Ashley Sutton

English 202C

Description/Definition

March 3, 2015

How Enzyme Linked Immunosorbent Assays Work

The Enzyme Linked Immunosorbent Assay or ELISA for short is a method of measuring

antibodies in a biological sample in which the antibodies that are being quantified are sandwiched

between two other antibodies, one of which has a HRP (horseradish peroxidase) molecule conjugated to

it. They can be used to test a variety of biological samples such as blood plasma, milk, and saliva. ELISA

tests are an integral part of immunology research, and knowing how they work is an asset to any

reasearcher.

Figure 1: Example 96 well plate used for ELISAs

Plate Coating

The first phase of an ELISA test involves a coating antibody being bound to the bottom and sides

of the wells of a 96 well plate with a maxisorb coating that helps antibodies to bind. The type of

antibody used for a coating antibody is dependent on what antibody you are trying to bind. Lets assume

you were trying to bind Equine IgG. You would want to use a coating antibody that will bind to the

Equine IgG, therefore an anti-equine IgG antibody would be used, such as Goat Anti-Equine IgG. This

type of antibody is made by injecting horse IgG into a goat. Since the horse antibodies are recognized as

foreign by the goats immune system it produces antibodies against the horse antibodies. These

antibodies are collected and are the integral component in the first part of the ELISA test. Since these

antibodies are specific to equine IgG, they will not bind to other antibodies in the sample. The proper

concentration of coating antibody must be used so that the plate develops a color reaction sufficiently,

but does not overdevelop in the end of the process. The antibodies are allowed to bind for an hour and

then the plate is washed with a microplate washer to remove any unbound antibody.

Microplate well

Coating antibody

Figure 2: Well after plate coating

Plate Blocking

After the coating antibody has bound to the plate, the next step is to block the plate. A solution

of fish skin gelatin is used to block the plate. It binds in all the areas where the coating antibody has not

bound, covering the entire surface of the plate. This prevents the subsequent antibodies from sticking to

the open surface of the plate and prevents the plate from overdeveloping. It is at this point that the test

can be left overnight if desired. If there will be an entended time until the rest of the test is completed

the plate can be sealed in a ziploc baggie and left in the refridgerator for up to 24 hours. The fish skin

gelatin is allowed to bind for half an hour and then the plate is washed with a microplate washer to

remove any unbound fish skin gel. If the plate was left in the refridgerator then it must be warmed to

room temperature for an hour before washing the plate.

Sample Binding

Diluted sample (such as blood plasma) or standard then binds to the coating antibodies. The

sample must be diluted to an expected concentration within the range of the standards so that a

quantitative value can be determined. The coating antibodies will attach specifically to the equine IgG in

the sample and not to any other component within the blood plasma. ELISA tests are very specific, they

cannot be contaminated as easily as many other assays. The antibodies in the sample or standard are

allowed to bind for an hour and then the plate is washed with a microplate washer to remove any

unbound antibody.

HRP Conjugate Binding

Another diluted anti-equine IgG is used which binds to another portion of the equine IgG. It

must be diluted so that the proportion of antibody bound HRP will develop sufficiently but not

overdevelop. This second antibody is conjugated to an HRP (horseradish peroxidase) molecule. This HRP

molecule is derived from a raddish and its purpose is to cause a colorimetric reaction proportional to

how many of the antibodies bind. It is during this step that the antibody of interest is sandwitched

between two other antibodies. The antibody is allowed to bind for an hour and then the plate is washed

with a microplate washer to remove any unbound antibody.

Microplate well

Coating antibody

Fish skin blocking molecule

Microplate well

Coating antibody

Fish skin blocking molecule

Antibody to be quantified

Microplate well

Coating antibody

Fish skin blocking molecule

Antibody to be quantified

HRP conjugated antibody

Figure 3: Well after plate blocking

Figure 4: Well after sample binding

Figure 5: Well after HRP conjugate binding

Colorimetric Reaction

TMB reacts with the HRP molecule and turns the currently clear contents of the plate into a

shade of blue. The shade of blue in the plate is directly related to the amount of equine IgG in the well.

The more equine IgG in the well, the darker blue the color will be. An acid then turns the blue color to a

corresponding darkness of yellow which can be measured at 450 nm wavelength with a microplate

reader. The amount of 450 nm wavelength light that is absorbed by the well is relative to the darkness

of the color in the sample. A darker shade of yellow will absorb more light. The amount of light absorbed

by each sample is compared to a standard curve to determine the concentration of equine IgG in the

original sample.

Summary

In summary the process of ELISA testing is a 6 step process. The coating antibody binds, followed by the

blocking fish skin molecules, next the antibodies of the sample in question bind and then an HRP

conjugated antibody binds to that. The HRP causes a colorimetric reaction in TMB and then the color is

changed from a shade of blue to a shade of yellow so that it can be read in a microplate reader. This is

how antibody concentrations are determined in a number of biological samples.

Figure 7: Example completed ELISA test

(after Stop solution has been added)

Microplate well

Coating antibody

Fish skin blocking molecule

Antibody to be quantified

HRP conjugated antibody

Color change once TMB is added

Figure 6: Well after colorimetric reaction