Radioimmunoassay without extraction of L-triiodothyronine in human serum]

A radio-immunoassay of L-triiodothyronin is described. Blinding of T3 to TBG is prevented by salicylate (1%--w/v). The rabbit antiserum obtained by injection of a complexe T3-bovine serum albumin is diluted between 1/10000 and 1/20000. Bound and free hormone are separated by polyethylene glycol (17%--w/v). Serum T3-concentration in normal subjects averaged 132 +/- 29 ng/dl (SD). In hyperthyroid and hypothyroid patients, these values were respectively 351 +/- 118 ng/dl and 68 +/- 21 ng/dl.     

Radioimmunoassay of estradiol-17beta in bovine peripheral plasma with and without chromatography.

An assay of estradiol-17beta (E217beta) in bovine peripheral plasma is described. The plasma is incubated with an antiserum to E217beta-BSA and the gamma-globulin fraction precipitated with ammonium sulphate. After extraction with diethyl ether E217beta in the precipitate is estimated by radioimmunoassay using a specific antiserum against E217beta-6-BSA. Plasma concentrations of E217beta during the normal estrous cycle determined by this method and by a method involving Sephadex LH-20 chromatography range from 4 to 23 pg/ml.     

Radioimmunoassay of plasma testosterone without chromatography.

A simple and rapid radioimmunoassay for determination of testosterone in peripheral plasma is described. A crude extract of the plasma is assayed directly without chromatography using an antiserum raised against testosterone-3-(carboxymethyl) oxime-bovine serum albumin. Accuracy and precision are satisfactory. Specificity is sufficient for the most purposes as has been demonstrated by comparison with a radioimmunoassay including chromatography.     

Radioimmunoassay for ovine, caprine and bovine prolactin in plasma and tissue extracts

1. A radioimmunoassay for ovine prolactin is described based on the inhibition of the reaction between (131)I-labelled ovine prolactin and guinea-pig or rabbit antiserum to ovine prolactin. The extent of the reaction after a 4-day incubation period is determined by chromatoelectrophoresis or by adsorption of unchanged (131)I-labelled ovine prolactin on charcoal. The sensitivity is equal to 5.9ng. of prolactin/ml. of plasma with chromatoelectrophoresis, or 0.2ng. of prolactin/ml. of tissue extracts with the charcoal separation. 2. A complete cross-reaction demonstrated between ovine prolactin and caprine pituitary extracts allows the assay to be used to measure caprine prolactin. The partial cross-reactions between ovine prolactin and bovine prolactin and between ovine prolactin and bovine pituitary extract differ, and an alteration in the immunological activity of bovine prolactin during its isolation is suggested. Bovine prolactin in plasma may be measured against a bovine pituitary extract as standard. No cross-reactions were demonstrated with pituitary extracts from a number of other species. The extent of the contamination of ovine and bovine growth hormone preparations by their respective prolactins is shown. 3. Dilutions of ovine and caprine plasma inhibit the reaction between (131)I-labelled ovine prolactin and antiserum with the same characteristics as ovine prolactin. 4. The immunoreactive material in plasma fractionates on Sephadex G-200 and in sucrose density gradients as a single peak similar to that shown by freshly dissolved ovine prolactin. There is no evidence that ovine prolactin is bound to a plasma protein. 5. By suppressing prolactin secretion and assaying serial samples of plasma thereafter it is shown that the immunological activity of the surviving hormone becomes progressively altered with time. It is suggested that this alteration is usually not detected but introduces an element of uncertainty into the quantitative but not the qualitative value of the measurements obtained by reference to standard ovine prolactin.     

Radioimmunoassay for aldosterone in plasma without chromatography.

A radioimmunoassay without chromatography is described for the determination of plasma aldosterone. The high sensitivity of the method renders possible the detection of about 1 pg aldosterone/ml. The high specificity of the antialdosterone sera (rabbit) may be due to the procedure used for the preparation of aldosterone-21-hemisuccinate and to the intensive purification of the aldosterone-albumin conjugate. The validity of the method was tested by determination of plasma aldosterone in normal subjects and in patients suffering from primary hyperaldosteronism or Addison's disease. In cases of urgent diagnosis, the incubation period was reduced from 16 hours to 1 hour. The elimination of the chromatographic step makes the method suitable for clinical routine work and automatization.     

A simple progesterone radioimmunoassay without column chromatography

A radioimmunoassay for plasma progesterone without Chromatographie purification was developed. The 11-hemi-succinate of 11 -hydroxy-progesterone conjugated to bovine serum albumin was injected into rabbits to stimulate antibody production. The resulting antisera was used at a final dilution of 1:3500. The mean recovery of labeled progesterone added to 100 samples after ether extraction (88.9 ± 9.1%) was higher than the recovery obtained when column chromatography followed ether extraction (84.8 ± 7.5%). For comparison, plasma pools were assayed for progesterone with and without the use of columns. A female plasma pool (luteal phase) gave a mean of 546.3 ± 26.5 (SD) ng/100 mls (n = 5) without column chromatography and 557.2 ± 20.8 (SD) ng/100 mls (n = 5) with column chromatography. Another female plasma pool (follicular phase) gave a mean of 87.9 ± 9.6 (SD) ng/100 mls (n = 24) with column chromatography and 93.3 ± 8.6 (SD) ng/100 mls (n = 7) without column chromatography. A male plasma pool gave a mean of 22.8 ± 4.4 (SD) ng/100 mls (n = 13) with column chromatography and 21.8 ± 7.7 (SD) ng/100 mls (n = 3) without column chromatography. The intra assay and inter assay precision gave a coefficient of variation of 3.7 for six samples and 10.9 for 24 samples, respectively. The specificity of the antibody was determined by checking cross reactivity with 26 steroids. The sensitivity (25 pg) and accuracy were proven to be highly satisfactory.     

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

Thin-layer chromatography of tissue lipids without extraction

Frozen tissue sections were applied directly to silica gel plates, and the lipids were separated by developing the plates in different solvent systems. Quantitation of the lipid classes was achieved by direct transmission densitometry after the plates were treated with a chromic acid-sulfuric acid spray.     

Radioimmunoassay of plasma progesterone after oral administration of micronized progesterone

A 200 mg dose of micronized progesterone (P) was administered orally to six healthy volunteers. Serial blood samples were collected and plasma P was assayed according to three methods of different specificity. The results were compared to those obtained with a reference technique based on gas chromatography-mass spectrometry. The interference of P metabolites particularly 5 alpha- and 5 beta-dihydroprogesterone was shown to be important when no or inadequate chromatography was included in the method. Radioimmunoassay after chromatography on a Celite column yielded results similar to those of the reference method. After oral administration, plasma P reached a peak at 3.00 +/- 0.44 h (mean +/- SEM; range: 1-4 h). The mean peak level 4.70 +/- 1.15 ng/ml (mean +/- SEM; range: 2.40-10.10 ng/ml) was at the lower limit of the range defined for a normal functional corpus luteum. The levels returned to initial values by 24 h after the single oral dose. The separation of P from its metabolites by an adequate chromatographic system appears mandatory after oral administration of micronized P.     

Culture of ovine embryos in the absence of bovine serum albumin

Bovine serum albumin (BSA), a relatively impure protein, is routinely used as a component of embryo culture media. Since media containing BSA are chemically undefined, it would be desirable to replace BSA with substitutes of similar activity which are either chemically better defined and/or better standardized than BSA. Two commercial products, Ultroser((R)) G (USG) and Solcoseryl((R)) (SOL), were evaluated as replacements for BSA in culture with respect to the development of ovine embryos in vitro. A total of 126 late 8-cell and early 16-cell embryos were distributed among modified Brinster's medium for ovum culture (BMOC-2) containing either 1.5% BSA, 2.0% USG or 2.0% SOL. All three culture media supported development of ovine embryos. Results indicate that 8- and 16-cell embryos will develop into blastocysts in a BSA-free medium containing either USG or SOL. A higher number of embryos developed into blastocysts in media containing BSA than in media containing USG or SOL, and more blastocysts hatched in media containing BSA. Although the overall degree of embryonic development was more advanced in BSA-supplemented media, the concentrations of USG and SOL that were used in this study may not have been optimal for ovine embryo culture.     

Effect of time and temperature on bovine serum and plasma progesterone concentration

Whole blood, with and without anticoagulant, from 5 pregnant cows was incubated at 40°C for 0 (30 minutes after collection), 6 and 24 hours (hr) before the blood was centrifuged and the plasma or serum was frozen for later progesterone assay. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 1.7 ng/ml at 6 hr (P < 0.01) and to 2.8 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 3.9 ng/ml at 6 hr (P < 0.01) and to 4.4 ng/ml at 24 hr (P < 0.01). Whole blood samples with and without EDTA were also incubated at 4°C for 24 hr. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 4.2 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 4.7 ng/ml at 24 hr (P < 0.01). The incubation time and temperature of whole blood, from collection of blood to the separation of serum or plasma, significantly affects assayable concentration of progesterone.     

Changes in porcine,ovine, bovine and equine blood progesterone concentrations between collection and centrifugation

The aim of this study was to determine if different methods of handling porcine, ovine, bovine and equine blood between collection and centrifugation influence measurable progesterone levels. A 2 × 2 × 5 factorial experiment was conducted for each species with heparin (with or without), temperature of incubation (4 and 22°C) and time of incubation (0, 6, 12, 24 and 48 h) as the main effects. Following centrifugation, plasma and serum samples were stored at ?20°C until progesterone concentrations were determined by radioimmunoassay. Method of handling porcine and equine blood between collection and centrifugation did not affect the levels of progesterone. However, heparinized blood held at 4°C resulted in the most consistent levels of progesterone over time. Progesterone levels were fairly consistent across time in the ovine blood by all methods of handling except heparinized blood incubated at 22°C. By 24 h after collection, plasma progesterone concentrations decreased by 50% for the ovine blood incubated at 22°C with heparin. Decreases were detected by all the methods of handling the bovine blood between collection and centrifugation. The rate of decline, however, was considerably faster for blood held at 22°C than blood held at 4°C. At 12–48 h after collection, the concentrations of progesterone averaged only 5% of the time 0 sample for blood incubated at 22°C. In contrast, at least 30% of the progesterone values in the time 0 sample were detected between 12 and 48 h of incubation for the blood held at 4°C.     

Radioimmunoassay of 17-hydroxyprogesterone in serum with or without thin-layer chromatographic purification.

A radioimmunoassay for the measurement of 17-hydroxyprogesterone (17-OHP) is described. The antigen 11-desoxycortisol-21-hemisuccinate-bovine serum albumin has been used to produce two antisera of different antibody populations in the same animal. The thin-layer chromatographic system described can be used to separate all the cross-reacting steroids investigated from 17-OHP. A simplified method is also presented using preliminary solvent extraction only. The mean 17-OHP levels measured, using this method, were, for normal men, 0.86 +/- 0.19 ng/ml (SD), for normal females 0.30 +/- 0.19 ng/ml in the follicular phase and 1.72 +/- 0.18 ng/ml in the luteal phase of the menstrual cycle, and 0.45 +/- 0.17 ng/ml in normal postmenopausal women.     

Radioimmunoassay and enzymeimmunoassay of plasma progesterone as monitors of progesterone sponge treatment in ewes

The daily plasma progesterone (P) concentrations achieved during insertion of P (750 mg) sponges into two groups of ewes were examined. Group I received prostaglandin (PG) treatment, which was required to suppress the P production (to levels of < 0.3 ng hormone/ml plasma) from the corpora lutea (CL) of a previous superovulation treatment, following which these Group I ewes and the anestrous Group II ewes were sponge treated. Radioimmunoassay (RIA) and enzymeimmunoassay (EIA) were used to measure the P levels in both groups. Progesterone (750 mg) sponges with and without citric acid impregnation were inserted into all the ewes for 12 days (d). Citric acid lowered the P levels reaching the plasma from the sponges, but it did not mask the characteristic profile (during the treatments) determined by the states of the ewes (single PG and double PG injected, Group I or in the anestrous Group II). The plasma P levels in Group I and II ewes rose to at least 7.0 ng/ml at intervals during treatment. The duration and magnitude of the P concentrations in the plasma were higher in the single PG compared with the double PG ewes during sponge insertion in Group I. The anestrous Group II ewes showed two major peaks (Day 1, P<0.01 and Days 11 to 12, P<0.05) during sponge treatment. A P level > 2.0 ng/ml was maintained over the entire treatment in the single PG and in the anestrous hormone-treated ewes, and was of shorter duration (7 d) in the double PG-treated animals. These endogenous patterns in P profiles of the ewes indicate that the hormone level during sponge insertion varies in magnitude and duration, parameters determined by the physiological/endocrinological state of the ewes at the start of the treatment. The EIA correlated significantly (P<0.001) with the RIA for the measurement of P concentration, when analyzed daily on an individual animal basis.     

Size exclusion chromatography for extraction of serum bile acids

A major problem in the measurement of serum bile acids is their quantitative extraction from the high molecular protein matrix. In our hands, the standard techniques of adsorption and reversed-phase chromatography have yielded incomplete recovery for different bile acids (33-93%) and poor reproducibility. In contrast, with the novel extraction procedure of size exclusion chromatography, recovery was nearly quantitative (75-104%) and reproducibility was satisfactory. The described method allowed for a reliable determination of serum bile acids in healthy subjects and patients with liver cirrhosis. We conclude that size exclusion chromatography for serum bile acid extraction is more reliable than alternative techniques, because the separation by size is independent of solubility, charge, and polarity.     

Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: its application for pregnancy diagnosis.

A sensitive and specific double-antibody RIA for a bovine pregnancy-associated glycoprotein (bPAG) is described. The limit of detection was 0.2 ng/ml. The assay was specific for bPAG in that pituitary and placental gonadotropic hormones and other placental or serum proteins assayed in serial dilutions did not cross-react. The RIA allowed measurement of bPAG in placental extracts, fetal serum, fetal fluids, and serum or plasma of pregnant cows. About 20% of unbred heifers and nonpregnant cows had detectable levels ranging from 0.30 +/- 0.09 to 0.50 +/- 0.17 ng/ml (mean +/- SD), and 15% of bull sera showed higher concentrations (3.01 +/- 1.73 ng/ml) of bPAG or bPAG-like protein. Variations among animals was observed in fetal serum bPAG concentrations. Bovine PAG was detected in maternal peripheral blood at Day 22 of pregnancy (mean +/- SD, 0.38 +/- 0.13 ng/ml) in some animals and at Day 30 in all pregnant cows. Peripheral serum bPAG levels increased progressively to 3.60 +/- 1.73 ng/ml (mean +/- SD) at Day 30 of pregnancy, to 24.53 +/- 8.81 ng/ml at Day 120, and to 1551.91 +/- 589.68 ng/ml at Day 270. Peak concentration of bPAG was 2462.42 +/- 1017.88 ng/ml and it occurred 1-5 days prior to parturition. After delivery, bPAG concentrations decreased steadily to 499.63 +/- 267.20 ng/ml at Day 14 postpartum (pp), 10.12 +/- 7.84 ng/ml at Day 60 pp, and 1.44 +/- 1.08 ng/ml at Day 90 pp. The undetectable concentration (less than 0.20 ng/ml) was reached by Day 100 +/- 20 pp. An investigation undertaken in Holstein heifers, Holstein cows, and Hereford cows used as recipients for purebred Holstein embryos supplied evidence of the influence of breed of recipient and sex of fetuses on peripheral concentrations of bPAG. A herd of 430 Holstein-Friesian heifers that had received transferred embryos were bled at Day 35 postestrus (pe) for measurement of bPAG. The bPAG was detected in 287 of 430 serum samples analyzed. By rectal palpation performed at Day 45 pe, 267 heifers with detectable levels of bPAG at Day 35 pe were confirmed to be pregnant as were 3 of 143 heifers previously diagnosed as not pregnant by RIA. These results suggest that detection of this placental-specific antigen in the serum could be used as a specific serological method for early pregnancy diagnosis in cattle from 28 days after breeding.     

Radioimmunoassay of plasma and salivary progesterone during the menstrual cycle

We have developed specific and sensitive radioimmunoassays for progesterone in saliva (SP4) easily applicable in routine practice to assess ovarian function. One of them uses [³H]progesterone, the other 3-CMO-[¹²⁵I]histamin as tracer. The reliability criteria of both techniques have been controlled and the results obtained with the two methods in 50 saliva samples are correlated well. Our clinical study has been chiefly done with [³H]progesterone. In the present study we reported the values obtained daily during the greatest part of the menstrual cycle in 14 normal cases and they are in agreement with previous reports. The use of [¹²⁵I]tracer, by increasing the sensitivity of the RIA and shortening the duration of the assay, improves the use of SP4 as a practical non-invasive sampling alternative for many clinical situations involving evaluation of the ovarian function.