Elisa test

Elisa test

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  1 Amjad Khan Afridi7t h August, 2016 ELISA ( Enzyme Linked Immunosorbent Assay) Introduction ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins. It is this binding and immobilization of reagents that makes ELISAs so easy to design and perform. Having the reactants of the ELISA immobilized to the microplate surface makes it easy to separate bound from nonbound material during the assay. This ability to wash away nonspecifically bound materials makes the ELISA a powerful tool for measuring specific analytes within a crude preparation. A detection enzyme or other tag can be linked directly to the primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It also can be linked to a protein such as streptavidin if the primary antibody is biotin labeled. The most commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes have been used as well, but they have not gained widespread acceptance because of limited substrate options. These include β-galactosidase, acetylcholinesterase and catalase. A large selection of substrates is available for performing the ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal-detection (spectrophotometer, fluorometer or luminometer). ELISA Formats ELISAs can be performed with a number of modifications to the basic procedure. The key step, immobilization of the antigen of interest, can be accomplished by direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. The antigen is then detected either directly (labeled primary antibody) or indirectly (labeled secondary antibody). The most powerful ELISA assay format is the sandwich assay. This type of capture assay is called a “sandwich” assay because the analyte to be measured is bound between two primary antibodies – the capture antibody and the detection antibody. The sandwich format is used because it is sensitive and robust.
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  2 Amjad Khan Afridi7t h August, 2016 ComparisonofDirect and Indirect ELISADetection Methods Direct ELISA Detection Advantages  Quick because only one antibody and fewer steps are used.  Cross-reactivity of secondary antibody is eliminated. Disadvantages  Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags.  Labeling primary antibodies for each specific ELISA system is time- consuming and expensive.  No flexibility in choice of primary antibody label from one experiment to another.  Minimal signal amplification. Indirect ELISA Detection Advantages  A wide variety of labeled secondary antibodies are available commercially.  Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.  Maximum immunoreactivity of the primary antibody is retained because it is not labeled.  Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification.  Different visualization markers can be used with the same primary antibody. Disadvantages  Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signal.  An extra incubation step is required in the procedure. Fluorescent tags and other alternatives to enzyme-based detection can be used for plate-based assays. Despite not involving reporter-enzymes, these methods are also generally referred to as a type of ELISA. Likewise, wherever detectable probes and specific protein binding interactions can be used in a plate- based method, these assays are often called ELISAs despite not involving antibodies.
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  3 Amjad Khan Afridi7t h August, 2016 Direct vs. Indirect Detection ELISA Strategies Among the standard assay formats discussed and illustrated above, where differences in both capture and detection were the concern, it is important to differentiate between the particular strategies that exist specifically for the detection step. However an antigen is captured to the plate (by direct adsorption to the surface or through a pre-coated "capture" antibody, as in a sandwich ELISA), it is the detection step (as either direct or indirect detection) that largely determines the sensitivity of an ELISA. Diagram of common ELISA formats (direct vs. sandwich assays) Common ELISA formats. In the assay, the antigen of interest is immobilized by direct adsorption to the assay plate or by firs t attaching a capture antibody to the plate surface. Detection of the antigen can then be performed using an enzyme- conjugated primary antibody (direct detection) or a matched set of unlabeled primary and conjugated secondary antibodies (indirect detection) Sandwich Assays A. Sandwich Assays having wells, in which each wells having coated An Antibodies and an Antigen. B. In this method we use 4 wells for a single test. i. Positive Control ii. Sample well iii. Negative Control iv. Blank C. We use specific solutions for each specific Wells. D. After the end of the process we absorb and measure the wells by Plate Reader or Strip Reader, that either test is positive or negative.
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  4 Amjad Khan Afridi7t h August, 2016 Requirements: 1. ELISA Testwell 2. ELISA Plates 3. Test tubes 4. Syringe 5. swab 6. centrifuge 7. Specimen dilution 8. Positive control 9. Sample ( Blood Sample ) 10.Negative control 11.RHP-Conjugate 12.Buffer solution 13.Distilled water 14.Chromogen “A” 15.Chromogen “B” 16.Stop solution 17.Micro pipettes and Tips 18.Container 19.Aluminum foil ( Cover) 20.Incubator 21.Plate reader or strip reader 22.Dropper 23.Permanent Marker 24. Tissuepaper 25. Gloves 26. Mask Sandwich Assays Procedure 1. Bring all the reagents to a roomtemperature . 2. Labeled the wells with specific ID number.
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  5 Amjad Khan Afridi7t h August, 2016 3. Pour 20 µL specimen dilution in each wells except Blank well. 4. Add 100 µL sample in sample well ( S ) . 5. Add 100 µL PositiveControl solution in positive well ( P), while add 100 µL Negative Controlsolution in negative Well ( N). 6. Covered and Labeled each wells and mix it carefully. 7. Next , replaced all the wells in the incubator and incubated the wells at 37 C for 60 minutes to react each an Antibodies with their specific an Antigen . 8. After incubation apply HRP- Conjugate to each wells except Blank ( B) well. 9. Again covered and mix the wells and incubated for 30 minutes at 37 C. 10. After incubation washed each wells five times with Buffer Solution to removed unbounded an antigens and other particles. Each washing steps should be 30 seconds. 11.After washing apply 50 µL Chromogen“A” and 50 µL Chromogen“B” in each wells, covered the well and incubated for 30 minutes at 37 C. 12.After incubation add stop solution in each wells to stopped the Chromogen“A” and Chromogen “B” reactions. 13.Next, Measureand absorb the wells results by Plate Reader or Strips Reader, either resultNegative or positive. 14.Result : Comparethe colors of the wells with Negative Control (N). If the colors of all the wells are similar to the Negative control well (N) the result would be known as Hbs Positive, if the colors are not similar to Negative Control well (N) , the result would be consider as Hbs Negative (N). 15.Note : the color change can not always be detected by naked eyes. Demonstrated and particle by Hakeem Zada