Enzyme Linked ImmunoSorbant Assay (ELISA)

Enzyme immunoassay combine the specificity of antibodies with the sensitivity of simple spectrophotometric enzyme assays by using antibodies or antigens coupled to an easily assayed enzyme that also possesses a high turnover number. ELISA is replacing RIA, despite the latter already being established, extensively automated and sometimes more sensitive.

ELISA may be used for assaying antigens by either a competitive or a double antibody method and for assaying a specific antibody by an indirect method. All these methods require the preparation of a calibration curve during the assay.

The ELISA divided into three types, they are

a) Competitive method b) Double-antibody method c) Indirect method

a) Competitive method: A mixture of a known amount of enzyme - labelled antigen and an unknown amount of unlabelled antigen is allowed to react with a specific antibody to attach to a solid phase. After the complex has been washed with buffer, the enzyme substrate is added and the enzyme activity measured. The difference between this value and that of a simple lacking unlabelled antigen is a measure of the concentration of unlabelled antigen.

Major disadvantage: this method is that each antigen may require a different method to couple it to the enzyme; this is not so for the double-antibody method.

b) Double-antibody method: The unknown antigen solution is reacted with specific antibody attached to a solid phase, washed and treated with enzyme labelled antibody (directed against a different epitope, if monoclonal antibodies). After a further wash the enzyme substrate is added. The amount of enzyme activity measured under standard conditions directly proportional to the amount of antigen present. Advantage: It is that only one procedure required coupling the enzyme to all preparations.

c) Indirect method: The method may be used to measure antibody levels. The putative antiserum is reacted with specific antigen attached to a solid phase. Any specific antibody molecules bind to the antigen and all other material is washed away. Exposure of the complex to enzyme labelled anti-immunoglobulin antibody results in binding to any specific antibody molecules adsorbed from the original serum. The complex is washed and the substrate for the enzyme added, resulting in activity proportional to the amount of specific antibody in the serum.

 

ELISA, in which enzyme labelled antibody binds an antigen, leading to substrate breakdown and colour change, can be used to detect primary antigen-antibody reactions

ELISA uses an enzyme for labelling rattier than a radioisotope. Like the radioisotopes, the enzyme is covalently coupled to the antibody, in principle, this is the only difference between ELISA and RIA. ELISA measures bound enzyme activity rather than bound counts per minutes. Quantification requires measuring the colour intensity of the colored products generated by the enzyme and added substrate. The intensity of the colour is equivalent to the amount of labeled antibody bound to antigen. ELISA has replaced RIA in many clinical and basic science laboratories. RIAs are potentially hazardous and tedious, require bookkeeping, and involve radioisotopes. These reasons combined with the commercial availability of plate readers that can be measure the absorbance of 96 wells in less than a minute, account for ELISA's growing popularity. ELISA and RIA are similar in sensitivity. Theoretically, ELISA can be more sensitive than RIA because each enzyme molecule can generate hundreds of thousands of colored product molecules that can be measured. In contrast, ^12S I molecule decays only once. Once application of ELISA is to detect the presence and titre of specific antibody for the AIDS virus.

Applications of ELISA:

• Whilst ELISA can be used for the assay of virtually any antigen, hapten or antibody, it is used predominantly in clinical biochemistry laboratories to measure, for example, IgG, and IgE, oncofetal proteins, hematological factors, immune complexes and hormones such as insulin, oestrogen and human chorionic gonadotropin.
• Examples of its use in the study of infectious diseases include the detection of bacterial toxins, Candida albicans, rotaviruses, herpes simplex viruses and the hepatitis B surface antigen.
• ELISA has also been used extensively for the assay of antibodies in infectious diseases including: anti-viral antibodies, e.g. to Epstein-Barr virus and rubella virus; anti-bacterial antibodies, e.g. to Brucella, Rlckettsia and Salmonella species, anti-fungal antibodies, e.g. to Aspergillus and Candida species, anti-parasite antibodies, e.g. to Plasmodium, Schistosoma and Trypanosoma species, and autoantibodies, e.g. anti-DMA and anti thyroglobulin.

monoclonal antibodies

When an antigen is introduced into the circulatory system of a higher vertebrate, it stimulates specific B-lymphocytes to produce antibodies. The antibodies are the immunoglobulins and they circulate in the blood serum.

When the immune system of an animal encounters a new antigen, it responds to specific antigenic determinants called 'epitopes' located on the antigen. Thus, a protein antigen may possess several epitopes and would induce the formation of several different antibodies, each specific for one epitope. Such a polyclonal antibody response facilitates the localization, phagocytosis and complement-mediated lysis of antigens.

For most reasearch, diagnostic and therapeutic purposes, "Monoclonal antibodies", derived from a single clone and thus specific for a single epitope are preferable.

In 1975, "Georges Kohler" and "cesar Milstein" devised a method for preparing monoclonal antibody, which quickly became one of the immunology's key technologies. These work got Nobel Prize in 1984.

Basic concept Principle on the monoclonal antibodies production:

By fusing a normal activated, antibody producing B-cell with a myeloma cell(a cancerous plasma cell), they were able to generate a hybrid cell, called a 'hybdridoma', that possessed the immortal-growth properties of the myeloma cell and secreted the antibodies produced by the B-cell. The resulting clones of hybridoma cells,which secrete large quantities of monoclonal antibodies can be cultured indefinitely. The development of techniques for producing monoclonal antibody gave immunologists a powerful and versatile research tool.

Production &Selection of hybridoma cells:

One common method requires the use of myeloma cells that are deficient (because of previously selected mutation) for one of the nucleotide salvage pathways, making them unable to grow in HAT medium (named for its three components -Hypoxanthine, Aminopterin, and Thymidine). If the mixture of hybridomas and unfused parental cells is placed in this medium, the parental myeloma cells can not survive. The B-cell X myeloma cells can survive because the B-cell contributes the missing enzyme for the salvage pathway. Although unfused B-cells are able to survive in HAT medium, these cells do not live for extended periods in vitro and thus die out.

HAT selection depends on the fact that mammalian cells can synthesize nucleotides by two different pathways; the denovo and the salvage pathways. The denovo path way, in which a methyl or formyl group is transferred from an activated form of tetrahydrofolate, is blocked by Aminopterin, a folic acid analogue. When the denovo path way is blocked, cells utilize the salvage pathway, which bypasses the aminopterin block by converting purines and pyrimidines directly intoYiucleotides for synthesis of DNA and RNA. The enzymes catalyzing the salvage pathway include Hypoxanthine-Guanine Phosphoribosyl transferase(HGPRT) and Thymidine kinase(TK). A mutation in either of these two enzymes blocks the ability of the cell to use the salvage pathway. HAT medium contains aminopterin to block the denovo pathway and hypoxanthine and thymidine to allow growth by the salvage pathway. Therefore, cells that lack either HGPRT or TK will die in HAT medium, because they lake the ability to use the salvage pathway to acquire essential intermediates for the synthesis of nucleic acids.

In hybridoma technology, the myeloma cells are actually double mutants. As mentioned above, they lack the enzyme HGPRTase and therefore are deselected in HAT. They have lost the ability to produce immunoglobulins (Ig- mutants).

Applications of Monoclonal Antibodies:

1. Improved diagnostic reagents: For the assay of wide range of compounds, including hormones, antibiotic interferons, and blood-clotting factors, antibody-antigen reactions are widely used. For blood typing and diagnostic microbiology, the ability of antibodies to agglutinate or precipitate cells have been used; the homogeneity of Monoclonal antibodies reduces reaction times and the likelihood of non-specific cross-reactions between the antibody and antigen is lessened by the use of Monoclonal antibodies. It is possible to diagnose pregnancy or ovulation more precisely with the Monoclonal antibodies.

2.Protein purification: Monoclonal antibody affinity columns are prepared by coupling monoclonal antibodies to a cyanogen bromide-activated chromatography matrix, for example, sepharose. Monoclonal antibodies immomabalized in this way are particularly valuable for the purification of proteins. Since the monoclonal antibodies has a unique specificity for the desired proteins, the level of contamination by unwanted proteins, usually, is very low. Even when the concentrating of the desired protein, in a mixture of proteins, is very low, Monoclonal antibodies has the capacity to combine with it and to remove the whole of it. For example, when the concentration of interferon was less than 0.02%, the anti-interferon monoclonal antibodies enable the recovery of 97% of the interferon by immunoaffinity chromatography.

3.Improved sensitivity and reproducibility of existing immunoassays or new assays for

Histocompatibility antigens, Complement component, Human growth hormones, Interkeukins, Oestrogen, Blood clotting factors, Sperm antigens, Blood group antigens, Progesterone, Fibronectin, Interferons, Gastrin

4. Therapy:

• Correction of drug over dose
• Reduction of risks associated with bone marrow transplants
• Detection of tumour metastases
• Treatment of cancer (directly, or by targeting “cytotoxic drugs”.

5. Diagnosis of:

• Sexually Transmitted Diseases (STDs)
• Cancer(by detection of Onco -foetal antigens)