Development of a quantitative immuno-PCR assay and its use to detect mumps-specific IgG in serum.
Determination of the immune status of individuals to vaccine-preventable diseases requires an assay that can detect antibodies that may be present at very low levels, especially when natural or vaccine exposure may have been many years previously. Immuno-PCR (iPCR) has recently been described as an ultrasensitive method for the detection of antigens and we have adapted the method for the quantification of antibodies to mumps virus. The procedure used was similar to an indirect ELISA except that the detecting antibody (anti-human IgG) was chemically conjugated to a short capture oligonucleotide rather than an enzyme. The capture oligonucleotide was then detected by the addition of target DNA, which was designed to hybridise to the capture oligonucleotide and function as a template for real-time PCR. The quantity of target DNA detected by the PCR depended upon the level of specific antibody in the test sample. We found that the sensitivity (and specificity) of the iPCR assay did not exceed that of the conventional ELISA. The sensitivity was limited by nonspecific binding of human IgG to the solid phase. Further development of reagents and assay formats is necessary to fully exploit the potential of quantitative iPCR, so that potential improvements in the sensitivity of anti-mumps IgG detection can be realised.
McKie
Anne
A
Virus Reference Division, Central Public Health Laboratory, 61 Colindale Avenue, Colindale, NW9 5HT, London, UK.
Samuel
Dhanraj
D
Cohen
Bernard
B
Saunders
Nicholas A
NA
D000914:Antibodies, Viral / Q000097:blood* / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000706:statistics & numerical data; D007074:Immunoglobulin G / Q000097:blood* / Q000235:genetics; D008969:Molecular Sequence Data; D009107:Mumps / Q000276:immunology*; D009109:Mumps virus / Q000276:immunology; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D007074:Immunoglobulin G; D009838:Oligodeoxyribonucleotides; D004247:DNA