Differences in serum GH cut-off values for pharmacological tests of GH secretion depend on the serum GH method. Clinical validation from the growth velocity score during the first year of treatment

BACKGROUND: The serum GH cut-off value for pharmacological tests of GH secretion (PhT GH) depends on the type of test and also on the method used for determining serum GH. Cut-off serum GH values as different as 5-10 ng/ml, have been reported, and have been validated biochemically. We have used the growth velocity (GV)-standard deviation score (SDS) during the first year of treatment with rhGH to validate these cut-offs on a biological basis. METHODS: Fifty pre-pubertal patients with short stature (height < or =-2 SDS and GV < or =-1.2 SDS) were studied. GH deficiency (GHD) was diagnosed in 39 patients, on the basis of clinical and auxological parameters and on the serum concentration of IGF-1, and non-GHD in the other 11 patients. Two PhT GH (arginine and clonidine) were carried out in the 50 patients. Serum GH was determined by two different methods: one detecting most of serum GH isoforms, named Total GH (HGH Bio-Tech, MAIA Clone), and another one, only detecting the 22 kDa GH, named 22K GH (GH-22K IFMA, Wallac). RESULTS: Basal data: all patients with GHD and with non-GHD had maximal serum GH response (MaxR) values below and above the cut-off, respectively, for the serum Total GH and 22K GH. The mean 22K GH/Total GH ratio was similar to previous publications. Post-rhGH treatment data: the two groups improved their height SDS during the first year of treatment, particularly patients with GHD. A receiver-operator curve was used to define the best threshold for post-treatment GV-SDS that separates GHD from non-GHD patients. This value was 1.91 GV-SDS. A negative correlation between first year treatment GV-SDS and pre-treatment serum GH MaxR was found for the two assays (p < 0.001). Then, the best cut-off GV-SDS, previously calculated with the receiver-operator curve (1.91 SDS) was used to interpolate the corresponding serum GH values, as determined by the two methods. For Total GH, the value was 10.8 ng/ml, and for 22K GH, it was 5.4 ng/ml. CONCLUSION: The cut-off values calculated by biological means to separate GHD from non-GHD were remarkably similar to those calculated biochemically (10.0 and 4.8 ng/ml, respectively) for Total and 22K GH. This is a biological validation for using different cut-off values, appropriate for each assay, to diagnose GHD.     

A rapid,quantitative immunofunctional assay for measuring human leptin

BACKGROUND: We have developed a rapid, sensitive and quantitative in vitro assay for leptin based upon its ability to bind to the soluble extracellular domain of the leptin receptor (sOB-R). Such an assay is theoretically capable of differentiating between physiologically active leptin molecules from those with modified, either enhanced or reduced, binding activity. METHODS: A preparation of sOB-R was immobilized to capture leptin from serum samples or standards. Anti-leptin antibodies that had been raised in rabbits were added in a second incubation step to identify leptin molecules bound to sOB-R. Signal detection was performed in a third incubation step by anti-rabbit IgG labeled with peroxidase. The immunofunctional assay (IFA) was clinically validated by the comparison of leptin levels in adolescents (n = 41, age range 9-18 years, BMI range 13.4-33.8 kg/m(2) and adults (n = 80, age range 18-77 years, BMI range 16.4-54.7 kg/m(2) measured using the IFA with data of an in-house RIA performed with the same standards and leptin antibodies. RESULTS: The functional sensitivity of the IFA was 0.4 ng/ml and comparable to the data of the RIA. Intra- and interassay coefficients of variation were below 12.5 % in both methods. Leptin levels correlated well with the BMI of the subjects studied (r = 0.70 for RIA, r = 0.72 for IFA; p < 0.0001) as well as between IFA (y) and RIA (x) (y = x -1.31 ng/ml; r = 0.97, p < 0.0001). The median of the quotient between IFA and RIA levels was 0.86 (quartile range 0.60-1.10) for all samples. CONCLUSIONS: So far, only at the most minor differences between leptin measurements using the newly developed IFA and those using a conventional RIA have been detected. Additional studies using the IFA method are required to investigate whether or not discrepant results with the IFA will be seen in various states of relative leptin resistance and whether or not such differences are of biological relevance.     

Response to long-term growth hormone therapy in short children with reduced GH bioactivity

BACKGROUND/AIMS: The aim of the present study was to investigate whether short children with normal growth hormone (GH) immunoreactivity, but reduced bioactivity (bioinactive GH) could benefit from rhGH treatment as GH deficient (GHD) patients. Methods: We evaluated 12 pre-pubertal children (8 M, 4 F), with GH deficiency-like phenotype showing normal serum GH peak levels (>10 ng/ml), measured by immunofluorimetric assay (IFMA-GH), in contrast with a reduced GH bioactivity (bio-GH), evaluated using the Nb(2) cells. We also evaluated 15 age-matched GHD pre-pubertal children (11 M, 4 F) with serum GH peak <5 ng/ml. Both groups were treated with rhGH therapy at the dose of 0.23 mg/kg/week s.c. RESULTS: Serum bio-GH/IFMA-GH ratio at peak time for each patient during the provocative test was significantly lower in bioinactive GH than in GHD children (0.29 vs. 2.05, p = 0.00001). Recombinant human GH therapy induced a significant (p < 0.001) increase in growth rate in both groups during the first 2 years. In the third year of treatment, while growth rate in GHD children is maintained, in bioinactive GH patients it decreases remaining, however higher compared to the pre-treatment one. CONCLUSIONS: Short rhGH therapy given to selected bioinactive GH children improve growth rate and might result in greater final adult height.     

Clinical significance of serum thyroglobulin and antithyroglobulin antibody in differentiated thyroid cancer after thyroid ablation

Serum thyroglobulin (Tg) is a suitable marker for differentiated thyroid carcinoma following total thyroid ablation. Between 1998 and 2003, serum samples from 715 papillary and 179 follicular tumor patients treated with total/nearly total thyroidectomy and radioiodine ablation therapy were collected. According to the "Guidelines for Oncotherapy in Hungary", serum Tg, antithyroglobulin antibody (TgAb), TSH and FT4 levels were measured in periods of 3 months following the first treatment and of 6 months after 2 years. In the present work the prognostic value of Tg and TgAb data of cancer patients with hormone substitution therapy were evaluated individually and retrospectively. Serum Tg and TgAb concentrations were measured with a highly sensitive immunoradiometric (IRMA) method, and with a second generation, broad epitope specificity competitive radioimmunoassay, respectively. TSH levels determined by fourth generation LIAISON kit were in a range of 0.05-0.10 mIU/L. Accuracy of measuring of Tg <1 ng/ml made it possible to select the low cut-off level (Tg <2 ng/ml) following total thyroidectomy. In the predominant part of TSH-suppressed patients (746/774, 96%) the serum Tg concentration was below the cut-off level of 2 ng/ml. The sensitivity of Tg determination in 59 TSH-suppressed thyroid cancer patients with lung and bone metastases was as high as 86 to 100%. On the contrary, the number of false negative data was high in cases with lymph node metastases of papillary cancer, and sensitivity did not exceed 62%. Specificity and sensitivity of Tg in TgAb negative patients were 91 to 100%. Based on our results it could be concluded that measuring of Tg and TgAb, using a current IRMA method and a second generation RIA kit, proved to be effective tools for the postoperative monitoring of differentiated thyroid tumours. It has to be noted that determination of TgAb is highly recommended for the adequate interpretation of serum Tg levels. Persistently high and/or increasing serum TgAb concentration with low Tg result had a diagnostic value during the follow-up and can be connected with the recurrence or persistence of the differentiated thyroid cancer.     

Bioassay of growth-stimulating activity of serum: comparison of lymphocyte and cartilage assays in normal and growth hormone-deficient children

The somatomedin and/or growth-stimulating activity of serum from hypopituitary children and short children with normal growth hormone (GH) response to stimulation tests were studied using different bioassays: thymidine incorporation into human activated lymphocytes; sulfate incorporation into chick embryo cartilage; and simultaneous thymidine uptake into the same cartilages. The results showed that lymphocyte assay is highly sensitive to small amounts of serum and is GH-dependent in children with low GH secretion. On the contrary, the cartilage assays need higher serum concentration and their GH-dependence appears only in subjects with normal or low-normal GH secretion. The lack of correlation between the results of the three bioassays suggests that they measure both somatomedins and different serum factors involved in the regulation of growth.     

Growth hormone (GH) peak after falling asleep reflects spontaneous nocturnal GH secretion, however is not corresponding to the results of GH stimulating tests in children with short stature

OBJECTIVE: Growth hormone (GH) secretion is characterized by a pulsatile, circadian rhythm, with the highest concentrations at night hours. Evaluation of nocturnal GH secretion may be truncated to 6 hours. Growth hormone stimulating tests are the standard method of assessment of GH secretion. In Poland, the assessment of GH peak during 2 hours after falling asleep was introduced as a screening procedure in children, suspected for GH deficiency. The aim of current study was to compare the results of a screening test with GH secretion during 6-hour nocturnal profile and with the results of GH stimulating tests, as well as with IGF-I secretion in children with short stature. Methods: In 72 short children, GH concentrations were measured every 30 minutes during first 6 hours after falling asleep and in two GH stimulating tests (the cut-off level of GH peak for all the tests was 10.0 ng/ml). Also, IGF-I concentrations were measured and expressed as IGF-I SDS for age and sex. Results: The screening test results correlated significantly with both GH peak in 6-hour profile and mean GH concentration, and the area under the curve (AUC) in 6 hour profile (r= 0.94, r=0.90 and r=0.89, respectively, p<0.05) but not with GH peak in stimulating tests (r=0.07, NS). There was no correlation between IGF-I secretion and any of the analyzed parameters of spontaneous and stimulated GH secretion. Conclusions: The results of screening test seem to reflect overnight GH secretion in short children, remaining, however, discordant with the results of GH stimulating tests and with IGF-I secretion.     

Effects of human growth hormone administration on growth rate and growth-stimulating activity of serum, measured by lymphocyte bioassay in hypopituitary children

The acute effect of human growth hormone (hGH) upon the serum bioassayable growth-stimulating activity was compared to the long-term effects of hGH on growth rate in two groups of hypopituitary patients aged 2-18 years. 12 patients had complete GH deficiency with GH peak below 3.5 ng/ml at two stimulation tests. 15 others, having both GH peaks below 8 ng/ml and at least one above 3.5 ng/ml, were considered as having partial GH deficiency. The growth-stimulating activity of serum was measured by its effect at concentrations 0.03 to 1.25% upon thymidine incorporation into lectin-activated normal human lymphocytes, named thymidine activity (TA). In patients with complete GH deficiency, the pre-treatment TA was positively correlated with the peak response of GH to stimulation tests. The increase of TA after 3-4 days of hGH treatment was positively correlated with the pretreatment TA level, and negatively correlated with the peak GH level. The effect of a 6-12 months therapeutic course of hGH upon the growth rate was positively correlated with the acute increase of TA. No such correlations were found in patients with partial GH deficiency. Many works have discussed the relationship of acute somatomedin responses and long-term clinical results of treatment with hGH in GH-deficient children. The present data, using a highly sensitive bioassay of serum stimulating activity, suggest that the degree of GH deficiency is an important factor to be considered. The response of GH-dependent serum growth factors to acute treatment with hGH could have more predictive value in cases with total lack of growth hormone than in cases of partial deficiency.     

Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography

Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml(-1))+192.53 and 1.05.10(-4) concentration (ng ml(-1))-1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.     

Partial growth hormone deficiency (GHD) in children has more similarities to idiopathic short stature than to severe GHD

INTRODUCTION: Assessment of growth hormone (GH) secretion is based on stimulation tests. Low GH peaks in stimulation tests, together with decreased insulin-like growth factor-I (IGF-I) secretion, confirm a diagnosis of GH deficiency (GHD). However, limitations in interpreting the test results and discrepancies between GH and IGF-I secretion in particular patients have both been reported. GH therapy should improve the prognosis of adult height (PAH). The aim of the study was to compare the deficit of height at diagnosis, IGF-I secretion and PAH in children with either decreased (in varying degrees of severity) or normal GH secretion in stimulation tests. MATERIAL AND METHODS: The analysis comprised 540 short children (373 boys, 167 girls), aged 11.7 +/- 3.2 years. In all the patients two GH stimulation tests were performed, IGF-I serum concentration was measured, bone age was assessed and PAH was calculated. According to the GH peak in the two stimulation tests, the patients were classified into the following groups: severe GHD (sGHD)--GH peak < 5 ng/mL (n = 44), partial GHD (pGHD)--GH peak 5-10 ng/mL (n = 190), idiopathic short stature (ISS)--GH peak at least 10 ng/mL (n = 306). RESULTS: A significantly greater deficit of height, lower IGF-I secretion and worse PAH were observed in sGHD than in both remaining groups, while all the differences between pGHD and ISS in the parameters analysed were insignificant. CONCLUSION: The results obtained indicate the necessity of applying another methods of qualifying short children for GH therapy other than GH stimulation tests with a cut-off value at a level of 10 ng/mL.     

Simple and sensitive method for the determination of celecoxib in human serum by high-performance liquid chromatography with fluorescence detection

A simple method is described for the determination of the cyclooxygenase-2 specific inhibitor celecoxib in human serum by HPLC using the demethylated analogue as internal standard. After protein precipitation with acetonitrile, samples were extracted with chloroform. Separation was achieved on a Prontosil C18 AQ column (150x3 mm I.D., 3-microm particle size) at a flow-rate of 0.35 ml/min using water-acetonitrile (40:60, v/v) as the mobile phase. Using fluorescence detection with excitation at 240 nm and emission at 380 nm, the limit of quantification was 12.5 ng/ml for a sample size of 0.5 ml of serum. The assay was linear in the concentration range of 12.5-1500 ng/ml and showed good accuracy and reproducibility. At all concentrations intra- and inter-assay variabilities were below 11% with less than 9% error. The method was applied to the determination of celecoxib for pharmacokinetic studies in man.     

Comparison of five techniques for the determination of protein content in mixed human saliva

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.     

Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine

Sensitive assays for the determination of cyclobenzaprine (I) in human plasma and urine were developed utilizing high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) and ultraviolet (UV) absorbance detections. These two analytical techniques were evaluated for reliability and sensitivity, and applied to support pharmacokinetic studies. Both methods employed a liquid-liquid extraction of the compound from basified biological sample. The organic extract was evaporated to dryness ,the residue was reconstituted in the mobile phase and injected onto the HPLC system. The HPLC assay with MS-MS detection was performed on a PE Sciex API III tandem mass spectrometer using the heated nebulizer interface. Multiple reaction monitoring using the parent → daughter ion combinations of m/z 276 → 215 and 296 → 208 was used to quantitate I and internal standard (II), respectively. The HPLC-MS-MS and HPLC-UV assays were validated in human plasma in the concentration range 0.1–50 ng/ml and 0.5–50 ng/ml, respectively. In urine, both methods were validatedin the concentration range 10–1000 ng/ml. The precision of the assays, as expressed as coefficients of variation (C.V.) was less than 10% over the entire concentration range, with adequate assay specificity and accuracy. In addition to better sensitivity, the HPLC-MS-MS assay was more efficient and allowed analysis of more biological fluid samples in a single working day than the HPLC-UV method.     

A nationwide attempt to standardize growth hormone assays

The Growth Hormone (GH) and Its Related Factors Study Committee of the Foundation for Growth Science, Japan, has been conducting a quality control study for 15 years to improve the equality of diagnosis of GH deficiency. It found that the greatest differences in measured GH values were due to the different potencies of the kit standards, which were primarily adjusted to WHO standards for human GH of pituitary origin. With the collaboration of kit makers and the Study Group of Hypothalamo-Pituitary Disorders of the Ministry of Health, Labor and Welfare, all GH kits in Japan have begun using the same recombinant human GH standard since April 2005. As a result the diagnostic cut-off peak GH has changed from 10 to 6 ng/ml.     

Evaluation of 24-hour growth hormone spontaneous secretion: comparison with a nocturnal and diurnal 12-hour study

Spontaneous growth hormone (GH) secretion in 116 short children was studied by sampling blood for GH measurement every 20 min over 24 h. We calculated 24-h mean GH concentration (MGHC), diurnal 12-h MGHC (dMGHC) and nocturnal 12-h MGHC (nMGHC). The children were subdivided into four groups: prepubertal children with 'classical' GH deficiency (group 1, n = 12, low responses to two provocative stimuli tests and MGHC less than 3 ng/ml), prepubertal children with 'nonclassical' GH deficiency (group 2, n = 36, normal GH responses to two provocative tests and MGHC less than 3 ng/ml), short normal children (normal GH responses to two provocative tests and MGHC greater than 3 ng/ml) at stage P1 of puberty (group 3, n = 41) and at stage P2 of puberty (group 4, n = 27). The values of MGHC, dMGHC and nMGHC were significantly higher in groups 3 and 4 than in groups 1 and 2, and in group 4 than in group 3. The values of MGHC and nMGHC were significantly higher in group 2 than in group 1. MGHC correlated highly with nMGHC and dMGHC (r = 0.97 and 0.94, respectively; p less than 0.001). On the basis of regression equations between MGHC and nMGHC or dMGHC, the study of the diagnostic accuracy showed values higher for nMGHC than for dMGHC: 94.1 vs. 89.6% for sensitivity, and 93.7 vs. 89.7% for specificity, respectively.     

GH values after clonidine stimulation measured by immunofluorometric assay in normal prepubertal children and GH-deficient patients

OBJECTIVE: To establish the cut-off values of GH measured by immunofluorometric assay, a more sensitive and specific assay, in normal prepubertal children and compare their values with those of proven GH-deficient patients. METHODS: 30 normal children (20 males) and 26 patients with known causes of GH deficiency were submitted to the clonidine test and their GH values were compared. A powdered clonidine tablet (0.1 mg/m(2)) was given orally and blood samples for GH measurements were drawn at times -30, 0, 60, 90 and 120 min. RESULTS: GH peak values presented a wide variation ranging from 1.7 to 25 micro g/l (mean +/- SD = 12.87 +/- 5.8 micro g/l) in the normal group. The cut-off values for the 5th and 10th percentiles of the distribution curve were 3.3 and 5.5 micro g/l, respectively. In the GH deficiency group, maximum GH levels after clonidine stimulation ranged from <0.1 to 2.1 micro g/l (0.56 +/- 0.58 micro g/l). CONCLUSIONS: The cut-off values obtained with the immunofluorometric method are lower than the ones obtained by radioimmunoassay. We suggest a cut-off value of 3.3 micro g/l (5th percentile) that ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GH deficiency when using this immunofluorometric method.     

Effect of pentobarbital and urethane on the release of hypothalamic somatostatin and pituitary growth hormone

Hypothalamic somatostatin release was investigated in the rat to elucidate the mechanism of anesthetic action on growth hormone (GH) release from the pituitary. Intraperitoneal injection of sodium pentobarbital (5 mg/100 gm B.W.) significantly elevated serum GH levels and increased hypothalamic somatostatin concentration from basal values of 0.98 +/- 0.01 to 1.21 +/- 0.06 ng/mg wet wt. In contrast, urethane (150 mg/100 gm B.W., IP) administration lowered serum GH levels and hypothalamic somatostatin concentration (0.64 +/- 0.04 ng/mg wet wt.). However, the mean concentration of pancreatic somatostatin showed no change in either case. In rats receiving passive immunization with 0.5 ml rabbit antiserum to somatostatin (SRIF-AS), serum GH levels were significantly increased (67.5 +/- 12.3 ng/ml) and did not differ from those in the group treated with normal rabbit serum (NRS) plus pentobarbital (101.3 +/- 18.5 ng/ml). However, serum GH levels in rats injected with SRIF-AS plus pentobarbital were increased to higher values than in rats given SRIF-AS alone. When urethane was administered to rats after passive immunization with SRIF-AS, urethane-induced suppression of serum GH levels was markedly inhibited (5.5 +/- 2.0 vs. 33.5 +/- 7.5 ng/ml). These results suggest a possibility that the changes in serum GH levels observed with pentobarbital or urethane administration may be induced at least in one part by somatostatin released from the hypothalamus.     

Development of a high-performance liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric assay for β-tigogenin cellobioside in human serum

A specific high-performance liquid chromatographic-atmospheric pressure chemical ionization tandem mass spectrometric assay for the quantitative determination of β-tigogenin cellobioside in human serum is described. Serum cleanup and acetylation of the analyte were required to achieve the desired lower limit of quantification, 10 ng/ml. The precision of the assay was better than 13% over a serum concentration range of 10–500 ng/ml.     

Kallikrein-binding protein is induced by growth hormone in the dwarf rat.

Rat kallikrein-binding protein (KBP), a member of the serpin family, is a tissue kallikrein inhibitor. It has been shown to be a potential pathogenic factor of diabetic retinopathy and may play a role in animal development and growth. To determine whether reduced KBP expression is involved in retarded animal growth, we examined the in vivo effect of growth hormone (GH) deficiency on the expression of KBP in the Lewis dwarf (dw/dw). We found that serum levels of functionally active KBP were reduced in the dwarf rat (P < 0.05) as determined by complex formation assay between serum KBP and (125)I-labeled rat tissue kallikrein. Enzyme-linked immunosorbent assay showed that KBP levels were significantly reduced in the serum of the dwarf rat compared to the Lewis rat (213.8 ng/ml vs. 413.8 ng/ml, n = 4, P < 0.01). The decreased KBP levels were confirmed by Western blot analysis. Moreover, treatment of the dwarf rat with recombinant human GH for 4 wk resulted in a significant increase in KBP activity (P < 0.01) and serum KBP levels compared with the untreated dwarf rat (549.8 ng/ml, n = 5, vs. 213.8 ng/ml, n = 4, P < 0.02). Northern blot analysis and densitometry showed that liver KBP mRNA levels were reduced by fivefold in the dwarf rat compared to the Lewis rat and the decrease was reversed by the GH treatment. These results indicate that the KBP levels are regulated at the RNA level. Furthermore, in vitro studies using cultured rat hepatocytes showed that GH may have a direct regulatory effect on KBP expression since KBP levels increased in the conditioned media of cells treated with GH. These results demonstrated that KBP is reduced in the genetic dwarf rat and is restored to normal by GH; therefore, KBP is a GH-dependent protein and may be a new target for studying the mechanism of pathological animal growth.     

A sensitive enzyme assay for biotin, avidin, and streptavidin

Reciprocal enzyme assays are described for the vitamin biotin and for the biotin-binding proteins avidin and streptavidin. The assays are based on the following steps: (a) biotinylated bovine serum albumin is adsorbed onto microtiter plates; (b) streptavidin (or avidin) is bound to the biotin-coated plates; (c) biotinylated enzyme (in this case alkaline phosphatase) is then interacted with the free biotin-binding sites on the immobilized protein. For biotin assay, competition between the free vitamin and the biotinylated enzyme is carried out between steps (b) and (c). The method takes advantage of the four biotin-binding sites which characterize both avidin and streptavidin. The method is extremely versatile and accurate over a concentration range exceeding three orders of magnitude. The lower limits of detection are approximately 2 pg/ml (0.2 pg/sample) for biotin and less than 100 ng/ml (10 ng/sample) for either avidin or streptavidin.     

Thermometric enzyme linked immunosorbent assay: TELISA.

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.