Optimization and validation of analytical conditions for bovine serum albumin using capillary electrophoresis.

A quick and reproducible capillary electrophoresis method was optimized and validated for the assay of bovine serum albumin (BSA). The effects of various parameters such as pH of buffer, concentration of buffer, capillary dimensions, use of coated capillaries, and additives such as surfactants and protein solubilizers were evaluated. The capillary coatings or additives did not give any advantage in reducing the surface adsorption of BSA on the capillary walls. The optimized conditions include use of borate buffer, pH 8.5 having a concentration of 150 mM in a 27 cm capillary with an aperture window of 100 x 200 microns for detection. The optimized method for the detection of BSA was validated. The interday and intraday coefficient of variation was not greater than 7.59% at BSA concentrations of 25-1000 micrograms/ml. The method developed was reproducible and accurate.     

Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer

Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (pH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear pH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.     

Antibodies reactive with the protein core of MUC1 mucin are present in ovarian cancer patients and healthy women

 Antibodies reactive with peptide epitopes on the core protein of MUC1 epithelial mucin have been demonstrated in some patients with adenocarcinomas. Because these epitopes can be exposed on MUC1 in the serum of healthy women, we measured concentrations of MUC1-reactive antibodies in the serum of healthy pregnant and non-pregnant women, and in patients with benign and malignant ovarian tumours. Antibodies were measured in an enzyme-linked immunosorbent assay utilising a synthetic peptide corresponding to a 105-amino-acid segment of the MUC1 tandem repeat region (5.25 repeats). MUC1-reactive antibodies were always of an IgM isotype and concentrations were highest in young healthy women and declined progressively with age (P = 0.0006) concomitantly with increasing serum MUC1 levels (P = 0.003). Regardless of age, antibody levels were lower in cancer patients than in healthy women (P<0.0001), but MUC1 levels were much higher in cancer patients (P<0.0001). Although high antibody levels were associated with greater survival in ovarian cancer (P = 0.015), multivariate regression analysis showed that this was not a significant independent prognostic indicator after consideration of the International Federation of Gynaecology and Obstetrics (FIGO) stage, histological type, serum MUC1 levels and age. Serial measurement of MUC1 and MUC1 antibodies during treatment in 18 patients with ovarian cancer and throughout pregnancy in 10 women showed a negative correlation between alterations in MUC1 and MUC1 antibodies. These results show that MUC1-peptide-reactive antibodies are present in the serum of healthy women and women with cancer and that they probably form immune complexes with MUC1, but provide no evidence for an augmentation of the humoral immune response to MUC1 in ovarian cancer Received: 8 January 1998 / Accepted: 26 February 1998     

Prevalence and avidity of human herpesvirus-6 specific IgG antibodies in pregnant women in Hungary

The prevalence, the level and the avidity of human herpesvirus-6 (HHV-6) specific IgG were examined in pregnant women and age-matched female blood donors. The study group consisted of 180 women (age 14-45); 60 women with normal pregnancy, 60 pregnant women with fetuses suspected of having any viral infection and 60 healthy blood donors with no history of pregnancy. Plasma or serum samples were tested for HHV-6 IgG antibodies by an immunofluorescence assay. Ninety-eight percent of blood donors and 97% of 120 pregnant women had IgG antibodies to HHV-6. The rate of seropositivity in women with normal pregnancies and women with fetuses suspected to have viral infection was the same. Pregnant women (n = 120) had significantly lower antibody titer than blood donors. No significant differences were found in the same respect between the two groups of pregnant women. Low avidity of IgG antibodies to HHV-6 was detected in 5% of pregnant women.     

复发性自然流产患者外周血中Th17及Treg细胞及其效应因子的变化

目的:探讨复发性自然流产患者外周血中Th17、Treg细胞以及相关细胞因子的变化。方法:选择复发性自然流产患者25例,正常妊娠妇女25例以及正常非妊娠育龄妇女25例,流式细胞术测定外周血中Th17和Treg细胞数量,ELISA法测定血清IL-10、TGF-β及IL-17的浓度。结果:复发性自然流产患者外周血中Th17细胞百分率显著高于正常妊娠以及正常非孕妇女(P0.05),Treg细胞百分率显著低于正常非孕妇女(P0.05),但与正常妊娠妇女相比无显著性差异(P0.05);复发性自然流产患者外周血IL-10及TGF-β水平显著低于正常妊娠妇女以及正常非孕妇女(P0.05),而IL-17水平显著高于正常妊娠妇女以及正常非孕妇女(P0.05)。结论:外周血Th17细胞数和IL-17水平的升高以及抑制性细胞因子IL-10和TGF-β水平的下降可能是复发性自然流产发生的重要原因。     

德州市孕期妇女麻疹抗体水平调查分析

目的了解孕期妇女麻疹抗体水平,为麻疹免疫策略提供依据。方法2005-2006年,在德州市随机抽取部分地区20-38岁孕期妇女进行血清麻疹IgG抗体检测。结果检测702人,麻疹IgG抗体阳性率为84．33％,保护率为37．75％,抗体几何平均滴度（GMT）为1：176．81。不同地区麻疹抗体阳性率和GMT的差异均有统计学意义（P〈0.01）;GMT呈现随年龄增长而增加的趋势（P〈0．05）。结论德州市孕期妇女麻疹IgG抗体水平较低。     

The presence of insertion elements<Emphasis Type="Italic">IS</Emphasis>861 and<Emphasis Type="Italic">IS</Emphasis>1548 in group B streptococci

The presence of insertion elements (IS) IS861 and IS1548 in the collection of 211 Streptococcus agalactiae strains isolated from pregnant women and dairy cows was assayed. IS861 was found in 67 human strains (59%) and 36 bovine strains (37%), IS1548 in 13 human strains (12%) and 16 bovine strains (16%). Two combinations, IS861+ IS1548- and IS861- IS1548-, were widely distributed in both human and bovine strains. The copy number and the restriction fragment length polymorphism (RFLP) of the two IS were determined in human group B streptococcus (GBS) strains. A minimum of 8 copies of IS1548 were detected in GBS strains while the copy number of IS861 varied from 1 to 9. The number of different hybridizing patterns with IS861 and IS1548 probes was 9 and 6, respectively. These hybridization patterns were divided into several clusters. All strains with IS were also clustered according to pulsed field-gel electrophoresis (PFGE) patterns. A correlation was found between the results of PFGE- and IS-based clustering.     

ELISA检测肿瘤患者血清64DP

据报道64DP是一种与肿瘤相关的人血清DNA结合蛋白质,其现有的定量测定方法略嫌繁琐粗糙。本文采用亲和层析法纯化的兔抗64DP抗体,建立了简便灵敏的酶免疫测定法(ELISA)。部分样本经ELISA和火箭电泳两种方法平行测定,证明两者有良好的相关性。对70例恶性肿瘤及86例正常对照血清的测定结果,肿瘤组平均64DP水平显著高于对照组,其中尤以肝癌、胃癌及肺癌的增高更为显著。对非肿瘤其它疾病患者的检测发现感染和外伤也能造成血清64DP的迅速增加。本研究表明血清64DP的改变不仅仅局限于恶性肿瘤,它具有急性期反应蛋白质的特征。这使64DP作为肿瘤标志物应用于临床受到一定限制。     

Immunoenzyme analysis of ovarian metastatic antigen-8]

Immunoenzymatic method for the determination of new ovarian-metastatic antigen-8 in human blood serum was worked out. OMA-8 level was studied in blood serum of 40 healthy women, 40 healthy men, 16 neonates, 33 pregnant women, 103 patients with genital tumours. It was found out that OMA-8 level in blood serum of healthy women changed between 2 to 15.4 micrograms/l, in men between 5-17.0 microgramS/l. OMA-8 concentration higher than 15.4 micrograms/ was considered elevated. The elevated OMA-8 level was marked in neonates (100%) and in pregnant (39.3%). High level was discovered in the amniotic fluid.     

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

妊娠糖尿病患者血清HbA1c与CRP水平的相关性分析

目的:为明确妊娠期糖尿病(gestational diabetes mellitus,GDM)患者血清糖化血红蛋白(glycosylated hemoglobin,Hb A1c)与C反应蛋白(C-reactive protein,CRP)的关系,本研究检测了GDM患者血糖、血清Hb A1c与CRP水平,并对Hb A1c与CRP的相关性进行了分析。方法:以68例2015年4月至2017年4月于我院诊治的GDM孕妇为研究对象,所有患者均符合《妇产科学》中关于GDM的诊断标准,另选取68例正常孕妇为对照组。采用葡萄糖氧化酶法检测血糖水平(空腹血糖及餐后2 h血糖水平),采用免疫凝集法检测和比较两组血清糖化血红蛋白(HbA1c)水平,采用免疫透射比浊法检测血清C反应蛋白(CRP)水平,并分析HbA1c与CRP的相关性。结果:GDM组患者空腹血糖(fasting plasma glucose,FPG)、2 h血糖(plasma glucose,PG)、血清HbA1c和CRP水平均显著高于正常组(P0.05),GDM患者血清Hb A1c和CRP水平呈显著正相关关系(r=0.654,P0.05)。结论:GDM患者血清HbAlC和CRP水平相较于正常孕妇有显著提高,且二者呈显著的正相关关系,二者联合检测可能作为GDM早期诊断的筛查的重要参考指标。     

Purification and characterization of early pregnancy factor from human pregnancy sera

Early pregnancy factor (EPF) is a pregnancy-associated protein detected in the maternal serum by using the rosette inhibition assay and by evaluating the suppression of adoptive transfer of contact sensitivity. Because of its inhibitory effect on the functional reactivity of immunocompetent cells, EPF is thought to be involved in immunoregulation of the maternal immune system during early pregnancy. EPF was purified six million-fold from the serum of pregnant women between 5 and 12 weeks of gestation. The specific activity of purified EPF was approximately 8 x 10(8) units/mg. The purification scheme involved sequential DEAE-cellulose chromatography, S-Sepharose chromatography, concanavalin A-Sepharose chromatography, heparin-Sepharose chromatography, Mono S fast protein liquid chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein has an apparent molecular weight of 21,500 as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 28,000 by gel permeation high pressure liquid chromatography. The isoelectric point of purified EPF moiety is 6.5. The biological activity was susceptible to the proteolytic enzyme trypsin, acidic pH conditions, organic solvents, and sodium dodecyl sulfate, but stable to heat treatment at 56 degrees C for 30 min and the reducing agent dithiothreitol. The biological and physicochemical properties of EPF appear to be distinct from other pregnancy-associated and immunoregulatory proteins.     

Pyridoxal 5'-phosphate disappearance from perfused rat jejunal segments: correlation with perfusate alkaline phosphatase and water absorption

The relative effects of perfusate alkaline phosphatase activity and net water absorption on 2 microM pyridoxal 5'-phosphate (PLP) luminal disappearance from rat jejunum were studied in a single-pass, in vivo perfused intestinal segment model. Perfusate consisted of unlabeled PLP in buffer (pH = 7.4). Net water flux was monitored by inclusion of [3H]polyethylene glycol. PLP was measured by the [14C]tyrosine apodecarboxylase assay. Single and multiple regression analysis of results during perfusion of 2 microM PLP in Krebs bicarbonate buffer demonstrated no correlation between perfusate alkaline phosphatase activity and net water absorption and significant correlations between PLP luminal disappearance and both perfusate alkaline phosphatase activity and net water absorption. Correlation for the latter was improved when disappearance results were corrected for variations in perfusate alkaline phosphatase activity. When perfusate buffers were selected to yield divergent rates of net water absorption, the one associated with greater net water absorption was also associated with greater PLP disappearance. That this could not be explained by changes in perfusate alkaline phosphatase activity was demonstrated both by assessment of the rate of decay of PLP added in vitro to exited perfusate incubated at 37 degrees C and by measurement of alkaline phosphatase activity under conditions defined by the buffers using a modified spectrophotometric assay. Conclusions were: (1) In vivo PLP luminal disappearance correlates significantly with both perfusate alkaline phosphatase activity and net water absorption; (2) these two factors appear to act as independent variables; and (3) future studies on PLP intestinal absorption will need to take both of these variables into account in the interpretation of results.     

The binding of pyridoxal 5'-phosphate to human serum albumin

Most of the pyridoxal 5'-phosphate (PLP) in plasma is bound to protein, primarily albumin. Binding to protein is probably important in transporting PLP in the circulation and in regulating its metabolism. The binding of PLP to human serum albumin (HSA) was studied using absorption spectral analysis, equilibrium dialysis, and inhibition studies. The kinetics of the changes in the spectrum of PLP when mixed with an equimolar concentration of HSA at pH 7.4 followed a model for two-step consecutive binding with rate constants of 7.72 mM-1 min-1 and 0.088 min-1. The resulting PLP-HSA complex had absorption peaks at 338 and 414 nm and was reduced by potassium borohydride. The 414-nm peak is probably due to a protonated aldimine formed between PLP and HSA. The binding of PLP to bovine serum albumin (BSA) at equimolar concentrations at pH 7.4 occurred at about 10% the rate of its binding to HSA. The final PLP-BSA complex absorbed maximally at 334 nm and did not appear to be reduced with borohydride. Equilibrium dialysis of PLP and HSA indicated that there were more than one class of binding sites of HSA for PLP. There was one high affinity site with a dissociation constant of 8.7 microM and two or more other sites with dissociation constants of 90 microM or greater. PLP binding to HSA was inhibited by pyridoxal and 4-pyridoxic acid. It was not inhibited appreciably by inorganic phosphate or phosphorylated compounds. The binding of PLP to BSA was inhibited more than its binding to HSA by several compounds containing anionic groups. It is concluded that PLP binds differently to HSA than it does to BSA.     

Proteome analysis reveals elevated serum levels of clusterin in patients with preeclampsia

Preeclampsia is a pregnancy-specific syndrome and a major cause of maternal mortality. The pathophysiology of preeclampsia is unknown, and no proteome analysis of preeclampsia has been reported. We sought to identify proteins associated with preeclampsia using a proteomic technique and performed two-dimensional electrophoresis (2-DE) on sera from six patients with preeclampsia and six normal pregnant women, followed by comparison of the SYPRO Ruby-stained 2-DE profiles. A group of overexpressed spots was identified in the limited study set. Overexpressed spots were identified as clusterin by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) followed by peptide mass fingerprinting, a protein database search, and Western blot analysis. Additionally, sera of 80 preeclamptic women and 80 normal pregnant women were processed by immunoassay methods to confirm changes in clusterin concentrations quantitatively. Immunoassays showed that clusterin levels in the 80 preeclamptic women were significantly higher than those in the 80 controls (mean +/- SD; 1.62 +/- 0.46 times reference level in preeclamptic women vs. 1.30 +/- 0.46 times reference level in controls, P < 0.001). Proteomic analysis of serum proteins is a promising tool for studying preeclampsia pathophysiology and identifying proteins associated with preeclampsia.     

血清妊娠相关血浆蛋白A在诊断异常妊娠中的临床意义

目的:探讨血清妊娠相关血浆蛋白A(PAPP-A)在诊断异常妊娠中的临床意义,分析其与异常妊振的关系。方法:选取299例5~13周的正常早孕妇为正常早孕组,同期选取稽留流产86例,先兆流产54例,异位妊娠76例为异常妊娠组,用酶联免疫吸附试验(ELASA)测定两组受试者的血清PAPP-A水平,分析两组受试者各个孕周内的血清PAPP-A水平的差异。结果:稽留流产孕妇在各个孕周内(9~13周)的血清PAPP-A水平显著低于同孕周内正常早孕孕妇(t值分别为9.500,8.113,3.511,9.538,8.504,P值均0.05);稽留流产孕妇总的平均血清PAPP-A水平亦低于常早孕孕妇(t=3.651,P值均0.05);异位妊娠孕妇在各个孕周内(9~13周)的血清PAPP-A水平显著低于同孕周内正常早孕孕妇(t值分别为7.976,9.030,9.941,11.625,14.079,12.569,P值均0.05),异位妊娠孕妇总的平均血清PAPP-A水平亦低于常早孕孕妇(t=28.168,P值均0.05);先兆流产孕妇(除孕8周)与正常早孕妊娠血清PAPP-A水平比较无显著统计学意义。结论:血清PAPP-A水平在异常妊娠如异位妊娠、稽留流产中显著降低,可作为诊断异位妊娠、稽留流产及先兆流产辅助诊断的生物学指标。     

The role of serum fructosamine as a screening test for gestational diabetes mellitus

The serum fructosamine concentration indicates the degree of glycation of serum proteins, particularly albumin, and reflects an average blood glucose level over the previous 1-3 weeks. Serum fructosamine, glycated haemoglobin (HbA1c), total serum protein, serum albumin, fasting plasma glucose and oral glucose tolerance test (OGTT) have been measured in 127 healthy control subjects, 102 type 1 and 152 type 2 diabetes mellitus patients and 106 nondiabetic pregnant women. Fructosamine concentration of 2.24 +/- 0.16 and 3.21 +/- 0.41 mmol/l (mean +/- S.D.) has been found in control subjects and diabetics respectively (P less than 0.001). During the second trimester a significantly lower fructosamine level (1.92 +/- 0.21 mmol/l) has been found in pregnant women, most likely due to the low serum albumin concentration (31.35 +/- 3.97 g/l). None of them had a fructosamine level above the normal limit of 2.55 mmol/l. On the other hand, 12 pregnant women showed a disturbed OGTT with normal fructosamine. If the serum fructosamine concentration was adjusted for 40 g/l albumin, then a mean fructosamine of 2.16 +/- 0.24 mmol/l was found in patients with gestational diabetes. Our results show that serum fructosamine has a similar diagnostic value as HbA1c for non-pregnant adults, but neither can replace OGTT for the diagnosis of gestational diabetes.     

Progesterone control of nuclear estrogen receptor: demonstration in hamster uterus during the estrous cycle and pseudopregnancy using a new exchange assay

Our previous studies showed that total nuclear estrogen receptor (Re) can be extracted and measured by exchange using 10 mM pyridoxal-5'-phosphate (PLP) at low temperature (0-4 degrees C). In order to further validate the PLP assay, we measured the Re concentration in uterine cytosol and nuclei by this method under physiological conditions, i.e. during the hamster estrous cycle and pseudopregnancy. In addition, we compared the Re results obtained by the PLP method with those obtained with two other assay procedures, i.e. the KCl and NaSCN methods. During the follicular phase of the estrous cycle, all three methods showed an elevation of nuclear Re in parallel with the increase in serum estradiol (E). However, the quantity of nuclear Re obtained with the PLP method was significantly greater than with the KCl method during the follicular phase. The surge of serum progesterone (P) during the ovulatory phase of the estrous cycle was followed by a dramatic fall in nuclear Re, and the greatest loss of nuclear Re during the ovulatory phase of the cycle was detected with the PLP and NaSCN methods. On a DNA basis, cytosol Re increased significantly between Day 3 and proestrus and subsequently fell during the ovulatory phase of the cycle. P treatment of proestrus hamsters resulted in a rapid (less than 4 h) loss of nuclear Re with little or no change in cytosol Re. Chronic P exposure during pseudopregnancy with serum E maintenance, resulted in a significant suppression of both cytosol and nuclear Re. Following P withdrawal, both cytosol and nuclear Re rose significantly (less than 4 h), indicating that this effect of P was readily reversible. The results demonstrate that the Re detected under physiological conditions by the PLP method responds to both E action and P action, and that the PLP assay provides a greater recovery of Re as compared to the KCl assay.     

A highly sensitive assay for ritodrine in human serum by hydrophilic interaction chromatography-tandem mass spectrometry

We developed a sensitive assay for ritodrine (RTD), a beta2-adrenergic agonist, in human serum. This method was based upon the selective and sensitive technique by a tandem mass spectrometry (MS/MS) using a hydrophilic interaction chromatography (HILIC) technique. This method involved a mixed-mode cation-exchange solid-phase extraction of RTD and isoxsuprine, the internal standard (IS), from serum with Waters Oasis MCX cartridges. The detection was made using a Micromass Quattromicro API LC-MS/MS system with electrospray ionization source in positive ion mode. The separation of the analytes was achieved within 4 min on a silica column with a mobile phase of ammonium acetate (10 mM, pH 4.5) and acetonitrile (10:90, v/v). Multiple reaction monitoring was utilized by monitoring 288.2-->121.1 for RTD, 302.2-->107.0 for IS. The calibration curve for RTD was linear over a range of 0.5-1000 ng/mL. When 50 microL serum was used for extraction, the lower limit of quantification was 0.39 ng/mL (97.5 fg on-column). The percent coefficient of validation for accuracy and precision (inter- and intra-day) was less than 9.8% and the recovery was ranged from 83.5 to 94.7% for RTD. This method enabled us to successfully determine RTD in maternal and fetal sera.