-ALP).
5. One bottle (#5) containing 9.5 ml of p-Nitrophenyl Phosphate (pNPP) substrate. Ready to use.
6. One bottle (#6) containing 15 ml of Wash Buffer (10xPBS-Tween). Dilute 10 fold with distilled or deionized water to 150 ml prior to use.
7. One bottle (#7) containing 5.5 ml Stop Solution, 3N NaOH.

4. Optional Equipment and Material Required

1. Pipettors capable of delivering 50 μl and 100 μl.
2. Microtiter plate reader (wavelength 405 nm)
3. Plate washer or squeezable wash bottle.
4. Timer.
5. Absorbent paper towels.

5. Assay Procedure

Let the components of the kit equilibrate to room temperature before use.

1. Carefully add 50 μl of standard or sample (dilute if Imidazole concentration is high) to the bottom of each well. Slightly tap the side of the strip holder to evenly distribute the sample.
2. Avoid touching the well with the pipette tip while adding 100 μl of IDZ-ALP conjugate (#3) to each well. Slightly tap the side of the strip holder to properly mix the sample and enzyme conjugate.
3. Incubate at room temperature for 40 minutes.
4. After incubation, dispose of the solution in the wells by inverting and shaking. Wash microtiter wells 3 times with wash buffer to remove the non-bound conjugate. Washing may be done manually as follows: use squeeze bottle to fill wells gently with wash buffer, dumping the wells between each wash by inverting and shaking. After the third wash, tamp holder with washed strips onto a piece of absorbent paper.
5. Add 100 μl of pNPP substrate (#5) to each well and incubate at room temperature for 20 min. To avoid contamination, place the needed amount of substrate into a test tube and only dispense from the tube itself.
6. Add 50 μl of Stop Solution (#7) to each well and tap the strip holder for proper mixing .
7. Read absorbance at 405 nm using ELISA reader.

6. Preparation of Standard Curve

1. Calculation
   1. Average the absorbance (OD_s) for each standard concentration of Imidazole including 0 μg/ml (OD₀).
   2. % of Inhibition = 100 -( OD_s / OD₀ ) x100
2. Plot values of % of Inhibition, step 6.1 (b), against their corresponding concentrations on Log₁₀ paper.
3. Calculate the imidazole concentration in the sample by interpolation and multiply by the sample's dilution factor to obtain the actual quantity of imidazole.