PCR-ELISA Protocol

• The principle of polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA) is to apply biotin-labeled primers when doing PCR amplification so that the PCR product can bind to the avidin-coated plastic plate. The digoxin-labeled probe is then hybridized with the PCR product, which can then be detected by an anti-digoxin ELISA.
• This experiment describes the procedure of PCR-ELISA.PCR-ELISA is an immunological method to detect PCR products, which is easier and less time-consuming than the routine use of electrophoresis and Southern blot. A large number of specimens can be processed at the same time, which facilitates automation.

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 labeled with biotin at the 5' end can usually be synthesized directly on a DNA synthesizer.

Hybridization of digoxin-labeled probes

• Dilute 0.1 μg of digoxin-labeled DNA probe in 90 μl 50 mmol/L Tris-HCl, 80 mmol/L KCl (pH 8.3).
• Take 10 μl of PCR product and add it to the tube, heat it to 90°C, and slowly cool it to 67°C. Centrifuge for 1 second and place in a 52°C water bath for 1 hour.

Immobilization of hybridization products onto plastic plates

• Use commercially available affinityin-coated enzyme labeling plates, or use ordinary enzyme labeling plates with affinityin-coated in the usual way.
• Add 100 μl of blocking solution (PBS containing 10 mg/ml BSA and 1 mg/ml fish DNA) to each well, and incubate for 1 h. Wash 3 times with PBST.
• Add the PCR product of digoxigenin probe hybridization directly to the enzyme plate and incubate at room temperature for 1 h. Wash 3 times with PBST.

• Dilute the anti-digoxin antibody in PBS, add 100 μl to each well, and incubate at room temperature for 1 h. Wash with PBST for 3 times.
• Dilute the enzyme-labeled secondary antibody in PBS, add 100 μl to each well, and incubate for 1 h at room temperature. Wash 3 times with PBST.
• Add 100 μl of enzyme substrate to each well and terminate the reaction after the color is suitable. Read the results on the enzyme marker.

• The sensitivity of this method is comparable to radioisotope labeling but avoids the dangers of isotopes.
• When making a large number of samples, the reaction solution should be prepared uniformly and then carefully dispensed into reaction tubes to avoid contamination and to keep the conditions consistent from tube to tube.
• Non-specific reactions are often due to the direct binding of digoxigenin-labeled DNA probes to the plate, so be sure to include a sufficient amount of ichthyogenic DNA in the blocking solution.

Nucleic Acid Extraction

Biofluids