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EXPERIMENT NO. - ELISA TEST AIM :ELISA Test CLINICAL BACKGROUND HIV is recognized as the etiologic agent of the acquired immune deficiency syndrome (AIDS). At present atleast two different syndrome (AIDS) viruses, HIV-1 and HIV-2 has been identified (1,2). HIV is disseminated world wide. Epidemiological studies reveal that HIV-2 is principally found in West African regions and has subsequently been detected in several European and South American countries. Recently divergent HIV-1 viruses have been identified which cannot be classified as existing HIV-1 subtypes. These viruses are designated as HIV-1 group 0(4,7). The first isolates have been reported in West Africa in 1987 & recently also in Europe and in the US. HIV-2 is related to HIV-1 in terms of morphology, cell tropism, ability to induce AIDS or AIDS related complex, & also overall genetic organization. Genomic analysis reveal that HIV-2 differs significantly from HIV-1 in terms of nucleotide sequences. Less than 60% amino acid identity is shared by the 'gag' and 'pot' genes and less than 37% amino identity is exhibited by the "env" genes. MATERIALS REQUIRED :Distilled water, sulphuric acid, precision pipettes (of 110L, 20-200 L), a multichannel pipette, micro-plate shaker, water bath, microplate washer, absorbent tissues, microplate reader. REAGENTS :HIV coated test well, negative control (human serum containing 0.1% sodium azide as preservative), positive control (normal human serum supplemented with antibodies to HIV inactivated by detergent treatment, and containing 0.1% sodium azide as preservative), a sample diluent, conjugate diluent, conjugate, TMB substrate solution, substrate buffer, wash solution, 4 adhesive plate scalers, 1 plastic minigrip bag. TEST PRINCIPLE :The wells of polystyrene microplate strips have been coated with synthetic peptides representing envelope HIV-1, envelope HIV-2 and envelope HIV-1 group O antigens. These coated proteins and peptides constitute the solid phase antigens. The test sample is incubated in such a well - viral specific antibodies in the sample, will bind to the solid phase antigens. Subsequently, an affinity purified rabbit antihuman Ig G (H chain specific) labeled with the enzyme horse raddish peroxidase (HRP) is added. Upon a positive reaction this labeled antibody becomes bound to any solid phase antigen/ antibody complex previously formed. Incubation with enzyme substrate produces a blue colour in the test well, which turns into yellow when the reaction is stopped with H2SO4. If the samples contain no HIV-1 or HIV-2 antibodies, then the labeled antibody cannot be bound specifically and only a low background colour develops. PROCEDURE :Before starting the assay adjust the temp. of the water bath to 37oC 1. Take the strip holder with the required number of strips, taking in account that for one strip, one negative and one positive control should be included. For more strips, at two negative and two positive controls should be included in each strip holder. During the test run, strips stay in the strip holder and can be marked an one edge. In case of automatic sample and regent addition. 2. Add 200 ml of diluent to each test well. 3. Add 10L of specimen or control to each appropriate test well. Depending on the type of automatic distributor, make sure specimen and controls are adequately mixed with the diluent. Continue the test procedure as prescribed from point S onward. In case of manual sample & regent addition. 2. Add 100L of diluent to each well 3. Add 10L of sample or control to each appropriate test well. Make sure specimen and control are adequately & completely mixed with the diluent or in microplate shaker for 1 minute at +/- 1000 rpm or manually by pipetting several times up & down. 4. Add 100L of diluent to each test well. 5. Cover the strips with and adhesive sealer. Incubate for 30 minutes at 37oC. 6. Wash each well 5 times. After the last aspiration the washing procedure is completely by inverting the plate and tapping it dry or absorbent tissue. 7. Add 200L prepared conjugate solution to each well; shake the strip holder carefully to mix. 8. Cover the strips with a new adhesive sealer. Incubate for 30 minutes at 37oC. 9. Wash each well 5 times. After the last aspiration the washing procedure is completed by inverting the plate and tapping it dry on absorbent tissue. 10. Add 200L prepared substrate solution to each well tap the strip holder carefully to mix. 11. Incubate for 30 minutes at 20-250 in dark. 12. To stop the reaction, add 50L sulphuric acid to each well in the same sequence & at the same time intervals as the substrate solution. Tap the strip holder carefully to ensure thorough mixing. 13. Blank the reader without strip holder & strips inside. Read (within 15 minutes after step 11) the absorbance of the solution in the wells at 450nm. PRECATIONS :1. All vessel used to prepare conjugate & substrate solution must be cleaned thoroughly & finally rinsed with distilled water. 2. Do not touch the top of the vessel or plates with your fingers to avoid contamination. 3. Make sure no air bubbles are present in wells. 4. Do not expose substrate solution to strong light during incubation. 5. Use a new pipette tip for each specimen allocated. Reference materials: ELISA (Enzyme-Linked Immunosorbent Assay) Introduction Enzyme-Linked Immunosorbent Assay (ELISA) is a useful and powerful method in estimating ng/ml to pg/ml ordered materials in the solution, such as serum, urine and culture supernatant. It's a kind of easy task to make ELISA if you have "good" antibodies against your concerned materials such as proteins, peptides and drugs. First, I will show the basic method in establishing ELISA using a "good" antibody. I will add some trouble shootings in the section. Second, I will show some improving methods for making ELISA, which is especially useful when using a "not so good antibody". Of course, some trouble shootings I will illustrate. Third, we will take a look at some remarks in making ELISA for the estimating serum/plasma materials, which is a little bit of different story from making ELISA for simple buffers. I hope this page will help you establishing a "good ELISA" to your concerning materials. Making ELISA using good antibodies Definition If you have "good" antibodies against your concerned materials, it is not so difficult to establish ELISA. It's a just around three-days working. However, you can't tell if your antibodies are good or not until accomplishment of an ELISA in most cases. If you use polyclonals, you can easily examine their ability. That is the "Ouchterlony" method. This old-fashioned double immunodiffusion method is really reliable and reproducible test in making ELISA. Put the antigen into the center well and put the serially diluted antisera into the outer wells. If you can see evident white band between the inner and outer wells at x 64 or more dilution point after the incubation 37 degree overnight, you are very lucky! You can use the antibody without any further treatment other than purification to Ig fraction. If you can see bands between x 2 and x 32 point, you should probably go to the second session for further treatment of your antibody. Note that these dilution points are a kind of arbitrary. It's depend on the antigen. So, I will say just try. If you cannot establish good ELISA by the first method, you can try the second. Making ELISA using not so good antibodies Definition It's a very common situation that you only have not so good antibody to your concerned material. It's necessary to reinforce your antibody in this case when using for ELISA. The best way to make your antibody a good one is the affinity purification using solid phased antigen, i.e., affinity column purification. First, I will show you how to make antigen-column using CNBr activated Sepharose 4B gel (Pharmacia), then I will show you how to use it effectively. Making ELISA for serum/plasma materials Definition When using ELISAs for the estimation of serum or plasma materials, many cares should be taken. Nevertheless, you will get misleading results. Never omit this step if you want to apply your ELISA to the clinical. Yes, it's really important!
