Factors influencing the measurement of oestrone sulphate by dipstick particle capture immunoassay.
Factors influencing the performance of a dipstick competitive particle capture immunoassay (PCI) for the steroid oestrone sulphate (OS) were investigated. Appropriate 'blocking' of the nitrocellulose dipstick membrane was necessary for the upward flow of microsphere particles. Traditional protein blocking agents including BSA, gelatin and casein were unsatisfactory while synthetic polymers and surfactants were effective in promoting microsphere movement. A simple buffer consisting of 1% aqueous NaCl containing 0.05% Tween 20 was suitable for carrying the components up the dipstick and facilitating the antibody-antigen interactions. Increasing microsphere diameters from 0.3 to 0.8 microm allowed the microsphere antibody coating concentration to be reduced which enabled lower concentrations of OS to be measured. However, upward flow rate and the maximum signal attainable was compromised as a consequence. Enlarging the dipstick membrane nominal pore size from 3 to 12 microm increased the speed of test dot development, but assay sensitivity suffered as a result in some instances. Changing the capture antigen markedly influenced the dose-response lines. No dose-response was achieved with OG-BSA as the capture antigen while OHS-BSA and OCMO-BSA as capture antigens produced dose-response lines with means +/- S.E.M. EC(50) values of 140 +/- 16 and 19 +/- 1 ng/ml, respectively.
Henderson
Keith
K
AgResearch, Wallaceville Animal Research Centre, PO Box 40063, Upper Hutt, New Zealand. keith.henderson@agresearch.co.nz
Stewart
Jeffrey
J
D000941:Antigens / Q000032:analysis / Q000276:immunology; D002364:Caseins; D003101:Collodion; D004958:Estradiol / Q000031:analogs & derivatives*; D004970:Estrone / Q000031:analogs & derivatives* / Q000032:analysis* / Q000276:immunology; D005780:Gelatin; D007118:Immunoassay / Q000379:methods*; D008567:Membranes, Artificial; D008863:Microspheres; D011142:Polyvinyl Alcohol; D011205:Povidone; D012710:Serum Albumin, Bovine; D012996:Solutions
D000941:Antigens; D002364:Caseins; D008567:Membranes, Artificial; D012996:Solutions; C041796:estradiol-17-glucosiduronate-(C-6)-bovine serum albumin conjugate; D012710:Serum Albumin, Bovine; D004970:Estrone; C002912:estradiol-6-(O-carboxymethyl)oxime; D004958:Estradiol; C016430:17 beta-estradiol hemisuccinate; D005780:Gelatin; D011142:Polyvinyl Alcohol; D003101:Collodion; D011205:Povidone; C017296:estrone sulfate