Enzyme-Linked Immunosorbent Assay ( abbreviated as ELISA ) , is a sort of immunochemical assay technique to observe the presence of a specific antibody or antigen in the trial samples. This method chiefly make usage of both immunological reaction ( the antibody-antigen reaction ) to observe the nowadays of specific antibody or antigen ) and biochemical reaction ( the enzyme substrate reaction ) to bring forth a seeable signal for both qualitative and quantitative measuring.
The ELISA method used in this experiment is an illustration of “ indirect ELISA ” method.
The ELISA home base Wellss are coated with optimized concentration of antigens before manus by agencies of charge interaction or with the aid of a spacer coating ( eg. L-lysine ) . Then the home base Wellss are washed with buffer solution, and a blocking measure is preformed by adding bovine serum albumen or casein, to barricade any uncoated infinite in the well before utilizing to observe antibodies in sample serum.
Then the sample serum is added to observe the nowadays of specific antibody, the antibodies will adhere to the antigens in the well ( in this experiment is the anti-DNA antibody ) .
Then a secondary antibody ( normally raised from a species against the antibody of the sample ) with enzyme-linked ( called conjugate ) was added to adhere. The enzyme used may be Alkaline phosphate or Horseradish peroxidase ( in this experiment is Alkaline phosphate ) ; this besides serve as signal elaboration measure as the enzymes conjugate chose used normally have more than one binding sites for the substrate added later.
Then a substrate is added for the enzyme to bring forth a colour reaction ( in this experiment is the PNPP which produce a xanthous colour ) to bespeak the nowadays of the specific antibody in the sample.
The higher the concentration of the antibody in the trial sample, the stronger the colour developed. We can utilize a spectrometer ( an ELISA reader in this experiment ) to mensurate the colour quantitatively alternatively of utilizing our oculus, which is more nonsubjective and accurate.
Washing with buffer ( normally a mild detergent ) is applied between stairss to take unbind antibodies to avoid non-specific binding of antibodies.
Normally positive and negative controls will be paralleled tally with the trial sample to formalize the consequence.
The cut-off point between a positive or negative consequence is normally determined statistically with known criterions. In add-ons, with a consecutive dilution of a known criterion ( known concentration of the specific antibody want to observe in the trial ) , we can besides happen the sum of the specific antibody in the trial sample from the graph of optical density against concentration of the known criterion. Therefore, the ELISA method can bring forth both qualitative and quantitative consequence in observing the specific antibody in trial sample.
ELISA is a comparatively high sensitive and specific trial for observing serum protein, the presence of specific antibody or antigen ; and besides considers as a high-throughput immunochemical assay. The usage of ELISA besides includes endocrines and infective antigens ( including virus and bacterium ) . The most common illustration is observing HIV in patient samples. In add-on, it has the advantage of utilizing non-radioactive substances, is safer than those radio-immunoassays.
“ Sandwich ELISA ” ( or “ direct ELISA ” ) is used to observe antigen in sample serum, is less-common. With known measure of gaining control antibodies coated to the well, the antigens in the sample will adhere to the antibodies to organize complex. Then enzyme-linked primary antibodies will be applied to observe the nowadays of the antigens.
“ Competition ELISA ” is a different method from the indirect and sandwich ELISA, in which the kit contains enzyme-linked antigen instead than enzyme-linked antibody. The labelled antigen will vie with the antigen in the sample for the antibody adhering sites in the well. Therefore, more antigens in the sample will give weaker signal as less labelled antigen can adhere to the antibody adhering sites in the well. The advantage of this method is can be used to observe antigen in impure samples.
“ Change by reversal ELISA ” is a new engineering utilizing immunosorbent polystyrene rod with stick outing nose cones. This ogives will dunk into the sample, therefore a higher sample volume can be used to better the sensitiveness. Furthermore, the nose cones can be sensitized with different reagent to observe different antibodies or antigens at the same time for multi-target checks.