The polymerase chain reaction and the enzyme-linked immunosorbent assay using antipeptide antibody: Practical problems in determining the presence of viruses by modern technology.

摘要

Individuals with antibody to hepatitis B virus (HBV) and horses with antibody to equine infectious anemia virus (EIAV) remain infectious for the virus. Until the present time there has been no test with adequate sensitivity except animal inoculation to assess the infectivity. The advent of automated instruments for the synthesis of peptides and deoxyribonucleic acids (DNA) has made possible new methods of testing. The polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) were investigated as sensitive tests for virus protein and DNA.; HBV-DNA and EIAV proviral DNA in serum were tested for by PCR. Parameters affecting the sensitivity of the test were also investigated. These included Mg{dollar}sp{lcub}2+{rcub}{dollar} concentration, primer concentration, enzyme concentration, annealing temperature, and the nature of the template. The optimal conditions for each set of primers and template varied. Some general findings were: Mg{dollar}sp{lcub}2+{rcub}{dollar} concentrations greater than 5.0mM resulted in loss of PCR product; primer concentrations in the 0.2 to 0.5{dollar}mu{dollar}M range were optimal; higher annealing temperatures lead to greater specificity of the product; and the sensitivity of the PCR was increased by an increased number of cycles if the template concentration is low. Contamination is a serious problem that may make the results of sample testing by PCR questionable.; Rabbit antibody to a synthetic peptide from the p26 gag protein of EIAV was used as a capture antibody in an ELISA. All results were negative. The negative results were attributed to failure of the anti-peptide antibody to recognize the peptide as it is expressed in the virion.