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ELISA assays are immunochemical methods for determination of substances such as peptides, proteins, antibodies and hormones, in which a crucial element of the detection is an antigen-antibody interaction. The acronym stands for Enzyme Linked ImmunoSorbent Assay. These assays utilize antibodies that are covalently linked to an enzyme. These enzymes can catalyze an easily visualized reaction, such as a color change, when substrate is added.

First I would like to bring everyone up to speed on the immunology and chemistry involved. One of the body's most important defense mechanisms against infection is the production of antibodies, or immunoglobulins. In mammals, there are 5 major classes of immunoglobulins (IgA, IgD, IgE, IgG, and IgM).  The major class of immunoglobulins in blood is IgG (gammaglobulin) and this class constitutes about 10% of total serum proteins. These proteins circulate in the bloodstream, where they make up part of the gammaglobulin fraction of blood plasma. There are about 10 to 12 mg of IgG per ml of serum. The antigens of an infecting agent such as a bacterium, virus, or foreign protein can stimulate an immune response and the production of antibodies.

The immunoglobulin genes are a highly evolved system for maximizing protein diversity from a finite amount of generic information. The diversity is essential for gaining immunity to the large variety of infections organisms and forgien substances that cause disease. Only vertebrates show an immune response. If a foreign substance, called an antigen enters the bloodstream the immune system responds with the production of proteins capable of recognizing and termination of the antigen. B and T lymphocytes, so named because they mature in the Bone Marrow or Thymus gland, are capable of gene rearrangement. This mechanism produce proteins, which are needed to produce an immune response.

Lymphocytes in the human blood constitute 20-30 percent of the nucleated blood cells; in lymph they constitute 99 percent. Until they come into contact with an antigen, lymphocytes continually cycle from the blood to the lymph to the blood, making a complete round once or twice a day. The lymph circulation is filtered through the spleen and various lymph nodes, where all type of different lymphocytes take up temporary residence. Immunoglobulin proteins or antibodies, which are secreted from B lymphocyte cells, can recognize and bind to various antigens. Since antigens can be almost anything, the immune response must have an amazing memory of structural recognition. Vertebrates must have the potential to produce immunoglobulins of vast diversity in order to recognize every antigen.

An antibody only binds to a particular type of antigen and no other. In a normally functioning vertebrate, if the immune system has come in contact with an antigen, it will produce antibodies against that antigen as a defense. The response is usually graded to the aggressiveness of the foreign contaminate. These antibodies will remain in the bloodstream, sometimes for prolonged periods, and can be detected. There are many laboratory procedures designed to detect the presence of antibodies and antigens based on their specific interactions. ELISA or Enzyme linked immunosorbant assay is used to detect and quantify specific serum antibodies (serum is blood plasma without the clotting factors). In ELISA, serum to be tested is exposed to specific antigens. Serum antibodies that combine with their antigens are detected by treating the test system with a conjugate, or another antibody linked to an enzyme. This antibody enzyme complex serves as a marker and attaches only to a specific substrate. When a substrate for the enzyme is added to the assay, a reaction between the substrate and the conjugate is usually indicated by a color change, but other substrates can be used i.e. radio labeled markers, florescence dyes, etc. If no serum antibodies were present to bind with the antigen, no conjugate interaction would take place, no color change will occur at the time of detection.

A majority of the noncovalent interactions dealing with antigen/antibody binding are hydrophobic bonds or interactions. These interactions are due to the strong tendency of water to exclude nonpolar groups, and are formed when uncharged, nonpolar amino acids in the antigen interact with nonpolar and uncharged amino acids in the antibody molecule.

Hydrogen bonding also contributes, if a hydrogen atom is bonded to a small, electronegative atom such as oxygen, nitrogen or fluorine (in biological systems, one will find hydrogen bonds involving oxygen and nitrogen), that electronegative atom will pull the hydrogen electrons toward itself. As a consequence, hydrogen will carry a partial positive charge. This partial positive charge will attract it to neighboring electronegative atoms, and this attraction forms the basis of the hydrogen bond.
 

Ionic bonds are the result of attractive forces between oppositely charged polar functions, such as carboxyl groups, and amino groups. They occur when a negatively charged group (such as the acid group on the end of a glutamate side chain) is near a positively charged group (such as the amino group on the end of a lysine side chain).
 

Van der Waals forces are the result of temporary shifts in electron distribution over a molecule. Thus, the induced electrical interactions between molecules may flux between partial positive and partial negative charges. These changes can have an opposite effect on a neighboring molecule; they allow attractions to occur between the positively charged nuclei and the electron density of an incoming atom. This type of interaction can contribute to antigen/antibody bind.

Bimolecular recognition events that occur through structural complementary are mediated by weak chemical forces. These interaction need to be sufficiently weak to be readily reversible. The average kinetic energy of molecules at 25^oC is 2.5kJ/mol; the energy is only several times greater than the dissociating tendency due to the thermal motion of the molecules. This mean that the bonds in your body are constantly forming and breaking at physiological temperature, and have a profound influence on the biological structures they build.

The amino acid sequence of IgG varies in a species-dependent manner, even for very similar proteins. We can inject IgG from one animal into a different host, the host will produce antibodies that recognize the foreign IgG molecules. 

We Inject a host with an antigen of interest. This may take place over multiple injections. Then allow host to produce an immune respone over an given interval. Collect samples of blood, separate and isolate proteins. Run assay.

Immunoassays are a powerful technique for detecting and measuring antigens and antibodies.  Immunoassays can be classified in many different ways based on the steps involved:

• antibody capture
• antigen capture
• two-antibody sandwich

Many types of immunoassays can be used to detect and quantitate both antigens and antibodies, but there are differences in the signal strengths of the labels, and the amount of background for each of these types of assays. I will cover 3 basic types of assays.

1. Sandwich Method
2. Direct Method
3. Indirect method (Going over full Protocol)

*A brief Overview

The "sandwich" technique is so called because the antigen being assayed is held between two different Antibodies. In this method:

1. Allow for passive absorption of Antibody to plate by incubation in defined buffer .
2. Wash
3. The test sample, containing the antigen being measured, is then added and allowed to react with the bound antibody.
4. Wash.
5. A known amount of enzyme-labeled antibody is then allowed to react with the bound antigen. Any excess unbound enzyme-linked antibody is washed away after the reaction.
6. Wash
7. The substrate is then added and the reaction between the substrate and the enzyme produces a color change. The amount of visual color change is a direct measurement of specific enzyme-conjugated bound antibody, and consequently antigen present in the specimen tested.

For antibody detection, only instead of the antibody being adsorbed to the surface, it is the antigen that is adsorbed.

1. Allow for passive absorption of antigen to plate by incubation in defined buffer.
2. Wash
3. Addition and Incubation of enzyme labeled antibody
4. Wash
5. Addition and Incubation of substrate
6. Wash
7. Read Assay

The Antigen is attached directly to the solid phase and is reacted directly with the enzyme labeled antibody.

For antibody detection:

1. Allow for passive absorption of antigen to plate by incubation in defined buffer.
2. Wash
3. The antiserum containing the antibody is then added to the well and allowed to bind.
4. Wash.
5. Second enzyme-labeled antibody is added. The enzyme-labeled antibody complex binds to a specific site on the bound antibody.
6. Wash
7. Addition and Incubation of substrate.
8. Wash
9. Read Assay

• Sodium carbonate (Na₂CO₃) buffer
• PBS-T (0.05% Tween-20)
• 3% BSA in PBS-T (Blocking solution)

ELISA Procedure

1. Prepare buffer solutions.
2. Prepare a solution of the antigen in sodium carbonate buffer.
3. Add antigen,
4. Cover the plate and incubate for three hours at room temperature or overnight at 2-6°C.
5. Remove unbound antigen by washing three times with the PBS-T buffer.
6. Block the remaining adsorption sites by adding 3% BSA/PBS-T to each well. Incubate.
7. Wash the plates with PBS-T.
8. Prepare a dilution of test sample.
9. Add the sample dilutions into the wells.
10. Incubate.
11. Wash with PBS-T to remove the unbound antibodies.

Detection Using Horseradish Peroxidase

(ELISA) is used for the quantitative determination of erythropoietin. The assay makes use of two antibodies in a double-antibody sandwich method - a monoclonal antibody specific for human Epo which is coated on the plate and a second polyclonal capture antibody conjugated to horseradish peroxidase. A chromogen added to the reaction is oxidized by the peroxidase enzyme to yield a colored reaction complex. The absorbance of the complex is directly proportional to the level of Epo present.

Quantitative determination of secreted human growth hormone (hGH). The assay is based on a sandwich ELISA principle using an anti-hGH coating antibody and a (DIG)-labeled capture antibody to hGH. An antibody to DIG which is conjugated to horseradish peroxidase is used as the detection antibody. The peroxidase enzyme catalyzes the cleavage of a chromogenic substrate to yield a product which can be measured spectrophotometrically.

How does ELISA assist in future product development? Celera Genomics has finished the human genome-sequencing phase and has started the assembly of the genome. With the completion of the sequencing of the entire Human genome. The race is on to understand and patent the proteins and the gene sequences. Any company that can get a head start in this new market can realize extreme profit potential, a statement by CELERA GENOMICS CEO sums this up, "This is expected to allow our subscribers worldwide to utilize our database to make important medical advances."

This will be of immense benefit to medical science. It will help us to understand and eventually treat many of the more than 4000 genetic diseases that afflict mankind, as well as the many diseases in which genetic predisposition plays an important role. Its not just genetic diseases that will benefit, understanding the proteins that genes produce will have a profound effect on the production of new drugs. New technologies emanating from the genome project will also find application in other fields such as agriculture and the environmental sciences. It is anticipated that the private sector will derive great benefit, and the DNA sequence information will undoubtedly be a major tool in most areas of basic and applied biological research.

The gene involved in cystic fibrosis, the most common lethal hereditary disease among Caucasians, was identified; a diagnostic test is available to identify gene carriers among high-risk families, and the first human gene therapy efforts are under way in federally approved clinical trials.

The expression of a Gene produces a protein; a protein can have a huge effect on function or dysfunction of an organism, i.e. insulin. Does an altered gene produce too little protein, a flawed protein, or not protein at all? Scientists need to understand just how the protein change causes the disease. As the mechanism of the disease becomes clear, scientists can devise new approaches to treatment involving either the protein or the gene. Understanding a relatively rare inherited disorder may also bring important insights into more common and complex diseases. To make up for the genetic error, scientists may try to replace a missing or ineffective protein with a drug or with the normal protein. Another option is gene therapy. Gene therapy involves "infecting" cells with a virus into which they have inserted normal genes. Others methods are non-viral, or even inject DNA directly into cells. For example, a patient's bone-marrow cells may be removed, treated with normal genes, and returned to the patient.

How this applies to ELISA, the assay can detect the presents of specfic proteins, and has the ability to quantify specific proteins and other targets of interest in a biological system. In Conjunction with other assays, these tools will help in the production of innovative new drugs.

ELISA has replaced a number of more cumbersome and time-consuming "classical" serological techniques, and has also widened the scope of the detection methods for antigens, specific antibodies, and viruses and their related markers of infection.