Competitive Protein Binding Analysis Research Articles

Simultaneous measurements of free thyroxine and triiodothyronine fractions in human serum by means of equilibrium dialysis (author's transl)

Simultaneous measurements of serum free thyroxine (T4) and triiodothyronine (T3) fractions were studied using a modification of equilibrium dialysis described by Sterling and Brenner. To 1.2 ml of the serum to be assayed, 131I-T4 and 125I-T3 were added in a concentration of 2 mug/dl and 25 ng/dl, respectively, both of which were preliminarily dialysed according to Schussler and Plager. Half ml of the serum with tracers added was dialysed against 9 ml of phosphate buffer (ionic strength 0.15, pH 7.4) for 18 hours at 37 degrees C and 0.1 ml was reserved from the rest in a counting tube (in duplicate). After the completion of dialysis, the dialysate was mixed with 1 ml of pool serum and the contaminating inorganic iodide (in the form of 131I or 125I) was eliminated by adsorption on anion exchange resin. The radioactivity of 3 ml of the dialysate and 0.1 ml of the preserved serum (with the tracers added) was counted and the free (or dialysable) fractions were expressed as a ratio of the count of the former divided by that of the latter adjusted to an equal volume by calculation. The amount of either T4 or T3 added as tracers had no influence on free T4 or T3 fraction unless either of them was added to a concentration of 10 mug/dl. When 125I-T3 of low specific activity (50 muCi/ug) was used as a tracer, free T3 fraction measured simultaneously with free T4 fraction tended to be higher than that measured with a single tracer. When 125I-T3 of higher specific activity (300 muCi/mug) was employed, free T3 fraction obtained with two methods did not differ significantly. Using serum T4 and T3 concentrations measured by competitive protein binding analysis and radioimmunoassay, respectively, the free T4 and T3 concentrations were estimated with sera of normal, hyperthyroid, hypothyroid and uncomplicated pregnant subjects.

Competitive Protein Binding Analysis for Thyroxine Using Sephadex Column (Tetralute)

Competitive Protein Binding Analysis for Thyroxine Using Sephadex Column (Tetralute)

Competitive protein binding analysis of vitamin B 12 using vitamin B 12-free serum as a standard diluent

Vitamin B 12 (B 12) was assayed using pooled human serum as binding agent and DEAE-cellulose for separation of bound from free B 12. It was shown that the assayed B 12 concentration was dependent on a factor not recognised before, namely the amount of binding serum. This effect could be corrected by adding B 12-free serum to the standard solutions. Stability of pooled human serum was improved by previous separation of the large lipoproteins. The assay procedure was simplified by predosing of DEAE-cellulose and [ 57Co]B 12, extraction with glutamic acid to avoid centrifugation and using filtration as the method for separation of bound from free B 12. The correlation with the Lacto-bacillus leichmannii bioassay was determined. The influence of several drugs and hormones on the assayed B 12 concentration was investigated. Ranges for normal and B 12-deficient sera were established.

Competitive protein-binding analysis of vitamin B 12 using Lactobacillus leichmannii as a specific binder

Vitamin B 12 was assayed by the competitive protein binding method, using Lactobacillus leichmannii as binding agent. The intact bacteria were used as “solid phase”, and separation of bound and free vitamin B 12 was obtained simply by filtration of the bacteria. Filtration could be rapidly done using a disposable syringe provided with a filter. Optimal conditions with respect to the extraction of vitamin B 12 from serum and binding of the vitamin to the bacteria were determined. The effect of several drugs and hormones on the assayed vitamin B 12 concentration was investigated. The correlation with the Lactobacillus leichmannii bioassay was calculated.

329. Competitive protein binding analysis of norethindrone in human plasma and urine

A competitive protein binding method has been developed for the quantitative analysis of norethindrone (NE) in plasma and urine utilizaing the progesterone binding globulin of pregnant guinea pig serum. The method involves dilution of the sample with buffer and extraction with benzene. The ethinyl steroids are then converted to silver salts by extraction with 1N AgNO3. NH4C1 is added to dissociate the silver salt and the ethinyl steroids are back-extracted into benzene. This extract is evaporated to dryness under a stream of air and mixed with the binding solution (buffered pregnant guinea pig serum plus tritiated-progesterone). Following incubation at 45 degrees C for 5 minutes and then at 0 degrees C for 30 minutes the unbound steroids are removed by treatment with dextran-charcoal and an aliquot of the supernatant is taken for liquid scintillation counting. The resulting counts (converted to DPMs) are then compared with standards prepared with known concentrations of NE in blank plasma or urine. The standard curve is linear from 10 to 500 ng. Normal male and female volunteers receiving single 5-mg doses of NE showed peak plasma levels of 20 and 42 ng/ml respectively in 2-4 hours after dosing. The plasma half-life of NE was 4-6 hours. About 2% of the dose was recovered as NE (free and conjugated) in the urine within 48 hours.(FULL TEXT)

Protein-Binding Assay for Plasma Testosterone, after Purification by Column Partition Chromatography

Abstract We describe a relatively simple, specific, and practicable method for measuring plasma testosterone by competitive protein-binding analysis. An ether extract is purified by a single chromatographic step on a micro-scale Celite—ethylene glycol column. Serum from women in the third trimester of pregnancy is used as the binding protein, and an ammonium sulfate precipitation step is used to separate the free and protein-bound testosterone. The method has a consistently low blank [0.59 ± 0.26 (SD) pmol/sample] and shows good precision. The mean testosterone concentration in normal men and menstruating women was 17.1 ± 4.0 (SD) and 1.2 ± 0.4 (SD) nmol/liter, respectively.

Eine einfache routinemethode zur analyse von 11-desoxy-cortisol in kleinen plasmamengen mit der competitiven proteinbindungsanalyse

The paper concerns a research specifically on the determination of 11-desoxy-cortisol via competitive protein binding analysis. Via the systematic search for the separation coefficients of the various corticosteroids a solvent mixture was chosen for II -desoxycorticosteroids. For special purposes a thin-layer Chromatographie procedure to separate IIdesoxycortisol was attempted preceding the competitive protein binding analysis. The clinical usefulness was made apparent by studies using the Metopiron test and the II-desoxycorticosteroids on plasma from normal persons and pregnant women.

Serum Thyroxine: Results Compared for a Commercial Radioimmunoassay and a Commercial Competitive Protein Binding Analysis

Abstract Two commercial isotopic methods for measuring serum thyroxine—radioimmunoassay and competitive protein binding analysis—have been compared with regard to recovery of thyroxine added to sera, within-run and run-to-run reproducibility, and results for patients’ sera. For the latter the relationship is expressed by the equation CPBA = 082 RIA + 0.33. The correlation coefficient was 0.92.

The determination of corticosterone concentration in rat plasma by competitive protein binding analysis

A competitive protein binding assay for the determination of corticosterone in rat plasma is described with the criteria of sensitivity, specificity, precision and accuracy evaluated in some detail. Rat plasma corticosterone binding globulin was used as the source of binding protein to assay the mass of corticosterone in ethyl acetate extracts of rat plasma. Steroids other than corticosterone which might interfere with the determination were shown either to be ineffective in displacing the radioligand or were present in such small quantities as to be insignificant in normal rats. The procedure involved the extraction of 50 μl samples of plasma, one fifth of the extract being assayed by means of a standard curve with a range of 0–6 ng. The sensitivity of the assay was 0.77 ng/50 μl (1.54μg/100 ml) and the precision over the most commonly encountered concentration range, 10.0–20.0 μg/100 ml, was ±0.81 μg/100 ml ( N = 76).

The group separation of plasma and urinary steroids by column chromatography on sephadex LH-20

Using Sephadex LH-20 column chromatography and solvent systems composed of cyclohexane and ethanol, a Chromatographic method is reported for the separation of free steroids in biological material. These systems are in routine use for the purification and separation of plasma corticosteroids prior to competitive protein binding analysis. The systems are also used for group separation of urinary steroids before gas Chromatographic analysis. Routine measurements of aetiocholanolone and pregnanetriol in urine by gas chromatography are carried out in this laboratory following Sephadex LH-20 chromatography. These systems have been particularly useful in the ‘profile’ analysis of urinary steroid metabolites in patients with endocrine disorders.

Work simplification of a competitive protein binding analysis for serum thyroxine a study of some analytical variables affecting accuracy and precision

Some variables affecting the accuracy and precision of a previously described work simplified procedure for the competitive protein binding analysis of serum thyroxine have been investigated. Ethanol is preferred to ethanol-ammonia mixtures for the extraction of thyroxine from serum as it introduces the minimum interfering material into the assay. The use of various inhibitors of thyroxine binding to proteins other than thyroxine binding globulin has been investigated. None showed any advantage over the use of barbiturate alone. Conditions for obtaining maximum slope in the standard response curve are considered, with respect to buffer composition, concentration of binding protein, and time for equilibration and resin separation of protein bound and non protein bound thyroxine. Theoretical assessment of the errors in the analysis indicates that at low serum thyroxine concentrations, the error in interpolating results from the standard response curve constitutes a major part of the overall analytical error, while at medium or high concentrations, the error in the recovery step is more important.

Work simplification of a competitive protein binding analysis for serum thyroxine, and a study of some laboratory factors affecting assay results

The work simplification of competitive protein binding (CPB) analysis for serum thyroxine is described. Modification and design of apparatus specifically for use in the assay adds gieatly to the convenience and reliability with which large workloads can be handled. Forty patient samples plus controls can be easily analysed by one person in 4 h. The simplified CPB working method is accurate, but, in comparison with an automated protein bound iodine (PBI) method, is somewhat inferior in precision and shows a slightly greater cost per analysis. Serum samples for CPB analysis were stable if stored at + 4° or — 20° for periods of up to 14 days. Storage at room temperature for two days or longer, however, resulted in an increase of about 19 nmoles/1 in the assay result. Haemolysis at the levels occasionally encountered in laboratory specimens was without any detectable effect on the assay result.

Physiological, Environmental, and Hormonal Factors at Insemination Which May Affect Conception1,2

Rectal and uterine temperatures of lactating dairy cows were recorded at insemination and 14.5±4.6h (mean±standard deviation) later for the first three services. Blood samples were collected from a subcutaneous abdominal vein at the later time. Plasma corticoids and progesterone were quantified by competitive protein-binding analysis. Climatological data were obtained throughout the experiment from September through August. Means and standard deviations of 226 rectal and uterine temperatures at insemination were 38.4±.04C and 38.6±.03C and later, 38.1±.03C and 38.3±.03C. Mean corticoid and progesterone (ng/ml) at the later time were 8.02±.32 and .28±.06. Uterine temperature at insemination; maximum, minimum, and average temperature the day of insemination; and maximum, minimum, and average temperature the day after insemination were associated with variation in conception. Uterine temperatures and average ambient temperature on day of insemination were inversely related to fertility. Progesterone at the later time was related to environmental temperatures on day of insemination. Plasma concentrations of progesterone and corticoids, sampled at the later time postinsemination, had no detectable association with conception.

Assessment of in vitro thyroid function tests in 100 consecutive patients referred to a thyroid clinic.

SUMMARYIn a series of 100 consecutive patients referred to a thyroid clinic the total serum thyroxine (serum T‐4) measured by competitive protein binding analysis had a much higher diagnostic accuracy than either the serum protein‐bound iodine (PBI) or the triiodothyronine serum uptake (T3 serum uptake) when used alone. In contrast to previous reports, the free thyroxine index (FTI) calculated from the serum PBI and the T3 serum uptake gave disappointing results. The FTI calculated from the serum T‐4 and the T3 serum uptake correlated well with thyroid status, but equally good results were given by the effective thyroxine ratio (ETR) which, although expensive, has the advantage of being a single procedure.Primary hypothyroidism can be diagnosed with a high degree of accuracy from a single venepuncture by demonstrating a low total serum thyroxine and an elevated plasma thyrotrophin (TSH). There were no false negative results. Fortysix of the fifty euthyroid patients had plasma TSH levels less than 12 μU/ml. The four euthyroid patients with TSH levels greater than 12 μU/ml had evidence of impaired thyroid reserve.

Determination of the binding of thyroxine to plasma proteins by competitive protein-binding analysis

Binding capacities for thyroxine-binding globulin (TBG) and thyroxinebinding prealbumin (TBPA) and the equilibrium constants KTBg and Ktbpa were studied by competitive protein-binding techniques. A tracer dose of [ 125I]thyroxine is equilibrated with the serum. Unlabelled thyroxine is added in concentrations from o to 200 μg/100 ml. Subsequent separation of free and bound hormone is carried out on Sephadex columns. These data are analysed by means of a Scatchard diagram with the bound/free hormone ratio as ordinate and the bound hormone as abscissa. The results agree with those obtained with other techniques. In our experience, this method is very useful for clinical evaluation.

Determination of serum testosterone and androstanediol by competitive protein binding

A method for the simultaneous measurement of serum testosterone (T) and androstanediol (Ad) utilizing aluminum oxide thin layer chromatography and competitive protein binding analysis is presented. The method separates not only T from Ad, but also the androstanediol isomers. The primary Ad found was 5α-androstane-3α, 17β diol. Levels of both Steroids were determined in normal adults and children, and in a variety of endocrine disorders. The average T Ad ratio was higher in female children than other controls except adult males. However, results were too variable and number of patients insufficient to draw definite conclusions as to the value of determination of this ratio in patients with androgen disorders.

Androstenediol levels in human peripheral plasma.

ABSTRACT Unconjugated androstenediol has been identified and measured in the plasma of normal adults. Identification of the steroid in ether extracts of plasma was based upon its affinity for “testosterone binding globulin,” chromatographic mobility before and after derivative formation, and identity after acetylation with carrier androstenediol diacetate upon recrystallization. By means of competitive protein binding analysis, peripheral plasma androstenediol levels were determined to be 68.2 ± 24 (sd) ng/100 ml in normal women, 124 ± 45 ng/100 ml in normal men. The androstenediol concentration in men averages twenty percent of the testosterone level, that of women is nearly twice their testosterone level. Since available data indicates that androstenediol is moderately androgenic, this steroid appears to contribute significantly to total androgenicity in women and may play a role in certain hyperandrogenic states.

Competitive Protein Binding Analysis in the Clinical Laboratory'An Introduction

Competitive Protein Binding Analysis in the Clinical Laboratory'An Introduction

Use of Normalized Serum Thyroxine (T4N)

The measurement of serum thyroxine is an indicator of the total hormone present in the serum; it is directly dependent on the protein bound portion of thyroxine. This portion is altered by various physiological states, sex hormone uses, and pregnancy, so that thyroxine determinations under these conditions may be unreliable. The dual competitive protein binding analysis reintroduces the patient's serum into the test system, a procedure which provides additional competitive binding sites that have a corrective effect on serum thyroxine values altered by physiological conditions but will not be affected by abnormal or pathological thyroid functional states. Clinical data and thyroid function tests in normal subjects and hyperthyroid and hypothyroid patients, pregnant women, and women receiving estrogens revealed that the normalized tetraiodothyronine (T₄N) determinations correlated better with the patient's thyroid status than did earlier thyroid function tests.

A simple procedure for the combined determination of plasma estrogen and androgen concentrations by competitive protein binding analysis

A simple method for the measurement of estradiol-17β, estrone, testosterone and androstenedione in 4–6 ml of human male and female plasma is described. By ether extraction of alkalinized plasma followed by a single celite column Chromatographic step or by two column Chromatographic steps on Sephadex LH 20, purified extracts are obtained for competitive protein binding radioassay. Cytosol from six-day pregnant rabbit uteri (according to Korenman) was used for estrogen assays, and human plasma from the third trimester of pregnancy (according to Sciarra) was used for androgen assays. The plasma values of testosterone and androstenedione were comparable with those obtained after t.l.c. and competitive protein binding. The specificity and sensitivity of the method enabled determinations of plasma estrogen to be made not only in normal adults, but also in hypogonadal patients where estradiol-17β values are at very low levels. The accuracy of these determinations was confirmed by repeated measurements of blanks which range from 0 to 5 pg/ml, and by recovery experiments of known amounts of added estradiol-17β.