This techniques used PCR amplification of a Marker Dna segment attached to an Antibody for detection oaf the Antigen for which this antibody is specific. The protocol of immuno-PCR may be stated in simple terms as follows.
(1) Test samples suspected of containing the antigen are added to microtitre plate wells and the antigen is immobilized on the surface of wells.
(2) The free binding sites on the microtire well surfaces are suitably blocked.
(3) The antibody specific to the antigen to be detected is added to the wells it forms antigen antibody complex, this occurs only in those wells where the antigen is present.
(4) The molecular linker add and bind the free region of the antibody A chimera is the most versatile molecular liker this chimaeric linker binds to the Fe domain of antibodies due to the protein A sequences. IT also binds to biotinylated DNA molecules due to the streptavidin mioety. The linker molecular is already complexed with the marker DNA molecule when it is added to the microtitre wells.
(5) A segment of the marker DNA is amplified by PCR.
The PCR products are analyzed by gel electrophoresis for a large scale application the PCR products can be labelled using flourochromes and happiens which permit their rapid even automated detection in the case of LCR.
The antigen antibody complex will be formed only in those microtitre wells which contain the target antigen. Therefore, PCR amplification will occur only in such wells in other products will contain the target antigen.
Immuno-PCR is highly sensitive it is several orders of magnitude more sensitive than ELISA. It can detect rare antigens and diagnose pathological condition much earlier it is extremely versatile that it can be applied to even single cells, can yield quantities estimates of the antigen and is amenable in addition is is relatively simple.
(1) Test samples suspected of containing the antigen are added to microtitre plate wells and the antigen is immobilized on the surface of wells.
(2) The free binding sites on the microtire well surfaces are suitably blocked.
(3) The antibody specific to the antigen to be detected is added to the wells it forms antigen antibody complex, this occurs only in those wells where the antigen is present.
(4) The molecular linker add and bind the free region of the antibody A chimera is the most versatile molecular liker this chimaeric linker binds to the Fe domain of antibodies due to the protein A sequences. IT also binds to biotinylated DNA molecules due to the streptavidin mioety. The linker molecular is already complexed with the marker DNA molecule when it is added to the microtitre wells.
(5) A segment of the marker DNA is amplified by PCR.
The PCR products are analyzed by gel electrophoresis for a large scale application the PCR products can be labelled using flourochromes and happiens which permit their rapid even automated detection in the case of LCR.
The antigen antibody complex will be formed only in those microtitre wells which contain the target antigen. Therefore, PCR amplification will occur only in such wells in other products will contain the target antigen.
Immuno-PCR is highly sensitive it is several orders of magnitude more sensitive than ELISA. It can detect rare antigens and diagnose pathological condition much earlier it is extremely versatile that it can be applied to even single cells, can yield quantities estimates of the antigen and is amenable in addition is is relatively simple.
