PCR-ELISA Protocol

Experiment Summary

This experiment introduces the procedure of PCR-ELISA. PCR-ELISA is an immunological method to detect PCR products, which is simpler and less time-consuming than the conventional method with electrophoresis and Southern blot, and can handle a large number of specimens simultaneously and facilitate automation.

Experimental Principle

The principle of PCR-ELISA is to apply biotin-labeled primers when doing PCR amplification so that the product of PCR can bind to an affinity (avidin)-coated plastic plate, and then hybridize the digoxin-labeled probe with the PCR product, which can then be detected by an enzyme-linked reaction against digoxin.

Experimental Steps

1. PCR amplification

DNA amplification is done according to the basic PCR technique, except that at least one of the primers used is labeled with biotin at the 5′ end. Primers with biotin-labeled 5′ ends can usually be synthesized directly on a DNA synthesizer.

2. Digoxin-labeled probe hybridization

Dilute 0.1 μg of digoxigenin-labeled DNA probe in 90 μl of 50 mmol/L TrisHCl, pH 8.3,80 mmol/L KCl. Take 10 μl of PCR product and add to the tube, heat to 90℃, and cool slowly to 67℃. Centrifuge for 1 s and keep warm for 1 h in a water bath at 52°C.

3. Fix the hybridization product to the plastic plate

1) Commercially available affinity-coated enzyme plates, or ordinary enzyme plates coated with affinity in the usual way.

2) 100 μl of closure solution (PBS containing 10 mg/ml BSA, 1 mg/ml fish sperm DNA) per well for 1 h at room temperature.

3) PBST wash 3 times.

4) PCR products of digoxin probe hybridization were added directly to the enzyme label plate and incubated at room temperature for 1 h.

5) PBST wash 3 times.

4. ELISA assay

(1) Dilute the anti-digoxin antibody in PBS, add 100 μl to each well and incubate for 1 h at room temperature.

(2) PBST wash 3 times.

(3) Dilute the enzyme-labeled secondary antibody in PBS, add 100 μl to each well, and incubate for 1 h at room temperature.

(4) PBST wash 3 times.

(5) Add 100 μl of enzyme substrate per well and terminate the reaction after the color is suitable.

(6) Read the results on the zymograph.

Cautions

The sensitivity of this method is comparable to that of radioisotope labeling, yet avoids the dangers of isotopes. When doing a large number of samples, the reaction solution should be prepared uniformly and then carefully dispensed into each reaction tube to avoid contamination and to make the conditions in each tube consistent. Non-specific reactions are often due to direct binding of digoxigenin-labeled DNA probes to the enzyme plate, so be sure to include a sufficient amount of fish sperm DNA in the closure solution.

* For research use only. Not intended for any clinical use.

Quick Inquiry

PCR-ELISA Protocol

Experiment Summary

Experimental Principle

Experimental Steps

1. PCR amplification

2. Digoxin-labeled probe hybridization

3. Fix the hybridization product to the plastic plate

4. ELISA assay

Cautions

Related Products and Services at Creative Biogene