| Background: Labeled immunoassay are the total conception of a series ofhighly and specific examine techniques, and have been widely applied in the basicand clinical fields. With the exertion of new theories and new techniques, the labeledimmunoassay have got great, development. During recent years, the most widelyapplied techniques have been radioimmunoassay (RIA) and enzyme immunoassay(EIA). Although sensitive and specific, both of these assays have certain drawbacksrelating to the label used. In RIA the most obvious drawbacks are the ineviTab.leradiation hazards and the short-life of the iodinated labels. In EIA toxicity andmutagenicity of some substrates are potential health risks, and the additional reactionwith substrate hampers the standardization of the assay. Conventionalimmunofluorescence and fluoroimmunoassay methods using organic fluorochromes,e.g, fluorescein isothiocyanate, have not achieved the high sensitivity of RIA and EIAmethods. This is mainly because of background problems caused by nonspecificfluorescent components in serum and other organic material and the short Stokes'shift of fluorescein isothiocyanate (20-30nm), which causes considerable interferencedue to scattering. These problems inherent in conventional fluorescence methods can be mostly avoided by adopting the principle of time-resolved fluoroimmunoassay(TRFIA), in which a fast light pulse is used to excite the label, and the fluorescence ismeasured at a certain delay time after the excitation. Thus, direct scattering andnonspecific background fluorescence with a decay time generally less than 10ns donot interfere with the assay. TRFIA requires fluorescent labels with a decay timepreferably over 1μs. The labels that fulfill this requirement consist of rare earth metal(Eu3+, Tb3+, Sm3+, Dy3+) chelates. The chelated ligand strongly absorbs theexcitation radiation and thansfers it to the chelated central atom. The emissionwavelengths are characteristic of the lanthanide used, although the intensity and thedecay are ligand dependent. A long Stokes' shift (up to 290nm) reduces backgroundfluorescence and helps to optimize the measurement of specific lanthanidefluorescence.Pregnancy associated plasma protein-A (PAPP-A) is a zinc-bindingmetalloproteinase which is found in the serum of pregnant women in 1974. It has amolecular weight of approximately 199 000 dalton. The gene for PAPP-A is inchromosome band 9q33.1. PAPP-A has been used in prenatal genetic screening andstudies of atherosclerosis. Women with low blood levels of PAPP-A at 8 to 14 weeksof gestation have an increased risk of intrauterine growth restriction, trisomy 21,premature delivery, preeclampsia, and stillbirth. PAPP-A is present in unstableatherosclerotic plaques, and circulating levels are elevated in acute coronarysyndromes which may reflect the instability of the plaques. PAPP-A may be a markerof unstable angina and acute myocardial infarction (heart attack).Estriol(E3) is one of the three major naturally occurring estrogens, the othersbeing estradiol and estrone. Estriol is a female sex steroid hormone produced almostexclusively by the placenta during pregnancy, and is the major estrogen produced inthe normal human fetus. During pregnancy the production of estriol depends on an intact maternal-placental-fetal unit. Steroid precursors from the maternal circulationare first converted to progesterone in the placenta. Progesterone is subsequentlyconverted to dehydroepiandrosterone sulfate (DHEA-S) in fetal adrenal tissue andthen 16-hydroxylated in the fetal liver. Subsequentially, 16-hydroxy-DHEA-S isconverted to estriol in the placenta. In non-pregnant circumstances the majority ofestriol is derived from 17-estradiol. Estriol exists in biological matrix in unconjugated(-9%) and conjugated forms (～91%). The half-life of estriol in the maternalbloodstream is only 20-30 minutes. Its measurement, therefore, offers a convenientand quick evaluation of current fetal status. A sudden decrease in fetoplacental E3production will result in a rapid fall in unconjugated E3 in the maternal serum. Thereare several potential advantages to measuring unconjugated E3 rather than total serumor urinary E3. Unconjugated estriol levels are free from effects related to maternalrenal or hepatic disease, and are not altered by the administration of certain antibiotics.Unconjugated E3 more accurately reflects fetal outcome in diabetic pregnancies and,since no hydrolysis of unconjugated E3 is required, a more rapid turnaround for thetest result is possible.Objective: To develop reagents for quantitative determination of PAPP-A anduE3 in serum by time-resolved fluoroimmunoassay(TRFIA), The self-made kit wascompared with PE Cop. kits in sensitivity, specificity, stability, etc.Methods: In this dissertation, a solid-phase two-site sandwich time-resolvedfluoroimmunoassay for quantifying PAPP-A in serum is developed by using twomonoclonal antibodies(one of the antibodies is labeled with Eu as a tracer and theother is immobilized to microtiter plates as the solid phase). A competitivesolid-phase immunoassay for determination of uE3 in serum samples usingtime-resolved fluorescence is described.Microwell coating and blocking: microtiter plates were coated with 200μL/well coating antibody diluted in 50 mmol/L carbonate buffer(pH 9.6) at 4℃for 16 hours,and then blocking at 4℃for 24 hours.Labeling: 0.5 mg protein added to ccentifugal fliter decvices purchased fromMillipore, centrifugate at 5000 rpm for 3 min. and repeated 6 times. Mix protein andEu gently and incubate overnight at 4℃.Coupling: Dissolve up to 50 mg E3 in 1ml DMSO, then 160mg KOH and 50mgbromoacetic acid are added to it. After mixing round for 2 hours, we add 8mldeionized water. Then add the 500μl of HCL solution to the aolution above for 24hours at 4℃. Finally, centrifugate at 10000 rpm for 5 min, we can gain E3-CME. Add10mg E3-CME, 4.4mg N-Hydroxysuccinimide and 7.9mg EDC to 1.5ml DMSO,Dissolve up to 40 mg of lyophilized BSA in 4ml of NaHCO3 buffer. Then twosolution was blended for 24 hours.Purification: separation of the labeled protein from unreacted chelate isperformed by gel filtration(Superdex 200). Elution buffer is Tris-HCl based, e.g, 50mmol/L containing 0.9% NaCl. In gel filtration the collected fraction size is 1ml/vial.The gel filtration elutate can be monitored by UV-absorbance at 280 nm. The Euconcentration of the fractions can be measured by making a 1:1000-1:10000 dilutionin DELFIA Eahancement Solution.Calibration of standards: renference standards of PAPP-A and uE3 were werecalibrated using calibrator purchased from PE.Indexes of reagents: the indexes consisted of analytical sensitivity, accuracy,precision, cross reactivity, stability and correlation.Rusults: The measure range of home-made PAPP-A TRFIA kit was 6-10000mIU/L. Its assay sensitivity was 1.2mIU/L and the recovery rate was 97.9%. Thecross reaction with alpha-fetoprotein, Progestin and Human chorionic gonadotropinwas 0.071%, 1.409% and 0.727%, respectively. The intra-and inter-assay coefficients of variation were 2.30％-4.37％and 2.41％-4.79％, respectively. The correlationcoefficient of 31 blood samples detection results between home-made andcommercially available PAPP-A kit (PerKin Elmer) was 0.991.The measure range of home-made uE3-TRFIA kit was 0.20-180nmol/L. Its assaysensitivity was 0.20nmol/L and the recovery rate was 107.6％. The cross reactionwith E2, Progestin and Progestone was 0.24％, 0.36％and 0.40％, respectively. Theintra-and inter-assay coefficients of variation were 1.33％-3.53％and 0.58％-5.95％, respectively. The correlation coefficient of 149 blood samples detection resultsbetween home-made and commercially available uE3 kit (PerKin Elmer) was 0.925.Conclusion: Time-resolved fluoroimmunoassay using lanthanide labels arecharacteristic of high sensitivity, least interference, good precision and wide dynamicrange. The labeling procedure is simple and the labels are stable. In conclusion,TRFIA will find new wide applications in clinical diagnosis. |
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