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 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 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 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.     

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 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 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 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.     

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%.     

Radioimmunoassay of Plasma 16a-Hydroxydehydroepiandrosterone

Abstract

This report describes a radioimmunoassay method for the measurement of unconjusted 16a-hydroxydehydroepiandrosterone (16a-DHEA) in plasma. The assay uses a specific anti16a-DHEA antiserum which cross reacted completely with dehydroepiandrosterone (DHEA) and to a lesser extent with androstenetriol and androstenediol. A chromatographic system, using celite microcolumns, separated 16a-DHEA from the cross reacting steroids prior to its measurement. Using this simple technique, we have measured 16a-DHEA accurately in small aliquots of plasma from newborns and adults of both sexes. The highest levels of 16a-DHEA were found in newborn infants with a mean value of 16.1 ng/ml. Pregnant women at term had a mean level of 3.2 ng/ml. The levels of 16a-DHEA were low in adults of both sexes with mean values of 0.66 and 0.87 ng/ml for male and female subjects respectively     

Radioimmunoassay of Plasma 16a-Hydroxyprogesterone

Abstract

This report describes a radioimmunoassay method for the measurement of unconjugated 16a-hydroxyprogesterone (16a-P) in plasma. Utilizing an antiserum against 16a-P combined with a one step celite microcolumn chromatographic system, we have measured this steroid accurately in small a liquors of plasma from both male and female subjects. The mean value ± S.D. for nine adult male subjects was 0.69 ± 0.16 ng/ml. Plasma 16a-P was significantly higher (p <0.01) during the luteal (1.20 ± 0.24 ng/ml) than during the follicular phase (0.57 ± 0.18 ng/ml).     

Radioimmunoassay of Testosterone in Murine Plasma

Abstract

This report describes a radioimmunoassay method for measuring unconjugated plasma testosterone (T) and its application to the measurement of T in murine plasma. The parameters of assay reliability, sensitivity, and accuracy were established. Di-hydrotestosterone, the only steroid which showed significant cross-reaction with the testosterone antibody used, was not found in significant quantities in male mice of 19 inbred strains examined. This suggested that chromatographic separation of T from other plasma steroids was unnecessary for the measurement of T in males of this species. Biological levels of T in normal adult male mice of 19 inbred strains showed a range of 0.64 to 21.9 ng/ml plasma and a mean of 5.10 ng/ml. Castration reduced testosterone levels to about 4% of the normal level; castration plus adrenalectomy reduced testosterone to levels below the sensitivity of this method. These investigations demonstrated the validity and reliability of this method for measuring uncanjugated plasma T in male mice.     

Radioimmunoassay for Thromboxane B2

Abstract

A radioimmunoassay for a prostaglandin endoperoxide metabolite, thromboxane B₂, was developed. The antibodies were very specific for this compound, and the method had a sensitivity of 10 pg. Platelets from a human subject were aggregated by addition of collagen and the synthesis of prostaglandin endoperoxides with time was monitored by parallel assay of prostaglandin E₂, F_2α ^and thromboxane B₂.     

Charge-transfer complexes of barbiturates and phenytoin

Simple and sensitive spectrophotometric methods are described, for the first time, for the determination of sodium salts of phenobarbital (1), thiopental (2), methohexital (3) and phenytoin (4). The methods are based on the reaction of these drugs as n-electron donors with the sigma-acceptor iodine and various pi-acceptors: 7,7,8,8-tetracyanoquinodimethane; 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; 2,3,5,6-tetrachloro-1,4-benzoquinone; 2,3,5,6-tetrafluoro-1,4-benzoquinone; 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone; tetracyanoethylene and 2,4,7-trinitro-9-fluorenon. Depending on the solvent polarity, different coloured charge-transfer complexes and radicals were developed. Different variables and parameters affecting the reactions were studied and optimized. The formed complexes were examined by UV/VIS, infrared and (1)H-NMR. Due to the rapid development of colours at ambient temperature, the obtained results were used on thin layer chromatograms for the detection of the investigated compounds. Beer's plots were obeyed in a general concentration range of 1-400 mug ml(-1) for the investigated compounds with different acceptors. Interference from some co-formulated drugs was also studied. No interference was observed due to additives commonly present in the pharmaceutical preparations. The proposed methods could be applied successfully to the determination of the investigated compounds in pure and pharmaceutical dosage forms with good accuracy and precision, the recoveries ranged from 98.7+/-0.5 to 101.1+/-0.5%. The results were compared favourably with the official methods.     

Double-antibody homogeneous fluorescence immunoassay of phenytoin

A homogeneous fluorescence immunoassay suitable for quantifying 5–25 mg l^?1 phenytoin (5,5-diphenyl-2,4-imidazolidenedione) in serum is described. The fluorophor-labeled phenytoin and unlabeled phenytoin (analyte) compete for a limited number of anti-phenytoin antibody binding sites in the presence of anti-fluorophor antibodies. The label employed is a sulfonamido derivative of 2-naphthol-8-sulfonic acid (2-8) which, at neutral pH, undergoes excited-state proton transfer. Binding of anti 2-8 antibodies to the drug-fluorophor conjugate results in quenching of the conjugate base emission at 480 nm. The relative standard deviations are about 5%.     

Simultaneous Radioimmunoassay of Plasma Testosterone and Dihydrotestosterone

Abstract

This report describes the simultaneous radioimmunoassay of plasma testosterone (T) and 5a-dihydrotestosterone (DHT), using as binding reagent a highly specific antiserum which reacted significantly with T and DHT. Utilizing a one step celite microcolumn to separate T from DHT, we have measured these two androgens accurately in small aliquots of plasma from both male and female subjects, Plasma concentrations of T and DHT expressed as mean ± S.D. in ng/ml were respectively: 1) in male subjects: a. normal adult: 4.9 ± 1.6 (T) and 1.3 ± 0.8 (DHT); b. hypogonadal: 0.24 ± 0.08 (T) and 0.20 ± 0.10 (DHT); c. prepubertal: 0.14 ± 0.02 (T) and 0.05 ± 0.02 (DHT). 2) in female subjects: a. normally menstruating, irrespective of time of cycle: 0.22 ± 0.07 (T) and 0.34 ± 0.17 (DHT); b. postmenopausal: 0.29 ± 0.08 (T) and 0.15 ± 0.07 (DHT); c. ovariectomized: 0.24 ± 0.06 (T) and 0.09 ± 0.01 (DHT). When plasma levels of T and DHT were measured during a complete menstrual cycle, the levels of these steroids were found to be higher during the luteal than during the follicular phose. The ratio of plasma T:DHT concentrations was five times higher in normal adult male (3.1) than in menstruating female subjects (0.6).