Advantages of soybean peroxidase over horseradish peroxidase as the enzyme label in chemiluminescent enzyme-linked immunosorbent assay of sulfamethoxypyridazine.
An indirect competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) of sulfamethoxypyridazine (SMP) was developed. The conjugates of streptavidin with cationic horseradish peroxidase (HRP) and anionic soybean peroxidase (SbP) were used in CL-ELISA for the detection of biotinylated anti-SMP antibodies. For streptavidin-HRP conjugate-catalyzed chemiluminescence measured 20 s after the initiation of the enhanced chemiluminescence reaction (ECR), the limit of detection (IC(10)), the IC(50) value, and the working range in CL-ELISA of SMP are 0.3, 12.4, and 1.2-85.0 ng/mL, respectively. An increase in the time interval between the ECR initiation and the luminescence measurement results in the loss in the quality of analytical measurements because of the time-dependent quenching of chemiluminescence typical of the HRP-catalyzed ECR. In the case of SbP-based CL-ELISA of SMP, the limit of detection, the IC(50) value, and the working range (0.025, 0.17, and 0.045-0.63 ng/mL, respectively) are better than those for HRP-based CL-ELISA. Furthermore, the analytical parameters of SbP-based CL-ELISA remain unchanged during a long period of time (for at least 30 min). The recovery values from four spiked milk samples with different concentrations of SMP in SbP-based CL-ELISA vary from 70 to 130%.
Sakharov
Ivan Yu
IY
Chemical Enzymology Department, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, Moscow, Russia. sakharovivan@gmail.com
Berlina
Anna N
AN
Zherdev
Anatoly V
AV
Dzantiev
Boris B
BB
D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D009195:Peroxidase / Q000737:chemistry*; D013025:Glycine max / Q000201:enzymology*; D013421:Sulfamethoxypyridazine / Q000032:analysis*
D006735:Horseradish Peroxidase; D009195:Peroxidase; D013421:Sulfamethoxypyridazine