Preparation, purification and characterization of anti-digoxigenin antibody HRP conjugate for using in PCR-ELISA DIG detection system

There are several methods used to visualize the end product of PCR amplifications. One of these methods in ELISA-based detection system (PCR-ELISA) which is very senstive and can be used to measure the PCR products quantitatively by colorimentric methods. In this technique, copies of DNA segment from genomic DNA are amplified by PCR with the incorporation of digoxigenin -11-dUTPSamples are analyzed in a microtiter plate format by alkaline denaturation and hybridization to biotibylated allele-specific capture peobes bound to streptoavidin- coated plates. Using the developed anti-digozigenin antibody horseradish peroxidase conjugate and the substrate 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification and characterization of anti-digoxigenin antibody HRP conjugate for using in PCR-ELISA DIG detection system. Several biochemical protocols and modifications were used to increase the sensitivity and specificity of this conjugate for e more efficient and cost-effective product.

Keywords:

Anti-digoxigenin antibody HRP conjugate , PCR-ELISA