Immuno-Polymerase chain reaction (Immuno-PCR or IPCR)

Presented by Shadman tariq sadiq

(91150000630) PhD study

Supervised by Proff.dr. Guven Ozdemir

Introduction Immuno-PCR is an antigen detection system with the

exponential signal enhancement of a PCR , in which the polymerase chain reaction (PCR) is used to amplify a segment of marker DNA that has been attached specifically to antigen–antibody complexes this mean a combines the advantages of both ELISA and PCR to detect antigen.

The specific antibodies are labeled with a DNA marker. By using the amplification steps of the PCR, the sensitivity can be better compared to the “classic” ELISA

Who developed this method

??

This method is developed by

Sano, T., Smith, C. L. & Cantor, C. R. in (1992)

Their research published in science journalThe name of research was

(( Immuno-PCR: very sensitive antigen detection by means of specific antibody–DNA conjugates ))

The concept of I-PCRThe concept of immuno-PCR (polymerase chain reaction),

illustrated in Fig. 1, is quite simple, and is similar to (ELISA) and radioimmunoassays (RIA).

1- In the original immuno-PCR scheme, a molecular linker, which has a bispecific binding affinity for antibody and DNA, is used to attach a marker DNA molecule specifically to an antigen–antibody complex.

2-A segment of the attached marker DNA is amplified by PCR with appropriate primers, and the resulting PCR products are analyzed.

3- The presence of specific PCR products demonstrates that the marker DNA molecules are attached specifically to antigen–antibody complexes, indicating the presence of antigen.

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Fig. 1

Different Between ELISA and PCR

• The immuno-PCR is based on the same principle than an ELISA:- Capture of antigen (direct on the plate or indirect by a capture molecule)- Recognition of the antigen by a detection antibody- Revelation of antigen/antibody complex

• The difference is that in the case of the immuno-PCR, the detection of the antigen/antibody complex is performed using a DNA reporter and not an enzyme.

• Also immuno-PCR technology has a sensitivity greater than ELISA .

• The immuno-PCR provides an ultrasensitive technology which combines the molecular specificity of antibodies with the sensitivity of the PCR.

• The use of a DNA reporter allows to perform a signal amplification step by PCR amplification, which is impossible to achieve in the case of a ELISA system.This brings a 10 to 1000 times sensitivity increase when compared to a classical ELISA !This increase in sensitivity can be extremely useful for example in the case of highly diluted sample.

The main steps of an immuno-PCR assay are as follows:

1. Immobilization of antibodies specific for the protein target to the surface of a vessel2. Washing to remove unbound antibody3. Addition of sample4. Washing to remove unbound sample5. Addition of a second specific antibody, coupled to a DNA molecule6. Washing to remove unbound antibody7. DNA amplification and detection

Advantages of Immuno-PCR:

- Sensibility dramatically improved in comparison to classical ELISA- Possibility to quantify- Reducted volumes and quantities- No specificity lost

Benefits of IPCR

1- biological and biomedical sciences such as diagnoses, toxins, cytokines, hormones..etc

2-powerful method for detecting low quantities of protein antigens.

3-microbial diagnostic.

Thanks for listening