Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.
There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) 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 use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.
Gill
Pooria
P
Department of Medical Biotechnology, Tarbiat Modarres University, Tehran, Iran.
Forouzandeh
Mehdi
M
Rahbarizadeh
Fatemeh
F
Ramezani
Reihaneh
R
Rasaee
Mohammad Javad
MJ
D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D000936:Antigen-Antibody Complex / Q000032:analysis; D000937:Antigen-Antibody Reactions; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004076:Digoxigenin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006735:Horseradish Peroxidase / Q000737:chemistry*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D011817:Rabbits; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000008:administration & dosage / Q000737:chemistry
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011933:Reagent Kits, Diagnostic; D012710:Serum Albumin, Bovine; D004247:DNA; D006735:Horseradish Peroxidase; D004076:Digoxigenin