Enzyme-linked Immunosorbent Assay

ELISA ELISA (Enzyme-linked immunosorbent assay ) is a serological test commonly used in various immunology laboratories. ELISA was introduced in 1971 by Peter Perlmann and Eva Engvall to analyze the interaction of antigens with antibodies in a sample by using an enzyme as a reporter (label reporter).ELISA is an immunoassay method that uses enzymes as labels. The ELISA method is divided into 2 types of techniques, namely competitive and noncompetitive techniques. This non-competitive technique is divided into two, namely sandwich and indirect. Hormone examination uses competitive techniques and sandwich. The competitive method has the principle that labeled and unlabeled antigens are added to the sample and competition occurs to form a limited complex with specific antibodies in the solid phase. Basic principles of sandwich assayis a sample containing antigen reacted with the first specific antibody bound to the solid phase. Next, a second specific antibody labeled with the enzyme is added and the enzyme substrate is added. The advantages of the ELISA method are: o Quite sensitive o Reagents are relatively cheap and can be stored for long periods of time o Can check several parameters at once o Equipment is easy to obtain o Does not use radiation substances Disadvantages of the ELISA method: o Examination using enzymes as labels is quite complex because enzyme activity is influenced by various factors Enzyme-Linked Immunosorbent Assay (ELISA) is a biochemical technique that is mainly used in the field of immunology to detect the presence of antibodies or antigens in a sample. In simple terms, a number of unknown antigens are attached to a surface, then specific antibodies are washed on the surface, so that they bind to the antigen. These antibodies bind to a enzyme, and in the final stage, substances are added that the enzyme can convert into a signal that can be detected. In fluorescence ELISA, when light of a certain wavelength is shone on a sample, the antigen/antibody complex will fluoresce so that the amount of antigen in the sample can be concluded based on the amount of fluorescence using a spectrophotometer. Generally, ELISA is divided into two types, namely competitive assay which uses antigen conjugates- enzymes or antibody–enzyme conjugates, and non-competitive assays that use two antibodies. In the ELISA non-competitive assay, the second antibody will be conjugated with an enzyme as an indicator. This second technique is often referred to as the "Sandwich" ELISA. This test has several disadvantages, one of which is the high possibility of false positive results due to cross-reaction between one antigen and another antigen. False negative results can occur if this test is carried out during the window period, namely when the formation of antibodies against a virus has just begun so that the number of antibodies is still small and may not be detected. There are several methods for elisa, including: 1. Direct elisa 2. Indirect Elisa 3. Elisa sandwiches 4. Competitive Elisa In elisa, the previously detected material must be attached to the solid phase (in the microtiter well). Material (ag / ab) can stick to microtiter wells, because these wells have been coated with polysterine/polyvinylchloride. Elisa's basic principles consist of: A. Coating phase B. Ag-ab reaction phase C. Chemical reaction phase Tools and materials : By using wel elisa which has 96 holes. On the wel wall there is a material called poly phenyl chloride/poly bromide so that the test material can stick. The sticking process takes place overnight at temperature 40C. After that, an Elisa examination is carried out. Ways of working : 1. Sample preparation ➢Plasma with EDTA was centrifuged at 3400 rpm for 15 minutes. ➢Store at -200C ➢Take 10 µL plasma + 190 µL Calibrator Diluent RD5-26 (1x) (200x dilution obtained) ➢ Take back 10 µL of plasma which has been diluted 200x + 190 µL Calibrator Diluent RD5 -26 (1x) (400x dilution obtained) ➢The plasma sample that has been diluted 400x will later be calculated for the Mannan Binding Lectin concentration using the ELISA technique. 2. Standard MBL preparation ➢The first tube was filled with 900 µL Calibrator Diluent RD5-26 (1x) and 100 µL standard MBL solution (concentration 100 ng/mL) so that the concentration in the first tube was 10 ng/mL. ➢In tubes two to seven, doubling dilution was carried out successively starting from the first tube with solvent using 500 µL ➢Calibrator Diluent RD5-26 (1x) to obtain the concentration in tubes two to seven. 3. Measure the absorbance with an ELISA reader. ➢ Make a curve and linear regression where x is the concentration value (obtained) and y is the absorbance value. ➢ The linear regression equation obtained will be used to calculate the MBL concentration in each blood plasma sample (400x dilution) from each student that has been prepared previously ➢ Make a graph of the MBL concentration of the Plasma sample (ng/mL) for each practitioner.