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ELISA (Enzyme-Linked Immunosorbent Assay)

Introduction

Enzyme-Linked Immunosorbent Assay (ELISA) is a useful and powerful method in estimating ng/ml to pg/ml ordered materials in the solution, such as serum, urine and culture supernatant. It's a kind of easy task to make ELISA if you have "good" antibodies against your concerned materials such as proteins, peptides and drugs. First, I will show the basic method in establishing ELISA using a "good" antibody. I will add some trouble shootings in the section. Second, I will show some improving methods for making ELISA, which is especially useful when using a "not so good antibody". Of course, some trouble shootings I will illustrate. Third, we will take a look at some remarks in making ELISA for the estimating serum/plasma materials, which is a little bit of different story from making ELISA for simple buffers. I hope this page will help you establishing a "good ELISA" to your concerning materials.

Definition

If you have "good" antibodies against your concerned materials, it is not so difficult to establish ELISA. It's a just around three-days working. However, you can't tell if your antibodies are good or not until accomplishment of an ELISA in most cases. If you use polyclonals, you can easily examine their ability. That is the "Ouchterlony" method. This old-fashioned double immunodiffusion method is really reliable and reproducible test in making ELISA. Put the antigen into the center well and put the serially diluted antisera into the outer wells. If you can see evident white band between the inner and outer wells at x 64 or more dilution point after the incubation 37 degree overnight, you are very lucky! You can use the antibody without any further treatment other than purification to Ig fraction. If you can see bands between x 2 and x 32 point, you should probably go to the second session for further treatment of your antibody. Note that these dilution points are a kind of arbitrary. It's depend on the antigen. So, I will say just try. If you cannot establish good ELISA by the first method, you can try the second.

Definition

It's a very common situation that you only have not so good antibody to your concerned material. It's necessary to reinforce your antibody in this case when using for ELISA. The best way to make your antibody a good one is the affinity purification using solid phased antigen, i.e., affinity column purification. First, I will show you how to make antigen-column using CNBr activated Sepharose 4B gel (Pharmacia), then I will show you how to use it effectively.

Definition

When using ELISAs for the estimation of serum or plasma materials, many cares should be taken. Nevertheless, you will get misleading results. Never omit this step if you want to apply your ELISA to the clinical. Yes, it's really important!

Here's one good method for the improvement your polyclonal antibody. You should push here right now!

ELISA Activity

The ELISA

An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the first and most basic test to determine if an individual is positive for a selected pathogen, such as HIV. The test is performed in a 8 cm x 12 cm plastic plate which contains an 8 x 12 matrix of 96 wells, each of which are about 1 cm high and 0.7 cm in diameter. The next page illustrates how an HIV ELISA is performed.

An ELISA plate

ELISA Activity The ELISA An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the

The ELISA Method

ELISA Activity The ELISA An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the

Partially purified, inactivated HIV antigens pre-coated onto an ELISA plate

Patient serum which contains antibodies. If the patient is HIV+, then this serum will contain antibodies to HIV, and those antibodies will bind to the HIV antigens on the plate.

Anti-human immunoglobulin coupled to an enzyme. This is the second antibody, and it binds to human antibodies.

Chromogen or substrate which changes color when cleaved by the enzyme attached to the second antibody.

Positive ELISA Test

animation!
animation!

Negative ELISA Test

ELISA Activity The ELISA An HIV ELISA, sometimes called an HIV enzyme immunoassay (EIA) is the

False positives

It is entirely possible that an individual not infected with HIV has antibodies which may give a positive result in the HIV ELISA. This is called a false positive. One reason for this is that people (especially women who have had multiple pregnancies) may possess antibodies directed against human leukocyte antigens (HLA) which are present on the host cells used to propagate HIV. As HIV buds from the surface of the host cell, it incorporates some of the host cell HLA into its envelope. False negatives can occur during the window between infection and an antibody response to the virus (seroconversion).

ELISA data from three patients

It is entirely possible that an individual not infected with HIV has antibodies which may give

Positive

Negative

     

Control

Control

Patient A

Patient B

Patient C

1.689
1.689
Positive Negative Control Control Patient A Patient B Patient C 1.689 0.153 O.055 0.412 1.999 Control
  • 0.153 O.055

Positive Negative Control Control Patient A Patient B Patient C 1.689 0.153 O.055 0.412 1.999 Control
0.412
0.412
1.999
1.999
Control Assay 0.123
Control
Assay
0.123

Above is ELISA data from three patients. Numbers are expressed as optical density at 450nm. The cutoff value indicating a positive result is 0.500. Optical densities of 0.300 to 0.499 are indeterminate and need to be retested. Values below 0.300 are considered to be negative. In most cases, a patient will be retested if the serum gives a positive result. If the ELISA retests are positive, the patient will then be retested by western blotting analysis.