A rapid magnetizable solid phase radioimmunoassay kit for human placental lactogen

The radioimmunoessay of human placental lactogen (HPL) with separation of antibody bound and free hormone was achieved by the magnetizable solid phase coupled to antibody. The precision, accuracy, sensitivity and specificity of the method has been carefully checked in this study and the procedure of the assay was performed at room temperature. The above parameters were evaluated by recovery test (99%); within assays (4%), between assays (5%), sensitivity (0.04 g/ml) and there was no obvious cross reactivity with human growth hormone (hGH) and human chorionic gonadotropin (hCG).     

Pregnancy monitoring with enzyme-immunoassays for human placental lactogen and total oestrogens

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Pregnancy monitoring with enzyme-immunoassays for human placental lactogen and total oestrogens

     

Radioimmunoassay of human chorionic gonodotropin

A rapid and sensitive radioimmunoassay has been developed for the measurement of Human Chorionic Gonadotropin (HCG) in serum. Human chorionic gonadotropin has been labelled with¹²⁵I to attain a specific activity between 80–120 μCi/μg. Aqueous dioxane (74 vol. %) has been employed to separate the bound and freehormone in the radioimmunoassay. The sensitivity of this technique has been found to be ∼1.5 mIU/ml of HCG. The intra assay variation has been found to be less than 5% and inter assay variation has been found to be less than 12%.     

Peroxidase-catalysed rupture of the CF bond as and indicator reaction for enzyme immunoassay : Kinetic determination of human immunoglobulin G, α-fetoprotein and placental lactogen

A fluoride ion-selective electrode is utilized as a sensor for the kinetic determination of peroxidase label in enzyme immunoassays. The method is based on a sandwich enzyme-linked immunosorbent assay (ELISA) technique, the peroxidase-catalysed rupture of the covalent CF bond in 4-fluorophenol and the subsequent release of fluoride ions. The determination of human immunoglobulin G (lgG), human α-fetoprotein (AFP) and human placental lactogen (HPL) was investigated. The potentiometric measurement of the rate of release of fluoride ion within 5 min provided a direct correlation with the concentration of analyte present in the sample. The concentration ranges investigated for the analytes were IgG 30 μg l^?1–10mg l^?1, AFP 5–500 μg l^?1 and HPL 60 ng l^?1–1 mg l^?1. Under the given experimental conditions, the detection limits were IgG 30, AFP 12.8 and HPL 1 μg l^?1. Replacing the rate method with the fixed-time mode (15–30 min) did not improve the detection limits. The performance of the present method was found to be comparable to that of the spectrophotometric detection technique.     

Radioimmunoassay

Biologically active substances are often effective in nanogram or even smaller quantities. Investigations of their mode of action therefore require highly sensitive microanalytical methods of detection. Chemical, chromatographic or spectrometric methods frequently lack the required sensitivity; it was only after the discovery of the radioimmunoassay (RIA) that quantitative determination of minute amounts of substances in the presence of a millionfold excess of other compounds became possible. The radioimmunoassay combines the extreme sensitivity of detection of radioactively labeled substances with the high specificity of immunological reactions. The superiority of radioimmunological methods over other analytical techniques rests upon their sensitivity, specificity, facile application, and especially their broad general scope.     

Purification of human placental prostaglandin 15-hydroxydehydrogenase

The NAD(+)-linked prostaglandin 15-hydroxydehydrogenase, which is responsible for the physiological inactivation of prostaglandins by catalysing the first step in the catabolism, was isolated and purified 995-fold from human placenta. The introduction of two new chromatographic steps in the purification procedure is responsible for an achieved specific activity of 1791 mU/mg. The molecular mass of the enzyme, as estimated by fast protein liquid chromatography, was 24,500 dalton. Sodium dodecyl sulphate discontinuous gel electrophoresis of the denatured enzyme revealed a molecular mass of 24,000 dalton. These data suggest that the enzyme consists of a single polypeptide chain.     

Radioimmunoassay of Triazolam

Abstract

Two radioimmunoassay (RIA) assay methods (I, II) have been developed for triazolam (T, U-33,030) in serum and plasma. The parameters of equilibration time, serum blank, antibody specificity, extraction efficiency, and drug-binding to glass were studied. Of the various triazolam analogs tested for cross reactivity, only the hydroxy metabolites interfered significantly. At the levels normally found in plasma or serum, a background blank was encountered from constituents such as fatty acids, lysolecithin, lecithin, and cholesterol. However, serum or plasma samples could be analyzed with (I) by constructing standard curves in which blank serum from the same subject was used. An alternate method (II) was found which simultaneously extracted and precipitated the interferences. Both methods could be employed for analysis of plasma or serum samples. However, II detects T metabolites less efficiently than I. The within day and between day coefficients of variation for method I were found to be 5.7% and 3.9% respectively at the 8 ng/ml level. Method I is suitable for measuring large numbers of samples where blank subject serum can be obtained and where detection of T metabolites in addition to T would not present a problem.     

Radioimmunoassay of phenytoin

We describe a radioimmunoassay procedure for the measurement of phenytoin (5,5-diphenylhydantoin) in serum samples. Antiserum to phenytoin has been produced against phenytoin-valeric acid-bovine serum albumin conjugate.¹²⁵I labelled phenytoin-acetic acid-tyrosine methyl ester has been used as a tracer. The assay covers a range of 10–500 ng/cm³ and has a sensitivity of 0.25 ng. The assay is validated by specificity tests, precision profile and recovery tests.     

Radioimmunoassay of human growth hormone and its application in pituitary dysfunction studies

A simple, specific and sensitive Radioimmunoassay (RIA) has been developed for the measurement of Human Growth Hormone (HGH) in serum samples.¹²⁵I-labelled HGH has been used as a tracer and dextran coated charcoal system employed to separate antibody bound hormone from the unbound one. The assay offers sensitivity of 0.16 ng/ml with a reproductibility of 7% intrassay and inter-assay variations. Serum HGH levels were measured at fasting-resting state and during insulin stimulation test in (1) 15 normal subjects (controls and (2) 31 patients with stunted growth, whereas (3) in 7 acromegalic patients the same were measured at fasting-resting state and after oral glucose administration. This procedure has been used to distinguish dwarfs due to growth hormone deficiency from other conditions unrelated to pituitary disease and to confirm acromegaly.     

Cysteine carboxyl O-methylation of human placental 23 kDa protein

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.     

Radioimmunoassay of Plasma Corticosterone

Abstract

This report describes a radioimmunoassay method for the measurement of unconjugated corticosterone (B) in plasma. Utilizing an anti-Cortisol antiserum cross reacting with B combined with a one step celite microcolumn chromatographic system, we have measured this latter steroid accurately in small aliquots of plasma from both male and female subjects. The plasma levels of B obtained by this method agreed well with levels observed in adult men and women, using other techniques. During a menstrual cycle studied, wide fluctuations were observed with levels ranging from 1, 3 to 14, 4 ng/ml. Adrenal suppression decreased the levels of B below 1 ng/ml. The mineralocorticoid B is predominantly ACTH -dependent.     

Radioimmunoassay of Plasma Pregnenolone-Sulfate

Abstract

This report describes a radioimmunoassay method for the direct measurement of pregnenolone-sulfate (⁵Δ-P-S) in diluted plasma. Utilizing a specific anti-⁵Δ-P-S antiserum which reacted both with pregneolone (⁵Δ-P) and its sulfate (⁵Δ-P-S), we have measured ⁵Δ-P-S with accuracy in small aliquots of diluted plasma. Because the plasma concentration of ⁵Δ-P-S is relatively high, the effect of ⁵Δ-P and other steroids on the assay was negligible. The usable range of the standard curve was between 0.1 and 25 ng of ⁵Δ-P-S. The range of values obtained by this method for plasma ⁵Δ-P-S in peripheral maternal plasma and in the newborn compared favorably with the range of levels observed with other reliable but more cumbersome methods.     

Radioimmunoassay of Plasma Dehydroepiandrosterone

Abstract

This report describes a radioimmunoassoy method for measurement of unconjugated dehydroepiandrosterone (DHEA) in plasma. Utilizing a highly specific DHEA antiserum combined with a one step celite microcolumn chromatographic system, we have measured this steroid accurately in small aliquots of plasma from both male and female subjects. The mean value for both male and nonpregnant females was 2.7 ng/ml. The values for third trimester pregnant females ranged from 0.5 ng/ml to 12.5 ng/ml. When plasma DHEA was measured over a complete menstrual cycle, the levels were higher during the early and latter parts of that cycle than at midcycle. One male subiect, studied over a 24 hour period for diurnal variation, demonstrated highest DHEA levels during the early morning.     

Radioimmunoassay of Plasma Cortisol

Abstract

A radioimmunoassay method for the direct measurement of cortisol in plasma after precipitation of the proteins with ethanol is described. Utilizing a specific antiserum against cortisol, with minimal or no cross reaction with other steroids, cortisol was meas-red accurately in a volume of 0.001 ml of plasma. The range of levels that could be measured per ml of plasma varied from 10 to 500 ng. The mean value of plasma cortisol at 8 a. m. obtained in twelve subjects by this method was about half the mean levels reported by another competitive protein binding assay.     

Isolation and characterization of human placental chorionic villar extracellular matrix

The cell-free extracellular matrix of human placental chorionic villi has been prepared by a procedure employing extraction of the terminal villar fragments with the detergents Triton X-100 and sodium deoxycholate. The isolated human placental extracellular matrix retains an intact, but collapsed, histoarchitecture, as observed by scanning and transmission electron microscopy. It remains intact, in large part because of the presence of continuous sheets of villar basement membranes and associated interstitial collagen fibers and scattered patches of fibrin. The staining charcteristics and chemical composition of the isolated human placental extracellular matrlix are similar to those reported for basement membranes in several tissues and indicate the presence of collagen-like and glycoprotein components in this preparation. Gel electrophoresis of urea-SDS-mercaptoethanol extracts of the matrix showed that it consists of several polypeptide components of various molecuar weights, some of which are associated into high molecular weight complexes by disulfide bonds.     

Radioimmunoassay of Plasma 17-Hydroxypregnenolone

Abstract

This report describes a radioimmunoassay method for the measurement of unconjugated 17-hydroxypregnenolone (17-⁵δ-P) in plasma. Utilizing a highly specific 17-⁵δ-P antiserum combined with a one step celite microcolumn chromatographic system, we have measured this steroid accurately in small aliquots of plasma from both male and female subjects. The plasma levels of 17-⁵δ-P obtained by this method agreed well with levels observed in adult men and women, using two other techniques. Compared with the latters, however, the present method offered a tenfold greater sensitivity and a better precision.     

Radioimmunoassay of Plasma Dehydroepiandrosterone Sulfate

Abstract

This report describes a radioimmunoassay method for the direct measurement of dehydroepiandrosterone sulfate (DHEA-S) in diluted plasma. Utilizing a specific DHEA-S antiserum which reacted completely with DHEA, we have measured DHEA-S accurately in small aliquots of diluted plasma. Due to the relatively high concentration of DHEA-S in plasma, the effect of DHEA and other steroids on the assay was negligible. The usable range of the standard curve was between 10 and 500 pg DHEA-S. The range of levels that could be measured per ml of plasma varied from 0.02 to 5.0 pg. The range of values obtained by this method for plasma DHEA-S compared favorably with the ronge of levels observed in adult men and women, using various other techniques.