Typical Components of an Sandwich Immunoassa

Antagene Inc is a custom service laboratory specializing in antibody production and immunoassay development. Once developed, we will ship the finished test to you so it can be used on-site by your staff, or in the field by your customers. We will then manufacture the components of the test whenever you need more. A typical 96-well microtiter plate sandwich immunoassay kit may include the following components (there are several different formats of an immunoassay; only 1 is presented here), Click to view the components:

�Pre-coated, stabilized 96- well microiter plate
�Sample diluent
�Assay diluent
�Calibrator diluent
�Standards and Controls
�Conjugated detection antibody (in a working diluent)
�10X wash solution
�Sangle component substrate
�Stop solution

Pre-Coated, Stabilized 96-well Microtiter Plate:
For microtiter plate assays, the plates are provided ready-to-use. They are pre-coated with the capture antibody, blocked, stabilized, and packaged in a resealable foil pouch with a desiccant packet. Using our methods, the plates may have a real time stability of 1 year when stored at 2^o - 8^o C. With a standard curve (1 blank and 7 standards) and 3 controls, a 96-well microtiter plate format can test 21 samples in triplicate and 37 samples in duplicate.

Sample Diluent:
The sample diluent is used to ensure that the analyte in the sample matrix is measured accurately. It is used to dilute the sample within the target range of the assay.

Assay Diluent:
In addition to a specimen diluent, certain matrixes require the use of a special assay diluent (which is applied to the plate just prior to adding the samples, standards, and controls). Assay diluents are often paired with a specific type of sample (such as serum, or cell culture media) to eliminate interference and non-specific binding generated from the matrix of the sample itself. These interferences are especially noticeable when running neat samples. An assay diluent may not be required for every assay.

Calibrator Diluent:
A calibrator diluent is used to ensure that the standards and controls will be measured accurately. This diluent must compliment the target analyte, capture antibody, and resemble the matrix of the sample.

Standards and controls:
A known amount of the analyte (such as 1 ng) is included with a kit and run on every plate. The standard is often lyophilized. Once reconstituted, it is diluted several times to prepare a range of known values with which to compare and properly quantitate the unknown amount of analyte in a sample. Controls are used to confirm the readings of the standard and to compare readings from different plates. Because they contain a known amount of analyte, they should always read within a certain optical density (OD) value based on the standard.

Conjugated Detection Antibody:
For some assays, the detection or �top� antibody is often an affinity purified polyclonal antibody conjugated to HRP. The enzyme-antibody conjugate can often be supplied ready-to-use in its working concentration in a special conjugate diluent. The conjugate diluent stabilizes the conjugate and minimizes nonspecific binding of the conjugate onto the blocked plate or matrix residue. The working conjugate will often have a real-time stability of 1 year when stored at 2^o-8^o C .

10X Wash Solution:
This specially formulated buffer is used to rinse the plate after the sample and conjugate incubation periods, just prior to the addition of the next reagent. It minimizes matrix residue and non-specific binding interferences of the samples and conjugate.

Single Component Substrate:
This reagent reacts with HRP to generate a colored signal product. It can come in many formulations (powdered, 2-component liquid, etc.). Antagene recommends a low-background, high-signal-generating ABTS or TMB that needs no preparation prior to use.

Stop Solution:
An appropriate stop solution is added to the plate with the ABTS or TMB substrate and stops its reaction with HRP. By stopping this reaction after 20 minutes, the plates can equilibrate before reading, which increases the accuracy and sensitivity of the assay.