Date post: | 16-Apr-2017 |
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Immuno-Polymerase chain reaction (Immuno-PCR or IPCR)
Presented by Shadman tariq sadiq
(91150000630) PhD study
Supervised by
Prof.Dr. Güven Özdemir
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.
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 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, 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. By using the amplification steps of the PCR, the sensitivity can be better compared to the “classic” ELISA
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Differents Between ELISA
and PCR
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 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.
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 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 IPCR1- biological and biomedical sciences such as
diagnoses, toxins, cytokines, hormones..etc2-powerful method for detecting low quantities of
protein antigens.3-microbial diagnostic.