The full name of ELISA is called the Enzyme-Linked ImmunoSorbent Assay ( 5 ) . ELISA is used for naming legion different conditions. As Virtual Medical Centre stated that it is normally requested if it is suspected you have been exposed to viruses such as HIV and Hepatitis B or C, or bacteriums and parasitic infections such as Toxoplasmosis, Lyme disease and Helicobacter pylori ( 7 ) . Other utilizations of the ELISA include sensing of prohibited drugs such as cocaine and Methedrines ( 7 ) . ELISA is besides a really sensitive immunochemical assay and is normally the first trial performed on potentially Human Immunodeficiency Virus ( HIV ) -infected blood ( 4,8-9 ) . This trial involves an enzyme and antibody or antigen ( 5 ) . Consequences can be positive or negative.
2. How the trial is performed
The end of an ELISA is to happen out if a peculiar protein is present and how much does it incorporate in a sample ( 3 ) . There are a few ways for executing an ELISA. Here is one of the commonly used methods and the one important to observing HIV antibodies:
Enzyme-linked-immunosorbent serologic assaies are performed in 96-well home bases which allow high throughput consequences ( 2-3 ) . The underside of each well is coated with prepared HIV protein to which will adhere the antibody ( diagram 1 ) ( 2-3 ) .
hypertext transfer protocol: //www.biology.arizona.edu/immunology/activities/elisa/graphics/antigen.gif Partially purified, inactivated HIV antigens pre-coated onto an ELISA home base ( 2 ) .
Entire blood is allowed to coagulate and the cells are centrifuged out to obtain the clear serum with antibodies which is called the primary antibodies ( 2 ) . The serum is incubated in a well, and each well contains a different serum ( diagram 2 ) ( 2-3 ) .
The antibodies of the patient in the serum. If the patient is HIV positive, so this serum will incorporate antibodies to HIV, and those antibodies will adhere to the HIV antigens on the home base ( 2 ) .http: //www.biology.arizona.edu/immunology/activities/elisa/graphics/antibodies.gif
The serum is removed after a period of clip, and decrepit disciple antibodies are washed off with a series of buffer rinses ( 9 ) . A secondary antibody is added to each well to observe the edge antibodies ( plot 3 ) ( 2-3 ) .
Anti-human Ig coupled to an enzyme. This is the 2nd antibody, and it binds to human antibodies ( 2 ) .http: //www.biology.arizona.edu/immunology/activities/elisa/graphics/horse.gif
An enzyme such as peroxidase or alkalic phosphatise is attached to the secondary antibody ( 3 ) . These enzymes can metabolise colourless substrates called chromagens into coloring material merchandises ( plot 4 ) ( 2,9 ) .
Chromogen attached to the 2nd antibody ( 2 ) .http: //www.biology.arizona.edu/immunology/activities/elisa/graphics/chromagen.gif
After an incubation period, the secondary antibody solution is removed and decrepit disciple 1s are washed off as earlier ( 3 ) . The concluding measure is the adding up of the enzyme substrate and the production of color merchandise in Wellss with secondary antibodies bound ( 3,9 ) .
When the enzyme reaction is complete, the full home base is placed into a home base reader and the optical denseness is determined for each well ( 3 ) . The sum of coloring material produced is analogous to the sum of primary antibody edge to the proteins on the underside of the Wellss ( plot 5 ) ( 9 ) .
Positive ELISA Test Negative ELISA Test
3. Restrictions of this trial
A negative facet of the ELISA is the happening of false positive or false negative consequence. As the Virtual Medical Centre and Stowell stated an illustration such that the antibodies induced by a recent grippe injection can do a positive consequence and so although ELISA is an of import diagnostic trial for HIV, in assorted instances it is normally followed up by a collateral trial ( 7,9 ) . False positives may besides happen if you have an implicit in status such as Lupus or rheumatoid disease ( 7 ) . However, a negative consequence does non ever intend there is no infection as some antibodies are non produced at one time following infection ( 9 ) . The Virtual Medical Centre reported that the antibodies to HIV make non look in blood until 6 hebdomads after exposure to the virus so it is recommended that if the person suspect he or she has been exposed so the trial should be repeated after 6 hebdomads to 6 months ( 7 ) .
4. Other checks are routinely performed to observe HIV
There are a figure of trials besides ELISA can happen antibodies or familial stuff ( RNA ) to the HIV virus. These trials include the most common scrutiny used that is a Western smudge trial. A positive ELISA trial is ever followed by a Western smudge trial and a positive Western smudge confirms an HIV infection ( 6 ) . A negative Western smudge trial means the ELISA trial was a false positive trial ( 4,6 ) . However, negative trials do non govern out HIV infection ( 1,4 ) . There is a period called the window period-the clip between HIV infection and the visual aspect of anti-HIV antibodies that can be measured, therefore a negative HIV ELISA and Western smudge will non govern out HIV infection ( 6,8 ) . In add-on, the Polymerase Chain Reaction ( PCR ) finds either the RNA of the HIV virus or the HIV’s DNA in white blood cells infected with the virus ( 8 ) . The PCR trial is really utile to happen a really recent infection, find if an HIV infection is present when antibody trial consequences were unsure, and screen blood or variety meats for HIV before contribution ( 8 ) . However, PCR trial is non done every bit on a regular basis as antibody assay because it requires proficient accomplishment and expensive equipment ( 4,8 ) . In add-on, the Indirect Fluorescent Antibody ( IFA ) trial besides detects HIV antibodies ( 8 ) . It is used to corroborate the consequences of an ELISA merely like a Western smudge trial. But it is more expensive than a Western smudge trial and non often used ( 8 ) .