Enzyme-Linked Immunosorbent Assay - Essay Example

?The ELISA (enzyme-linked immunosorbent assay) is a biochemical method of determining antibodies and antigens present in a specimen. A specific antibody is introduced over a sample of an antigen so that binding may occur. The antibody is then linked to an enzyme. The plate is then washed. This step is extremely important because the immunuoglobulin is in excess, and therefore the color enzyme will distribute incorrectly. Finally, some type of detection method is used; usually a colour changing substance is added. This is the basic direct ELISA test, and it is usually utilized in medical testing and plant testing. It is an efficient and usually an extremely effective way to test for certain antibodies. The indirect ELISA approach is typically applied in medical testing because it looks for antibodies present in a given sample. Samples are taken from an individual and then these are tested for the presence of antibodies. This helps to determine if the individual’s body is already producing antibodies against certain antigens. After the sample is taken, antigens are introduced. If antibodies are present, they will form a bond that is invisible to the naked eye. An immunuoglobulin is added to the sample in order to verify if a bond was formed or not. The bond between the antibody, antigen and immunuoglobulin will remain on the plate after rinsing. The immunuoglobulin added must be of the same species. Finally, a colour indicator is added so that the bond between the antibody and antigen are identified properly. The accuracy of an ELISA test does rely on certain factors. If these factors are in any way compromised or overlooked, the ELISA test results may be unreliable. These factors may also contribute to a false positive or a false negative. The design of an ELISA test may sometimes affect its outcome, and an ELISA test is either direct or indirect. When using the single antibody approach, the laboratory must used a specific, enzyme-labelled reagent. The assay sensitivity affects both the direct and indirect ELISA tests because it measures the amount of accurate positives that are found. There are four types of sensitivities possible in an ELISA test: true positives, false positives, true negatives and false negatives. Whether or not a patient receives a false positive or a false negative is dependent upon how accurately the ELISA test was conducted, and whether a variable important to the outcome were compromised in some way. A false positive indicates that a person who does not in fact carry the antibodies against a specific type of antigen is tested as positive for those antibodies. Similarly, a false negative indicates that a person who does in fact carry those antibodies against a specific type of antigen, tests negative for them. This may or may not occur with an HIV-1 test if the standard protocol for the ELISA test is correctly or incorrectly followed. Unquestionably, it is of the utmost importance to follow standard ELISA test protocol. ELISA test errors may occur when the reagents used are mishandled or if their integrity is comprised in some way. The reagents for specific antigen tests are usually specific- they may not be interchangeable between different species. Additionally, certain antibodies require certain reagents in order to be effectively read; substitutions cannot be made. Temperature, preparation, preservation, and shelf-life of the reagent affects how its performance. Commercial availability limits the use of some reagents, and therefore the potential for testing for specific antigens. The handling of samples and the reagents used are extremely important to the outcome of the test. Checking expiration dates and properly storing reagents according to their individual requirements is imperative. Reagents must also be checked for signs of disintegration prior to their usage in an assay; examples include precipitation and discoloration. The substrates should always be colourless. The plates where the experiment is conducted must be properly handled. The plates must be large enough to accommodate the ELISA test reaction. Some tests require overnight incubation, and enough space for the reaction to occur. Also, the placement of the wells on the plate can affect the readings. Erratic or incorrect readings are possible if the reaction was conducted in one of the wells with a disadvantageous position. The material from which they are composed must still have retained its original integrity in order for a proper reaction to occur. For example, an older plate that may be slightly damaged (though damage is minimal) may not provide a perfect testing environment. It should be replaced. Also, individual trials should be absolute repetitions of one another, and if the same plates cannot be used, they should be identical. ELISA testing results are, in some cases, reliant upon how the experiment was conducted. It is important protocol be followed, and that there is an extreme attention to detail. Testing for HIV-1 is done primarily with the competitive ELISA method.. The competitive is very effective in determining if a patient is HIV-1 positive or not. Occupational transmission is possible with the HIV-1 virus, and procedure errors (for fear of contraction or transmission) may impede the protocol that should be followed. Consequently, studies have been done to show how heat transmission affects HIV-1 affectivity. The competitive ELISA is different from the indirect in that HIV-1 antigens are placed on a plate. The patient’s serum is then added to the plate. Anti-immunuoglobulin enzyme is added and it binds to the patient’s antibodies; it acts as “competition” to the patient’s antibodies. Next, a colour changing substrate is added so the results can be clearly detected. The competitive ELISA is advantageous for testing HIV-1 because of what may be considered a ‘lenient’ reagent need. Often, crude or impure samples are testable. Testing kits require that the patient’s serum, and therefore, their antibodies to be placed in the dish, rather than for the antigen. The personal and social reliance on a competitive ELISA for HIV is of the utmost importance. False positives and false negatives are extremely detrimental to a patient. A patient may receive a false positive if their serum contains antibodies against human leukocyte antigens (HLA). This is more common in women who have had multiple pregnancies. The HIV incorporates the HLA into itself during incubation. The test is designed to determine only for HIV antigens, and it will not be clear if it was the HLA that disrupted the results. Should this occur, a patient should seek additional health counseling. They should discuss their results and further testing with a health practitioner. False negatives are also possible with the competitive ELISA. Seroconversion occurs prior to the window of infection- when the patient’s immune system has not begun producing antibodies against the antigen yet. It is often recommend that a patient wait until the necessary amount of time, though hard as it may be, in order to avoid producing a false negative. Testing for HIV-1 is must easier, private, and most importantly, efficient. The three types of ELISA tests described are vital in testing patients for the presence of antibodies against antigens. The competitive ELISA is an essential part of HIV testing. If proper protocol is followed, the ELISA test produces reliable results. It is important to monitor the entire testing procedure so that usable, correct testing results are found. False positives and false negatives may occur due to various reasons. However, working attentively, repeating trials and ruling out extraneous variables (such as seroconversion) will produce accurate results. Read More