EIA kits & Reagents

This product group includes competitive enzyme immunoassays for prostaglandins and their metabolites, leukotrienes, cyclic nucleotides, steroids, and biologically active peptides. Larger proteins like endothelin and cytokines such as IL-1, IL-2, and IL-4 are measured using a sandwich immunometric enzyme immunoassay format.

Our EIA kits are designed to allow the user to run an assay quickly and conveniently with minimum prior setup. The only prerequisites are Milli-QTM water (or equivalent) and accurate pipetting equipment. Several of the kits (PGD2, Bicyclo PGE2, and the cyclic nucleotides) require derivatization of the samples prior to use. Special reagents to accomplish this are included with each kit.

The enzyme label that is common to nearly all our assays is acetylcholinesterase (AChE). AChE is a 537 amino acid protein with a molecular weight of approximately 80,000 Daltons. It functions in most vertebrates as a neurotransmission modulator, terminating impulses by the rapid hydrolysis of the acetylcholine released at the synapse. In keeping with the critical role that this plays in the coordination of fine motor movements, AChE has an extremely rapid substrate turnover rate which approaches the rate of a diffusion-controlled chemical reaction.


	AChE offers the following advantages:

Kinetic superiority

Hydrolysis of acetylthiocholine by acetylcholinesterase shows true first-order kinetics and proceeds rapidly with a turnover of 64,000 sec-1. That's nearly 3 times faster than HRP or alkaline phosphate. These characteristics afford a wider dynamic range and greater sensitivity than that available with other labeling enzymes.

Low background

Acetylthiocholine is a stable compound. Nonenzymatic hydrolysis to thiocholine in EIA buffer is essentially absent. This results in a very low background and an increased signal/noise ratio. Substrates like TMB used for peroxidase assays are inherently unstable under the assay conditions leading to excessive noise, a loss of sensitivity and increased coefficients of variation.

High Sensitivity and precision

The sensitivity is equal to or better than RIA, typically [puissance3]1 pg/well for the eicosanoids and in the fmol/well range for many peptides. Percent CV averages [puissance3]10 in a wide range for most analytes.

Versatility

Unlike peroxidase, which is a suicidal enzyme inactivated by the catalytic turnover process, AChE is completely stable. The level of bound enzymatic activity remains constant for many hours in the presence of substrate. Practically speaking, this allows the user to include both very dilute and very concentrated samples on the same plate, without having to sacrifice one by terminating the reaction in order to read the other. With AChE, one simply has to read the plate early in the development to obtain values for rapidly developing wells. Then the plate can continue to develop for up to 24 hours to get data from wells with low levels of bound enzyme. Even more reassuring is the knowledge that if an AChE plate is accidentally dropped, one needs only to wash it and add fresh substrate, and the development step will proceed identically a second time. In the case of a dropped or splashed HRP plate, there is only one recourse - buy a new kit and repeat the assay.


	Suggestions for further reading.

Antoine, C., Lellouche, J-P., Maclouf, J., et al. Development of enzyme immunoassays for leukotrienes using acetylcholinesterase. Biochim. Biophys. Acta 1075, 162-168 (1991).
Frolich, J.C. Measurement of icosanoids. Prostaglandins 27, 349-369 (1984).
Patrono, C. and Peskar, B.A. (eds.) Radioimmunoassay in basic and clinical pharmacology (Handbook of experimental pharmacology; Vol. 82). Springer-Verlag, Berlin (1987).

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