Radioimmunoassay of human placental lactogen

A sensitive and specific radioimmunossay procedure (RIA) has been developed for the measurement of Human Placental Lactogen (HPL). Pure HPL has been labelled with¹²⁵I and a specific activity of 100 μCi/μgm of HPL has been attained. Dextran-coated charcoal has been employed to separate the bound from the free hormone in radioimmuno-assay. The sensitivity of this technique has been found to be 0.2 ng of HPL. Intraassay and inter assay variations have been found to be less than 10%. This procedure has been adopted to establish the normal range of HPL in pregnant women at different periods of gestation, and to evaluate risk pregnancies.     

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.     

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 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 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.     

Immunonanogold-catalytic resonance scattering spectral assay of trace human chorionic gonadotrophin

Gold nanoparticles in diameter of 10 nm were used to label rabbit anti-human chorionic gonadotrophin (RhCG) antiserum to obtain a resonance scattering spectral probe (AuRhCG) for human chorionic gonadotrophin (hCG). The immunoreaction between AuRhCG and hCG take place to form hCG–AuRhCG immunocomplex in pH 5.0 citric acid–Na₂HPO₄ buffer solution. The immunocomplex solutions were centrifuged to obtain the supernatant solution. The AuRhCG in the supernatant solution exhibited strong catalytic effect on the particle reaction between Ag⁺ and hydroquinone to produce gold–silver composite particles in pH 3.4 citric acid–trisodium citrate buffer solution. There is a stronger resonance scattering (RS) peak at 423 nm for the particles. With the addition of hCG, the AuRhCG in the supernatant solution decreased, and the RS intensity at 423 nm decreased. The decreased RS intensity ΔI_423 nm was proportional to the concentration of hCG in the range of 2.5–208.3 mIU/mL with a detection limit of 0.83 mIU/mL. This method has been applied to the determination of hCG in urine samples, with satisfactory results.     

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 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.     

Purification of human chorionic gonadotropin hormone by anion-exchange high-performance liquid chromatography

Chromatographia - A high-performance liquid chromatography (HPLC) method was developed for the purification of 50mg crude human chorionic gonadotropin (HCG) hormone sample in one chromatographic...     

Biologic activity of human chorionic gonadotropin following reversed-phase high-performance liquid chromatography

Human chorionic gonadotropin (hCG) was analyzed by reversed-phase high-performance liquid chromatography (HPLC) using mobile phases previously described in the literature, as well as newly developed solvent systems. Fractions of hCG collected following reversed-phase HPLC were bioassayed by activation of adenylate cyclase to determine their biologic potencies. hCG retained only 10-60% of its biologic activity following reversed-phase HPLC, depending on the chromatographic conditions employed. A portion of the reduced biologic activity was attributed to dissociation of the alpha- and beta-subunits of hCG at the low pH of the mobile phases, since neutralization of the pH prior to lyophilization and bioassay increased the biologic potency of the chromatographed hormone. The remaining loss in biologic activity is presumably due to organic solvent denaturation.     

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 aflatoxin M1

Radioimmunoassay for the differential determination of aflatoxin M₁ has been developed. It is based on the use of antiserum having almost the same affinity to both aflatoxins B₁ and M₁ /K_a for aflatoxin B₁=2.0×10⁹ and K_a for aflatoxin M₁=2.1×10⁹/ and making possible their simultaneous determination. Aflatoxin B₁ content is determined specifically by a commercial RIA, and aflatoxin M₁ concentration is calculated as the differences between these two assays.     

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.