A novel liquid-phase immunoassay is described which involves: competitive binding of the analyte and labelled antigen to specific antibodies; enzymic transformation of the free ligand to a more hydrophobic derivative; and separation of the antibody-bound and free ligand by partition of the reactants into an organic and aqueous phase. The principles of the method, described as ligand differentiation immunoassay, have been used in conjunction with a tritiated antigen to establish a continuous-flow system for the measurement of oestriol-16α-glucuronide in urine. Conventional autoanalyser (AAII) modules have been used, and the pump tubes and glass connections are standard accessories. The equipment is relatively inexpensive (£1500 + detector) and the reagents are readily available. Each sample takes 55 minutes to be processed, and 30 results are obtained per hour.
References
1.
PollardAGarnettESWebbeC.An automated technique for assessment of thyroid status. Clin Chem1965; 11: 959–67.
2.
PollardAWaldronCB.Automatic radioimmunoassay. In: Automation in analytical chemistry, Vol 1. Technicon symposium. Mediad, New York, 1966; 49–58.
3.
BagshaweKDMitchellHD.Automated immunanalysis by radioimmunoassay. In: HunterWMCorrieJET, eds. Immunoassays for clinical chemistry.Edinburgh: Churchill Livingstone, 1983; 221–5.
4.
JohnstonHJBrannBERitchieRFGravesJ.The development of a totally automated radioimmunoassay system for human insulin. In: Automation in analytical chemistry, Vol. 2 Technicon Symposium. Mediad, New York, 1973; 69–72.
5.
LunerSJ.Continuous-flow automated radioimmunoassay using antibodies attached to red blood cells. Anal Biochem1975; 65: 355–61.
6.
CottrellDBTorenECMagnusonJAUpdikeSJ.Computer controlled automation of radioimmunoassay based on gel entrapment of antibody. Clin Chem1975; 21: 829–33.
7.
MarschnerIErhardtFHennerJScribaPC.A modular analyser system for double antibody radioimmunoassays. Z Klin Chem Klin Biochem1975; 13: 481–8.
8.
BowieLJ.Automated instrumentation for radioimmunoassay.Florida: CRC Press Inc., 1980.
9.
ForrestGC.A general review of automated RIA. In: HunterWMCorrieJET, eds. Immunoassays for clinical chemistry.Edinburgh, Churchill Livingstone, 1983; 211–20.
10.
BarnardGJRMatsonCMMakawitiDWKilpatrickMJCollinsWP.A novel liquid phase separation system for automated immunoassay. In: HunterWMCorrieJET, eds. Immunoassays for clinical chemistry.Edinburgh; Churchill Livingstone, 1983; 231–49.
11.
EdwardsCWRTaylorAABaumCKKurtzAB.Evaluation of two new separation techniques for steroid immunoassays. In: CameronEHDHillerSGGriffithsK, eds. Steroid immunoassay.Cardiff: Alpha Omega Publishing Ltd, 1975; 229–42.
12.
CaisMShimoniM.A feasible solvent separation for immunoassaysAnn Clin Biochem1981; 18: 317–23.
13.
MorrisR.Radioimmunoassay of oestriol in the urine of non-pregnant women. Ann Clin Biochem1981; 18: 163–8.
14.
SamarajeewaPKellieAE.The radioimmunoassay of steroid glucuronides: the estrogen C-3 glucuronides as haptens. Biochem J1975; 151: 369–76.
15.
BranchCMCollinsPOCollinsWP.Ovulation prediction: changes in the concentrations of urinary estrone-3-glucuronide, estradiol-17β-glucuronide and estriol-16α-glucuronide during conceptional cycles. J Steroid Biochem1982; 16: 345–7.
16.
AdlercreutzHLehtinenTKairentoA-L.Prediction of ovulation by urinary estrogen assays. J Steroid Biochem1980; 12: 395–401.
17.
BakerTSJennisonKKellieAE.A possible method for the detection of ovulation and the determination of the fertile period. J Steroid Biochem1980; 12: 411–5.
18.
AdlercreutzHLehtinenTTikkanenM.Preliminary studies on the determination of estriol-16α-glucuronide in pregnancy urine by direct radioimmunoassay without hydrolysis. J Steroid Biochem1976; 7: 105–7.
ForrestGC.Development and application of a fully automated continuous flow radioimmunoassay system. Ann Clin Biochem1975; 14: 1–11.
22.
BrookerGTerasakiWLPriceMG.Gammaflow: A completely automated radioimmunoassay system. Science1976; 194: 270–6.
23.
JohnsonLR.Automated radioimmunoassay on Aria II. In: BowieLJ, ed. Automated instrumentation for radioimmunoassay Florida: CRC Press Inc, 1980; 91–126.
24.
IsmailAAAWestPMGoldieDJ.The ‘Southmead system’, a simple, fully-automated continuous-flow system for immunoassays. Clin Chem1978; 24: 571–9.
25.
IsmailAAAWestPMGoldieDJPooleAC.An improved fully automated continuous-flow system for immunoassays. Ann Clin Biochem1981; 18: 287–94.
26.
GoldieDJWestPMIsmailAAAPooleACA fully continuous flow system for immunoassays. In: HunterWMCorrieJET, eds. Immunoassays for clinical chemistry.Edinburgh; Churchill Livingstone, 1983; 226–30.
27.
MakawitiDWMatsonCMBarnardGJCollinsWP.A novel radioimmunoassay for plasma testosterone. J Steroid Biochem1983; in press.
28.
MatsonCM. PhD thesis 1982, University of London.
29.
MakawitiDWAllenWEKilpatrickMJ.The measurement of oestrone sulphate in peripheral plasma as a predictive index of ovulation in the pony mare. J Reprod Fert1983; in press.
30.
MakawitiDWBarnardGJCollinsW.Monitoring ovarian function by a novel pseudohomogeneous radioimmunoassay. In preparation.
31.
BarnardGJRMatsonCMCollinsWP.Binding assays. UK Patent 1981 No; 8118291.
32.
SmithDSAl-HakiemHHLandonJ.A review of fluoroimmunoassay and immunofluorimetric assay. Ann Clin Biochem1981; 18: 253–74.
33.
KohenFKimJBBarnardGLindnerHR.An assay for urinary estriol-16α-glucuronide based on antibodyenhanced chemiluminescence. Steroids1980; 36: 405–19.
34.
StryerL.The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites. J Mol Biol1965; 13: 482–95.
35.
McCapraF.Chemiluminescence of organic compounds. Prog Org Chem1974; 8: 231–77.
