Immuno-Polymerase chain reaction (Immuno-PCR or IPCR) Presented by Shadman tariq sadiq (91150000630) PhD study Supervised by Prof.Dr. Güven Özdemir

Contents Introduction Who developed this method. The principle of I-PCR. Comparision between ELISA and PCR . Assemblage or forms of IPCR. The main steps of an immuno-PCR assay Advantages of Immuno-PCR Benefits of IPCR

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 that IPCR is a combines the advantages of both ELISA and PCR to detect antigen.

Who developed this technology

Sano, T., Smith, C. L. & Cantor, C. R. This method is developed by Sano, T., Smith, C. L. & Cantor, C. R. in (1992) Their research published in science journal The name of research was (( Immuno-PCR: very sensitive antigen detection by means of specific antibody–DNA conjugates ))

The principle of I-PCR The principle of immuno-PCR 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 is used to attach a marker DNA molecule specifically to an antigen–antibody 2-A segment of the attached marker DNA is amplified by PCR, 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.

Fig. 1

The specific antibodies are labeled with a DNA marker 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 www.genengnews.com

Comparision between ELISA and PCR

ELISA and PCR The immuno-PCR is based on the same principle of ELISA: - Capture of antigen (direct on the plate or indirect by a capture molecule) - Recognition of the antigen by a detection antibody 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 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.

Assemblage or forms of IPCR

Steps of IPCR

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 vessel 2. Washing to remove unbound antibody 3. Addition of sample 4. Washing to remove unbound sample 5. Addition of a second specific antibody, coupled to a DNA molecule 6. Washing to remove unbound antibody 7. DNA amplification and detection

Advantages of Immuno-PCR: - Sensibility dramatically improved in comparison to classical ELISA. - Possibility to quantify - Reduced 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.

Detection of microbial antigens Although PCR is widely employed to detect nucleic acid molecules in viral infections, proteins remain the major pathological mediators and are detected primarily by ELISA. In recent years, I-PCR assays have been exploited for the detection of bacterial,parasitic ,prions and viral protein molecules. Several laboratories have demonstrated high-sensitivity detection (100–700-fold increase) of antigens by I-PCR compared with ELISA (Table 1).

Table 1 cont…

Cancer detection in premalignant stage is directly related with increase survival rate. Several biomarkers have been investigated and characterized for monitoring changes inside the cancerous cells. Although enzyme-linked immunosorbent assay (ELISA) is the method of choice in clinical practice for detecting biomarkers in serum/urine samples. However, in certain malignancies the amount of biomarkers before reaching metastasis are too low to be detected by conventional ELISA. The seminal work of Sano et al. led to the development of highly sensitive and powerful detection method, the immuno-PCR (iPCR), which can detect very small amount of antigens/biomarkers. In spite of, several publications on iPCR sensitivity.

References Adler, M., Wacker, R., Niemeyer, C.M. (2003) A real-time immuno-PCR assay for routineultrasensitive quantification of proteins, Biochem. Biophys. Res. Commun. 308, 240–250. Cao, Y. (2002) In situ immuno-PCR. A newly ndeveloped method for highly sensitive antigen detection in situ, Methods Mol. Biol. 193, 191–196. Sano, T., Smith, C.L., Cantor, C.R. (1995a) Very Sensitive Antigen Detection By Immuno- PCR, in: Nelson, J.O., Karu, A.E., Wong, R.B. (Eds.) Immunoanalysis of Agrochemicals: Emerging Technologies, American Chemical Society Symposium Series Number 586, American Chemical Society, Washington, DC, pp. 175–185. Sano, T., Smith, C.L., Cantor, C.R. (1992) Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates, Science 258, 120–122. Hendrickson, E.R., Truby, T.M., Joerger, R.D., Majarian, W.R., Ebersole, R.C. (1995) High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction, Nucleic Acids Res. 23, 522–529. Joerger, R.D., Truby, T.M., Hendrickson, E.R., Young, R.M., Ebersole, R.C. (1995) Analyte ndetection with DNA-labeled antibodies and polymerase chain reaction, Clin. Chem. 41, 1371–1377.