hello friends today we are going to discuss about competitive Eliza P t2 Eliza is used to determine the concentration of either antigen or antibody is present in a particular sample let us discuss how this particular technique is used to determine the concentration of antibody suppose we have to determine the concentration of anti a antibodies from a serum sample that is the antibody which specifically binds to the a antigen for this we will require a antigen in this case means you must have the antigen specific to the antibodies whose concentration is to be determined for example if you want to determine the concentration of anti HIV antibodies from the sample you must have HIV antigen with you second and the most important thing that you must have is the enzyme linked and ta antibody this antibody it also binds to the a antigen like the antibodies which are present in the sample but these antibodies they are coupled to the enzyme the enzyme can be horseradish peroxidase beta-galactosidase or alkaline phosphatase all this enzyme how chromogenic substrates for example tmb that is tetra methyl benzene which is the subset of enzyme horseradish peroxidase gives blue color when converted into the product and its intensity can be determined by spectrophotometric method in the first step a microtiter well is coated with a antigen like this the coating is mediated with the help of coating buffer after this the sample containing and taa antibodies whose concentration is to be determined and a fixed amount of enzyme linked and T a antibodies are added to the well these two types of antibodies will compete with each other to bind the antigen present in the well now you might think which type of antibody is going to win this competition well the antibody with the greater concentration wins the competition now we don't know which kind of antibodies concentration is more let us assume both the situations if the concentration of antibodies from the sample is more than enzyme-linked antibodies as in this case this antibodies will occupy most of the antigens of the well on the other hand very few enzyme-linked antibodies will bind to the antigens but if the condition is reversed as in this case most of the antigens will be occupied by the enzyme-linked antibodies after this the well is washed to remove unbound antibodies this type is followed by addition of chromogenic substrate and incubation for a fixed period of time after incubation the reaction is stopped by adding strong acids now during this incubation the enzyme connected to the enzyme-linked antibody will convert this substrate into the color product now if the number of enzyme linked antibodies are less in the well as in this case less product will form and hence the color intensity and its absorbance value will be less and if the number of enzyme linked antibodies are more in the well as in the second case more product will form and therefore absorbance value will also be more so in short if the absorbance of the product is less concentration of antibodies in the sample is more and if the absorbance is more the concentration of the antibodies in the sample is less the actual concentration of antibodies present in the sample can be determined by extrapolating the value of absorbance on the standard graph like this so in this way the competitive iliza is used to determine the concentration of antibodies exactly reverse method is used to determine the concentration of antigens