ABSTRACT
This study addresses observations made in view of testing in practice the guideline in the European Pharmacopoeia (EP) on omitting the rat potency test for release of polio containing vaccines. In general, use of the guideline is valid and the D-antigen ELISA can indeed be used as an in vitro alternative for the in vivo test. However, the set-up of the ELISA is crucial and should include detection of antigenic site 1 in polio serotype 3 as destruction of that site by trypsin results in a reduced rat potency. Antigenic site 1 in polio serotype 2 may also be modified by trypsin, but the cleavage of viral protein 1 (VP1) did not affect the rat potency. Therefore, any antigenic site, except site 1, can be used for detection of polio serotype 2. It is advised to include testing of the effect of trypsin treatment in the EP-guideline. This allows polio vaccine manufacturers to check whether their in-house ELISA needs improvement.
Subject(s)
Biological Assay , Enzyme-Linked Immunosorbent Assay , Poliovirus Vaccine, Inactivated/immunology , Poliovirus/drug effects , Trypsin/pharmacology , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Biosensing Techniques , Capsid Proteins/drug effects , Chlorocebus aethiops , Dose-Response Relationship, Immunologic , Europe , Hot Temperature , Poliovirus/immunology , Poliovirus/isolation & purification , Poliovirus/pathogenicity , Practice Guidelines as Topic , Rats , Vero Cells , Virulence/drug effectsABSTRACT
A fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20 h-protocol was optimized by minimizing the total incubation time to 1 h, and by replacing the signal reagent 3,3',5,5'-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range.
Subject(s)
Antigens, Viral/immunology , Luminescent Measurements/methods , Poliovirus/immunology , Antigens, Viral/analysis , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , Luminescent Measurements/standardsABSTRACT
The reactivity of the N-acetoxy metabolite of 2-amino-5-phenylpyridine (Phe-P-1), a pyrolysis product of phenylalanine, towards 2'-deoxyguanosine (dG), 2'-deoxyguanosine-3'-monophosphate (dGMP) and DNA was studied and compared with that of the ortho-methyl derivative. Reaction of 2-acetoxyamino-5-phenylpyridine (N-OAc-APP) with dG resulted in substitution at the 8-position of this nucleoside by the ortho carbon of the amine. The major reaction, however, was acetylation of dG. In contrast, 2-acetoxyamino-3-methyl-5-phenyl-pyridine (N-OAc-MeAPP) mainly attacked the 8-position of dG by the exocyclic nitrogen and hardly any acetylation of the nucleoside occurred. The adducts were chromatographically isolated and characterized by their mass and NMR spectra. Upon reaction of N-acetoxy compounds with DNA and dGMP, formation of the same adducts was observed, besides the formation of minor amounts of unidentified compounds, as was established by 32P-postlabelling analysis. The amount of DNA-bound amine, formed by the interaction of N-OAc-APP with DNA, was approximately 15 times smaller than that observed after the reaction with the corresponding ortho-methyl derivative under the same conditions.
