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Elisa and VDRL test

The document discusses ELISA (enzyme-linked immunosorbent assay), a blood test used to detect antibodies. There are several types of ELISA that detect antibodies through an antigen-antibody reaction, with detection via an enzyme conjugate. ELISA can detect antibodies related to HIV, Lyme disease, syphilis, and other infectious diseases. The test involves drawing blood, linking an antigen to an enzyme, and detecting if the sample causes a color change indicating the presence of antibodies. The document also discusses the VDRL test, a non-treponemal test for syphilis detection that detects antibodies reaction to cardiolipin antigen.

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ELISA
ENZYME-LINKED IMMUNOSORBENT ASSAY • An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that detects and measures antibodies in blood. This test can be used to determine antibodies related to certain infectious conditions. Antibodies are proteins that body produces in response to harmful substances called antigens.
Types • Direct ELISA • Sandwich ELISA • Competitive ELISA • Reverse ELISA
Uses • HIV, which causes AIDS • Lyme disease • Pernicious anemia • Rocky mountain spotted fever • Rotavirus • Squamous cell carcinoma • Syphilis • Toxoplasmosis • Varicella-zoster virus, which causes chickenpox and shingles • Zika virus
• A blood sample is needed. Most of the time, blood is drawn from a vein located on the inside of the elbow or the back of the hand. • The sample is sent to a laboratory where the targeted antibody or antigen is linked to a specific enzyme. If the target substance is in the sample, the test solution turns a different color.
• Principle of ELISA: Based on Basic Immunology Response Lock and Key Concept: 1) Antigen (key) 2) Antibody (lock): –Key fits into the lock Enzyme conjugate substrates • Bound to a secondary antibody that binds with the antibody- antigen complex.
Equipment's • Microwell Plate: Flat bottom polystyrene plate, contains 8 x 12 wells holding 350 μL each. • Multipipette: An 8-channel 100 μL pipette is a good help for even small-scale work. • Washing Device: – Manually operated washing devices. – May be of use particularly when there is a risk that the samples tested in elisa contain infectious material, so must be collected for subsequent disinfection. • Microplate washer: – These are very efficient with unusually low carry-over contamination.
Reagents Used • Reagent Composition Coating Buffer 0.01 M Phosphate Buffer + 0.15 M NaCl (PBS) Diluting/Washing Buffer 0.01 M Phosphate Buffer + 0.50 M NaCl + 0.1% Tween 20 Blocking Buffer Bovine Serum Albumin (BSA) Enzyme Horse-redish peroxidase (HRPO) Chromogenic Substrate Trimethyl benzidine (TMB) Stop Solution 0.5 M H₂SO₄
VDRL TEST
• Venereal Disease Research Laboratory (VDRL) Test is a slide flocculation test employed in the diagnosis of syphilis. • Since the antigen used in this test is cardiolipin, which is a lipoidal extracted from beef heart, it is not a specific test. • This test is also classified as non-specific or non-treponemal or standard test. • The antibodies reacting with cardiolipin antibodies have been traditionally termed “regain”.
Principle • Patients suffering from syphilis produce antibodies that react with cardiolipin antigen in a slide flocculation test, which are read using a microscope. It is not known if the antibodies that react with cardiolipin are produced against some lipid component of Treponema pallidum or as a result of tissue injury following infection.
Requirements • Patient’s serum, water bath, freshly prepared cardiolipin antigen, VDRL slide, mechanical rotator, pipettes, hypodermic syringe with unbeveled needle and microscope. Known reactive and non- reactive serum controls are also required.
VDRL antigen • The cardiolipin antigen is an alcoholic solution composed of 0.03% cardiolipin, 0.21% lecithin and 0.9% cholesterol. The cardiolipin antigen must be freshly constituted each day of test. The working antigen is a buffered saline suspension of cardiolipin.
VDRL slide • This is a glass slide measuring 2 X 3 inch with 12 concave depressions, each measuring 16 mm in diameter and 1.75 mm deep.
Procedure • Patients’ serum is inactivated by heating at 56o C for 30 minutes in a water bath to remove non-specific inhibitors (such as complement). The test can be performed both qualitatively and quantitatively. Those tests that are reactive by qualitative test are subjected to quantitative test to determine the antibody titres.
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