ELISA : Theenzyme-linked immunosorbent assay (ELISA) is an important tool in for detecting the presence of an antibody or an antigen in a sample. The ELISA's key features are its high level of sensitivity and robustness. It allows easy visualization of results and can be completed without using any radioactive materials because of which it remains a desired assay of choice for routine scientific,medical and veterinary diagnostic assays.
Types of ELISA : There are two basic types known of ELISA:
1)Indirect ELISA : This method is used to determine the presence of a specific antibody in a given specimen. The steps of the general, indirect, ELISA for determining serum antibody concentrations are: 1)The sample containing a known antigen of defined concentration is added to the well of a microtiter plate where it adsorbs to the wells of the plate through charge interactions. 2) A concentrated solution of non-interacting proteins, such as Bovine Serum Albumin (BSA) or casein, is added to the plate wells to block the non-specific adsorption . 3) Test-antiserum is added to these walls and allowed to incubate; if the antibodies in test antiserum are homologous, they bind with adsorbed antigens forming antigen-antibody complex. The wells are again washed to remove any free antibodies. 4) Enzyme-conjugated (labelled) antibodies are now added which link to the antigen-antibody-complex formed in the previous step. 5) The plate is washed again to remove excess unbound enzyme-antibody conjugates. 6) A chromogenic substrate is applied which converts the enzyme to elicit a signal. 8) The result is viewed/quantified using a spectrophotometer.
2) Direct ELISA : This method employs monoclonal antibodies for detection of a particular antigen in a sample.In this approach, the steps involved are as follows : 1) An unlabeled antibody specific to the antigen of interest is immobilized in microtiter well. 2) The antigen is added and allowed to react with the immobilized antibody to form Ag-Ab complex. 3) An Enzyme conjugate is added (that is specific to the antigen) which forms a double antibody sandwhich 4) The unreacted antibodies are washed away. Thereby, enzyme subsrate is added to elicit a chromogenic or fluorogenic signal.
5) Results are quantified using a spectrophotometer, spectrofluorometer, or other optical device. This procedure is also referred to as, Double Antibody Sandwitch (DAS) ELISA.
Advantages and disadvantages of both Direct and Indirect ELISA :
A) Advantages of using direct ELISA : It is relatively quick as only one antibody is used. Hence, fewer steps are involved. Also, cross-reactivity of secondary antibody is eliminated.
B) Disadvantages of using direct ELISA : By labeling the primary antibody with enzymes, its Immunoreactivity might be affected. Also the labeling for every specific ELISA is time-consuming and a costly affair. Moreover, this method gives minimum signal amplification compared to indirect method.
C) Advantages of using indirect ELISA : Plenty of commercially available labelled secondary antibodies are available. This method generates a stronger signal amplification. Also, many different visualization markers can be used with the same primary antibody.
D) Disadvantages of using indirect ELISA: There are chances of cross-reactivity with the secondary antibody which may give non-specific signals. Also, steps are increased in this method. Few other variants of ELISA are also available and these are also often used. These are :
Direct Antigen coating (DAC) ELISA :
1) The antigen of interest is immobilized on the surface of a well. Now, antibody specific to the antigen is added and allowed to react with the adsorbed antigen. Unreacted molecules of the antibody are washed away leaving only the Ag-Ab complex. This constitutes the primary reaction. 2) In the secondary reaction, an anti-immunoglobulin (anti-Ig; an antibody that reacts with all the antibodies belonging to the same class) is added into the same vessel and allowed to react with the Ag-Ab complex which has been previously formed. 3) Now the anti-Ig will bind to the antibody component of the Ag-Ab complex. This anti-Ig is conjugated to an enzyme in such a way that its activity is not impaired. The enzymes usually used in this case is alkaline phosphatase. 4) The unreacted anti-Ig is washed away and finally the substrate of the enzyme is added along with the necessary reagents to develop color due to the enzyme activity. The intensity of the color developed is proportional to the enzyme concentration. Therefore, color intensity is used to determine the amount of antigen or antibody present. This method of ELISA is often referred to as Direct antigen coating (DAC) ELISA. This method is chiefly useful in estimating the amount of specific antibody such as HIV, present in the antiserum used for the primary reaction. This is the simplest procedure and can be completed within three procedure and can be completed within three hours.
Sandwich ELISA : This method measures the amount of antigen present between two layers of antibodies, hence called sandwich ELISA. The antigens to be measured must contain at least two antigenic sites that they can bind to the antibody, and at least two antibodies act in the sandwich. This type of ELISA is chiefly used in quantitation of proteins and polysaccharides. The steps involved in sandwich ELISA are described below : 1) Two antibodies that can bind to two different epitopes on the same antigen are required. 2) One antibody is first immobilized on a microtitre well and is referred to as capture antibody. The other antibody is labelled with a suitable enzyme [eg. (HRP), alkaline phosphatase (ALP) etc.] and is referred to as labelled antibody. 3) Nonspecific binding sites are then blocked on the surface. 3) Then a sample (standard and test) containing the antigen is allowed to react with the immobilized antibody in the well. 4) After that the well is washed, and again enzyme linked antibody is added and allowed to react with the bound antigen. 5) Unreacted labelled antibody is now washed out and the enzyme bound to solid support is estimated by adding a chromogenic substrate. 6) The color developed is measured spectrophotometrically, which is directly proportional to the antigen concentration.
Competitive ELISA :
1. Unlabeled antibody is incubated in the presence of its specific antigen. 2. These bound antibody/antigen complexes are then added to an antigen coated well. 3. The plate is washed, so that unbound antibody is removed. The more antigen is present in the sample, the less the antibody will be able to bind with the antigen in the well, hence it leads to Competition 4. The secondary antibody specific to the primary antibody is added. Then this secondary antibody is coupled to the enzyme. 5. A substrate is added and then the remaining enzymes elicit a chromogenic signal. For competitive ELISA, the higher the original antigen concentration, weaker will be the eventual signal .
Reverse ELISA : This variant of ELISA has been recently developed for investigating the West Nile virus envelope protein and how it is able to detect virus-specific antibodies. In this method an antibody is sandwiched by antigens. The advantage of Reverse ELISA over other ELISA techniques is that it has : (i) higher specificity and sensitivity and (ii) the ability to comparably titrate antibodies from different species. This newer technique uses a solid phase made up of an immunosorbent polystyrene rod with ogives. This entire device is immersed in a test tube containing the collected sample followed by incubation in the presence of a target followed by washing. A second incubation step is then carried out in the presence of a secondary conjugated antibody followed by second cycle of washing and then subsequent color development by addition of chromogenic substrate. Based on the platform of ELISA, this method uses an antibody microarray to capture native antigens. Autoantibody reactivity is then evaluated by differentially labeling patient IgG and incubating these antibodies with the native antigens that are immobilized on the antibody microarray.The essential steps involved in this procedure are discussed below: 1. Preparation: Purify and separate autoantibodies (IgG) from serum. Label them then with either radioactive tagging or a fluorescent dye. Ogives should be carefully coated with solution and an then a substrate indicator should be added later on to measure intensity of monoclonal antibodies present. 2. Extraction: Now, label autoantibodies with native protein on array. Native antigens bound to antibodies on array are extracted from microarray with unique monoclonal antibodies. Array should be incubated with native protein extracts such as cells, tissue, or body fluid, etc. 3. Analysis : Based on the intensity of the dye that appears in assay, we can analyze the concentration. When the process is complete, the assay is then evaluated by its intensity of color which is determined for each well. The amount of color produced co-relates to the amount of primary antibody bound to the proteins on the bottom of the microwells.
Advantages of ELISA:
1) ELISA tests are relatively accurate tests, is easy to perform and gives fast results. 2) It is considered highly sensitive and specific assay compared to radioimmuno assay (RIA) tests. 3) It doesnt involve the use of hazardous radioisotopes. 4) It doesnt need costly instrumentation. 5) Antigens of very low or unknown concentration can be detected since capture antibody only grabs specific antigen.
Disadvantages of ELISA : 1) One of the major disadvantage is that ELISA is capable of detecting only one antigen at a time, which becomes a limiting factor. 2) Results are dependent on the skills of the technician and the quality of the ELISA kit. 3) Enzyme/substrate reaction is short term so microwells must be read as soon as possible 4) Negative controls may indicate positive results if blocking solution is ineffective as secondary antibody or antigen (unknown sample) can bind to open sites in well. 5) It being an enzymatic reaction, even small quantities of non-specific binding of primary or secondary antibody might result in false signal. 6) As only monoclonal antibodies can be used as matched pairs which recognize one specific binding site i.e. epitope, this procedure can be expensive as monoclonal bodies are costlier than polyclonal bodies and are also difficult to find.
Applications of ELISA : The ELISA procedure has a broad range of applications from disease diagnosis like infectious diseases, autoimmune disease, certain cancers and Alzheimers. ELISA is also useful in detecting potentially hazardous allergens present in food. ELISA can also facilitate drug discovery by identifying drug targets, evaluating disease progression and allows rapid presumptive screening for certain classes of drugs.
1) ELISA has been used successfully in screening and diagnosis of Chagas and human hydatid disease. 2) ELISA has also been able to determine Ig-E based food allergies. 4) Brucella abortus- a highly contagious zoonosis caused by ingestion of unsterilized milk or meat from infected animals or close contact with their secretions, has been diagnosed effectively using ELISA. 5) ELISA has been also used in diagnosis of rotavirus. 6) ELISA has been used as a rapid assay in comparison to other assays in diagnosis of HIV.
7) ELISA has been used in detecting anti-malarial drug sensitivity affectively in case ofPlasmodium falciparum aldolase.
8) Autoimmune disorders have also been effectively diagnosed using ELISA. E.g. antibodies to F-actin (a protein forming the thin filaments in muscle fibres) have been proposed to increase specificity in the diagnosis of autoimmune hepatitis using ELISA. 9) ELISA enables the measurement of LDL receptors successfully.
10) Disorders related with celiac disease have also been successfully diagnosed using ELISA.