TECO Leptin. Human Leptin ELISA. Instructions for Use English. Catalogue No. TE1016 For Research Use Only.

Transcription

1 TECO Leptin Human Leptin ELISA Instructions for Use English Catalogue No. TE1016 For Research Use Only. TE1016_02e 11/2008 TECOmedical Group

2 Symbol Description Kit Instructions LOT Lot Number Exp Expiry Date 8 C 46 F 2 C 36 F Storage Temperature Manufacturer Caution: read instructions REF 96 TE 1014 Caution: irritant Tests TECOmedical AG Headquarters TECOmedical Group Gewerbestrasse Sissach Switzerland phone +41(0) fax +41(0) mail info@tecomedical.com web Technical Services: Germany phone France phone Benelux phone +31(0) USA/Canada phone Or contact our local representative in your country. 2

3 TECO Human Leptin ELISA Kit Reagents and Materials Supplied: Symbol Description Format Antibody Coated Wells 12 break apart strips of 8 wells (12 x 8 in total), in a frame, Ready to use Standard A 1 ng/ml - lyophilized Standard B 10 ng/ml - lyophilized Standard C 25 ng/ml - lyophilized Standard D 50 ng/ml - lyophilized Standard E 100 ng/ml - lyophilized Control 1 lyophilized, Range as indicated on data sheet Control 2 lyophilized, Range as indicated on data sheet Dilution Buffer Ready for use Antibody-HRP-Conjugate Ready for use TMB Substrate Ready for use Wash Buffer 20 times concentrated Stop Solution 0.2 M H 2 S M sulfuric acid, ready for use Cover for Microtiterplate, adhesive 1 plate 1 x 0.75 ml 1 x 0.75 ml 1 x 0.75 ml 1 x 0.75 ml 1 x 0.75 ml 1 x 0.5 ml 1 x 0.5 ml 1 x 25 ml 1 x 12 ml 1 x 12 ml 1 x 50 ml 1 x 12 ml 2 pieces Kit instruction 1 x 3

4 Storage Store kit at 2 8 C. Do not freeze. Store unused reagents at 2 8 C. Instructions for Use The TECO Human Leptin kit is a sensitive two-site sandwich enzyme linked immunosorbent in-vitro assay for the quantitative determination of leptin in human plasma and serum. Background Leptin, the product of the ob gene [1, 2], is a recently discovered (1994) single-chain proteohormone with a molecular weight of 16 kd, which is thought to play a key role in the regulation of body weight. Its amino acid sequence exhibits no major homologies with other proteins [1]. Leptin is almost exclusively produced by differentiated adipocytes [3 5]. It acts on the central nervous system, in particular the hypothalamus, thereby suppressing food intake and stimulating energy expenditure [2, 6 9]. Leptin receptors alternatively spliced forms exist that differ in length belong to the cytokine class I receptor family [10 12]. They are found ubiquitously in the body [10, 11, 13, 14] indicating a general role of leptin, which is currently not fully understood. A circulating form of the leptin receptor exists which acts as one of several leptin binding proteins [15]. Besides its metabolic effects, leptin was shown to have a strong influence on a number of endocrine axes. In male mice, it blunted the starvation-induced marked decline of LH, testosterone, thyroxine and the increase of ACTH and corticosterone. In female mice, leptin prevented the starvation-induced delay in ovulation [16]. Ob/ob mice, which are leptin deficient due to an ob gene mutation, are infertile. This defect could be corrected by administration of leptin, but not through weight loss due to fasting [17], suggesting that leptin is pivotal for reproductive functions. All these actions may, at least in part, be explained by the suppressive effect of leptin on neuropeptide Y (NPY) expression and secretion by neurons in the arcuate nucleus [6, 18, 19]. NPY is a strong stimulator of appetite [20, 21] and is known to be involved in the regulation of various pituitary hormones, e.g. suppression of GH through stimulation of somatostatin [22, 23], suppression of gonadotropins [23] or stimulation of the pituitary-adrenal axis [21]. The most important variable that determines circulating leptin levels is body fat mass [24 26]. Obviously, under conditions of regular eating cycles, leptin reflects the proportion of adipose tissue [27] showing an exponential relationship [37]. This constitutive synthesis of leptin is modulated by a number of non-hormonal and hormonal variables. Stimulators in both rodents and humans are overfeeding [28, 29], insulin [3, 5, 30 33] and glucocorticoids [5, 34 36]. Suppression has been shown for fasting [27], camp and beta-3-adrenoceptor agonists [35]. From these findings it becomes clear that leptin is an integral component of various metabolic and endocrine feedback loops [38]. It is important to note that serum leptin levels show a moderate circadian variation with a peak during the night at about 2 a.m. [37]. The leptin values at this time are about 30 to 100 % higher than the levels measured in the morning or early afternoon. This variation together with the influence of food intake needs to be taken into account, when blood samples are collected. 4

5 Under fairly standardized conditions, i.e. normal eating cycles and blood sampling in the morning or early afternoon, a single leptin measurement is informative. Leptin levels are high in most obese individuals suggesting the presence of leptin insensitivity [20, 26, 37, 38, 41, 42]. In a small percentage of patients, however, leptin levels have been found inappropriately low with respect to their fat mass. It remains for future studies to prove that these individuals represent a new pathophysiologic entity: leptin deficiency. Since leptin has also been shown to be of great importance for reproductive functions, possible new pathophysiologic mechanisms may be discovered relating infertility to insufficient leptin production. The discovery of leptin has released an avalanche of research activities seeking to understand the regulation and actions of this new hormone. Most importantly, it has provided a key to better understand the physiology of body weight regulation and to unveil possible pathophysiologic mechanisms in both obesity and nutritional disorders. Further, it may provide new insights into certain causes of infertility. The widespread importance makes leptin an interesting parameter for researchers investigating metabolic syndrome, obesity, cachexia and other metabolic disturbances related to individuals with nutritional disorders. This enzyme immunoassay kit is suited for measuring human leptin in serum or plasma, and conditioned adipocyte culture media for scientific research purposes. 5

6 References [1] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature. 1994; 372: [2] Halaas JL, Gajiwala KS, Maffei M, et al. Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 1995; 269: [3] MacDougald OA, Hwang CS, Fan H, Lane MD. Regulated expression of the obese gene product (leptin) in white adipose tissue and 3T3-L1 adipocytes. Proc Natl Acad Sci U S A. 1995; 92: [4] Rentsch J, Chiesi M. Regulation of ob gene mrna levels in cultured adipocytes. FEBS Lett. 1996; 379: [5] Wabitsch M, Jensen PB, Blum WF, et al. Insulin and cortisol promote leptin production in cultured human fat cells. Diabetes. 1996; 45: [6] Stephens TW, Basinski M, Bristow PK, et al. The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 1995; 377: [7] Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science. 1995; 269: [8] Pelleymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 1995; 269: [9] Levin N, Nelson C, Gurney A, Vandlen R, de-sauvage F. Decreased food intake does not completely account for adiposity reduction after ob protein infusion. Proc Natl Acad Sci U S A. 1996; 93: [10] Tartaglia LA, Dembski M, Weng X, et al. Identification and expression cloning of a leptin receptor, OB-R. Cell. 1995; 83: [11] Chen H, Charlat O, Tartaglia LA, et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996; 84: [12] Lee GH, Proenca R, Montez JM, et al. Abnormal splicing of the leptin receptor in diabetic mice. Nature. 1996; 379: [13] Lynn RB, Cao GY, Considine RV, Hyde TM, Caro JF. Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice. Biochem Biophys Res Commun. 1996; 219:

7 References [14] Considine RV, Considine EL, Williams CJ, Hyde TM, Caro JF. The hypothalamic leptin receptor in humans: identification of incidental sequence polymorphisms and absence of the db/db mouse and fa/fa rat mutations. Diabetes. 1996; 45: [15] Sinha MK, Opentanova I, Ohannesian JP, et al. Evidence of free and bound leptin in human circulation. J Clin Invest. 1996; 98: [16] Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neuroendocrine response to fasting. Nature. 1996; 382: [17] Chehab FF, Lim ME, Lu R. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet. 1996; 12: [18] Schwartz MW, Baskin DG, Bukowski TR, et al. Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes. 1996; 45: [19] Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG. Identification of targets of leptin action in rat hypothalamus. J Clin Invest. 1996; 98: [20] Campfield LA, Smith FJ, Burn P. The OB protein (leptin) pathway a link between adipose tissue mass and central neural networks. Horm Metab Res. 1996; 28: [21] Rohner-Jeanrenaud F, Cusin I, Sainsbury A, Zakrzewska KE, Jeanrenaud B. The loop system between neuropeptide Y and leptin in normal and obese rodents. Horm Metab Res. 1996; 28: [22] Chan YY, Steiner RA, Clifton DK. Regulation of hypothalamic neuropeptide-y neurons by growth hormone in the rat. Endocrinol. 1996; 137: [23] Pierroz DD, Catzeflis C, Aebi AC, Rivier JE, Aubert ML. Chronic administration of neuropeptide Y into the lateral ventricle inhibits both the pituitary-testicular axis and growth hormone and insulinlike growth factor I secretion in intact adult male rats. Endocrinol. 1996: 137:3-12. [24] Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS. Leptin levels reflect body lipid content in mice: evidence for dietinduced resistance to leptin action. Nat Med. 1996; 1: [25] Maffei M, Halaas J, Ravussin E, et al. Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med. 1995; 1:

8 References [26] Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 1996: 334: [27] Kolaczynski JW, Considine RV, Ohannesian J, et al. Responses of leptin to short-term fasting and refeeding in humans: A link with keto-genesis but not ketones themselves. Diabetes. 1996; 45: [28] Harris RB, Ramsay TG, Smith SR, Bruch RC. Early and late stimulation of ob mrna expression in meal-fed and overfed rats. J Clin Invest. 1996; 97: [29] Kolaczynski JW, Ohannesian J, Considine RV, Marco C, Caro JF. Response of leptin to short-term and prolonged overfeeding in humans. J Clin Endocrinol Metab. 1996; 91: [30] Saladin R, De-Vos P, Guerre-Millo M, et al. Transient increase in obese gene expression after food intake or insulin administration. Nature. 1995; 377: [31] Cusin I, Sainsbury A, Doyle P, Rohner-Jeanrenaud F, Jeanrenaud B. The ob gene and insulin. A relationship leading to clues to the understanding of obesity. Diabetes. 1995; 44: [32] Kolaczynski JW, Nyce MR, Considine RV, et al. Acute and chronic effects of insulin on leptin production in humans: studies in vivo and in vitro. Diabetes. 1996; 45: [33] Malström R, Taskinen M-R, Karonen S-L, Yki-Järvinen H. Insulin increases plasma leptin concentrations in normal subjects and patients with NIDDM. Diabetologia. 1996; 39: [34] De-Vos P, Saladin R, Auwerx J, Staels B. Induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake. J Biol Chem. 1995; 270: [35] Slieker LJ, Sloop KW, Surface PL, et al. Regulation of expression of ob mrna and protein by glucocorticoids and camp. J Biol Chem. 1996; 271: [36] Miell JP, Englaro P, Blum WF. Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects. Horm Metab Res. 1996; 28: [37] Sinha MK, Ohannesian JP, Heiman ML, et al. Nocturnal rise of leptin in lean, obese, and non-insulin- dependent diabetes mellitus subjects. J Clin Invest. 1996; 97:

9 References [38] Havel PJ, Kasim-Karakas S, Dubuc GR, Mueller W, Phinney SD. Gender difference in plasma leptin concentrations. Nature Med. 1996; 2: [39] Rosenbaum M, Nicolson M, Hirsch J, et al. Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab. 1996; 81: [44] Adresse NIBSC: Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain. [45] Blum WF, Juul A; Reference ranges of serum leptin. In: Leptin the voice of adipose tissue, Blum WF et al, eds., Johann Ambrosius Verlag, Heidelberg, 1997,s [40] Hassink SG, Sheslow DV, de Lancey E, Opentanova I, Considine RV, Caro JF. Serum leptin in children with obesity: relationship to gender and development. Pediatrics. 1996: 98: [41] Scholz GH, Englaro P, Thiele I, et al. Dissociation of serum leptin concentration and body fat content during long term dietary intervention in obese individuals. Horm Metab Res. 1996; 28: [42] Caro JF, Kolaczynski JW, Nyce MR, et al. Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet. 1996; 348: [43] Robinson CJ, Gaines-Das R, Woollacott D, et al. The first international standard for human leptin and the first international standard for mouse leptin: comparison of candidate preparations by in vitro bioassays and immunoassays. J Molecular Endocrinol. 2001; 27:

10 Assay Principle The TECO assay kit for human Leptin is a Sandwich-Assay using two specific and high affinity monoclonal antibodies. The Leptin in the samples binds to the first antibody coated on the microtiter plate. In the following step the second anti-leptin-antibody binds in turn to the immobilised Leptin. The second antibody is biotinylated and will be incubated in a mixture with a Streptavidin-Peroxidase-Enzyme Conjugate. In the closing substrate reaction the turn of the color will be catalyzed quantitatively depending on the Leptin-level of the samples. Materials Required and not Supplied Pipettes capable of dispensing 20 µl, 100 µl, 350 µl, 500 µl and 750 µl Graduated cylinders for reconstituting or diluting reagents Manual Aspiration System and multi-channel pipette or automatic washer for ELISA plates Distilled water Vortex mixer ELISA plate reader suitable for 96 well formats and capable of measuring at 450 nm (Reference: nm). ELISA Plate Shaker ( 350 rpm) (orbital shaker) Software package for data generation and analysis 10

11 Warnings and Precautions This kit is intended for in vitro research use by professional persons only. Follow the instructions carefully. Observe expiration dates stated on the labels and the specified stability for reconstituted reagents. Refer to Materials Safety Data Sheet for more detailed safety information. Material of human origin used in the preparation of this kit has been tested and found non reactive for HIV-1 and HIV-2 as well as for HCV antibodies and HbsAg but should, nonetheless, be handled as potentially infectious. TECOmedical AG is not liable for loss or harm caused by non-observance of the Kit instructions. 1. For Research Use Only. Not for use in diagnostic procedures. 2. Treat all specimen samples as potentially biohazardous material. Follow General Precautions when handling contents of this kit and any patient samples. 3. Disposal of containers and unused contents should be done in accordance with federal and local regulatory requirements. 4. Use the supplied reagents as an integral unit prior to the expiration date indicated on the package label. 5. Store assay reagents as indicated. 6. Do not use coated strips if pouch is punctured. 7. Test each sample in duplicate. 8. Use of multichannel pipettes or repeat pipettors is recommended to ensure the timely delivery of liquids. 9. a) 0.2 M sulfuric acid is caustic and can cause severe burns. b) handle TMB with care. Do not ingest. Avoid contact with skin, eyes, or clothing. If contact is made, wash with water. If ingested, call a physician. 10. As preservative is used (0.01 %) 2-Methyl-4-isothiazolin-3-one solution and (0.01 %) 5-chloro-2-methyl 2H isothiazol-3-one / 2-methyl-2H-isothiazol-3-one solution for the antibody, dilution buffer and washing buffer. Do not ingest. Avoid contact with skin, eyes, or clothing. If contact is made, wash with water. If ingested, call a physician. 11

12 Preparation of Reagents Microtiterplate coated with an anti-human Leptin Antibody 12 break apart strips of 8 wells (96 in total) in a frame and sealed in a foil bag. Fit strip wells firmly into the frame. After opening, immediately return any unused wells to the original foil package and seal. Store at 2 8 C until expiration date. thru Standards 5 vials of lyophilized Standard containing recombinant Leptin (1, 10, 25, 50 and 100 ng/ml). Reconstitute each Standard with 750 µl of Dilution Buffer. Keep reconstituted reagents at room temperature for 15 minutes and then mix them gently (no foam!) with a Vortex. After reconstitution, the Standards are stable 2 months at -20 C. Only 3 freeze/thaw cycle or keep in aliquotes. Store lyophilized at 2 8 C until expiration date. Control 1 1 vial of lyophilized control (human serum). Reconstitute with 500 µl of Dilution Buffer. Keep reconstituted reagents at room temperature for 15 minutes and then mix them gently (no foam!) with a Vortex. After reconstitution, the control serum is stable 2 months at -20 C (3 freeze/thaw cycles or store in aliquotes). For the exact value, refer to data sheet. Store lyophilized at 2 8 C until expiration date. Control 2 1 vial of lyophilized control (human serum). Reconstitute with 500 µl of Dilution Buffer. Keep reconstituted reagents at room temperature for 15 minutes and then mix them gently (no foam!) with a Vortex. After reconstitution, the control serum is stable 2 months at -20 C (3 freeze/thaw cycles or store in aliquotes). For the exact value, refer to data sheet. Store lyophilized at 2 8 C until expiration date. Dilution Buffer 1 vial of 25 ml, ready for use. Possible precipitation in the Buffer, resolve before using by mixing and/or warming. Store at 2 8 C until expiration date. Antibody-HRP Conjugate 1 vial of 12 ml, ready for use. Anti-human leptin conjugated to horseradish peroxidase (HRPO). Store at 2 8 C until expiration date. TMB Substrate 1 vial of 12 ml stabilized H 2 O 2 Tetramethylbenzidine. Ready for use. Store at 2 8 C until expiration date. Wash Buffer 1 vial of 50 ml of buffer. Possible precipitation in the Buffer, resolve before using by mixing and/or warming Bring the vial content to 1000 ml with distilled water. The diluted washing solution is stable for 4 weeks at 2 8 C. Store undiluted at 2 8 C until expiration date. 12

13 Stop Solution 0.2 M H 2 SO 4 1 vial of 12 ml 0.2 M H 2 SO 4. Ready for use. Store at 2 8 C until expiration date. Cover for Microtiterplate 2 pieces, adhesive. Preparation and Stability of Serum Samples Sample Type Serum and plasma samples are suitable (significant deviation of hleptin levels in corresponding Serum, Heparin or EDTA Plasma samples were not found), as well as cell culture, urine and saliva. Stability Maximum 2 days at room temperature Maximum 2 years at -20 C Maximum 5 freeze/thaw cycles Note Most of the time the use of undiluted samples, 20 µl per well, should be appropriate. It can be expected that patients with a BMI >30 will have Leptin levels over 100 ng/ml: the sample should then be diluted in the Dilution Buffer, e.g. 1:2. The hleptin concentrations may be completely different in body fluids of human origin other than serum or cell culture supernatants. 13

14 Assay Procedure All determinations (Standards, Control Sera and Samples) should be assayed in duplicate. When performing the assay, the Standards, Control Sera and the Samples should be pipetted as fast as possible (<15 minutes). To avoid distortions due to differences in incubation times, HRP Conjugate, Substrate Solution and Stop Solution should be added to the plate in the same order and with the same time interval as the samples. Allow all reagents to equilibrate at room temperature (20 25 C) for at least 30 minutes. 1. Prepare the frame and the required number of coated strips. 2. Allocate the wells of the Microtiter plate for Standards, Controls and Samples. 3. Pipette 100 µl Dilution Buffer into all wells. 4. Add 20 µl Dilution Buffer in duplicate (Blank). 5. Pipette 20 µl of each Standard ( thru ), Control sera ( and ) and samples into the corresponding wells. 6. Cover the wells with sealing tape and incubate the plate for 1 hour at room temperature on a plate shaker ( 350 rpm). 7. After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3 x with 350 µl diluted washing buffer (15 seconds incubation per cycle). After the last wash cycle tap the inverted wells gently on a dry absorbent surface to remove excess wash solution. 8. Pipette 100 µl of the Antibody HRP Conjugate in each well. 9. Cover the wells with sealing tape and incubate the plate for 30 minutes at room temperature on a plate shaker ( 350 rpm). 10. After incubation wash the wells 3 x with Washing Buffer as described in step Pipette 100 µl of the TMB Substrate Solution in each well. 12. Incubate the plate for 15 minutes, in the dark, at room temperature (20 25 C). 13. Add 100 µl of Stop Solution in each well. 14. Measure the colour reaction within 30 minutes at 450 nm (reference filter between nm). With strong colour reaction e. g. >3 OD, also measure at 405 nm. Protocols for the different automatic ELISA systems are available. 14

15 Result Analysis For the evaluation of the assay, the absorbance values of the blank should be below 0.25 and the absorbance values of Standard E should exceed 1.0. Samples, having higher absorbance values than Standard E, should be tested again with a higher dilution. A standard curve can be established by plotting Standard concentration on the x-axis (linear scale) against the absorbance of the Standards on the y-axis (linear scale). The Leptin concentrations in samples can then be read off the standard curve. A 4-parameter curve fit should be used for automatic data reduction. Typical Results (Example only, not for use in calculation of actual results) Standards Absorption at 450 nm ng/ml A B C D E L-Control H-Control For each assay, the results of the controls must be within the target range indicated for every lot.the QC protocol with target ranges is provided with the kit. If control values are not within the limits of the target range, the assay results should be considered questionable and the samples should be tested again. 15

16 Observed Values Serum leptin levels are mainly determined by body fat mass with low levels in lean individuals and high levels in obese subjects. In addition, there is a clear gender difference with higher levels in females at a given percentage body fat. Further, leptin levels are influenced by pubertal development. Any attempt, therefore, to give ranges of expected leptin levels must account for these relationships. Various methods for the estimation of body fat are available such as calculation of body mass index [weight (kg) divided by the square of height (m)] (BMI), bioelectric impedance assessment (BIA) or total body dual energy x-ray absorptiometry (DXA). Although the accuracy of BMI with respect to reflecting true fat mass is inferior to other more sophisticated methods such as BIA or DXA, BMI provides a number of advantages: It is independent of the regression models applied. It is easy to determine, only weight and height measurements are required. It is retrospectively mostly available. It is the most precise measure during short-term changes of fat mass, e.g. during fasting. Therefore, the following expectation ranges of serum leptin levels were referred to BMI as the major confounding independent variable and were stratified according to gender and pubertal development (45; see figures 1 8 and tables 1 9). After the age of 20 years, no significant age dependence was observed. These gender and age adjusted expectation ranges may be used to compare a measured leptin level at a given BMI with normal subjects to detect deviations. The best-fit regression lines for the various subgroups are exponential curves of the form: Leptin = a e (b BMI) The 5 th and 95 th percentiles are given by the following equations: and Leptin = a e (b BMI c) Leptin = a e (b BMI + c) respectively. In a semi-logarithmic plot (y-axis = log leptin), these curves give straight lines. The values for a, b and c are given in table 1 according to gender and pubertal stage and also for adults. Using these values, the expectation ranges of leptin levels can be easily extended to lower or higher BMI ranges if required. Example The 50 th percentile for boys at Tanner stages 3 and 4 is given by the following curve: Leptin = e ( BMI) The 5 th percentile is given by: Leptin = e ( BMI ) and the 95 th percentile is given by: Leptin = e ( BMI ) In a semi-logarithmic plot, these lines are parallel with an equal distance to the 50 th percentile. 16

17 Calculation of standard deviation scores (SDS; Z-scores) A convenient method to detect any deviation of a measured leptin level from the corresponding observed range is to calculate its standard deviation score by relating the leptin level at the patient s BMI to the average leptin value of the corresponding sex and age group and expressing its deviation by the x-fold standard deviation. This method may be considered as normalization to the normal reference cohort. Thus, the leptin values can be adjusted for BMI, gender and pubertal stage/age (i.e., the influence of gender, age and BMI are removed) and may be pooled for further analysis. Accounting for the logarithmic distribution of leptin levels, the leptin SDS can be calculated by the following equation: Leptin SDS = (ln(leptin) ln(a) b BMI) d In this equation, ln represents the natural logarithm (referring to the basis e). The constants a, b and d are given in table 1 according to gender and pubertal stage/age. Example: A boy at Tanner stage 3, BMI = 25 kg/m 2, measured leptin concentration = 5 ng/ml. Leptin SDS = (ln(5) ln (0.0181) ) = Constants a, b, c and d for calculation of leptin ranges and leptin SDS based on BMI. Groups of individuals were stratified according to gender and pubertal stage/age. TS= Tanner stage, n= number of subjects, a,b,c and d = constants as defined in the text. Cohort n a b c d Males TS 1& TS 3& TS Adults Females TS 1& TS 3& TS Adults Table 1 17

18 BMI (kg/m 2 ) Percentile (µg/l) Table 2: Girls Tanner stages 1 and 2 BMI (kg/m 2 ) Percentile (µg/l) Table 3: Boys Tanner stages 1 and 2 Figure 1: Observed ranges of human serum levels referring to BMI: Girls Tanner stage 1 & 2 (see text for details). Figure 2: Observed ranges of human serum levels referring to BMI: Boys Tanner stage 1 & 2 (see text for details). 18

19 BMI (kg/m 2 ) Percentile (µg/l) Table 4: Girls Tanner stages 3 and 4 BMI (kg/m 2 ) Percentile (µg/l) Table 5: Boys Tanner stages 3 and 4 Figure 3: Observed ranges of human serum levels referring to BMI: Girls Tanner stage 3 & 4 (see text for details). Figure 4: Observed ranges of human serum levels referring to BMI: Boys Tanner stage 3 & 4 (see text for details). 19

20 BMI (kg/m 2 ) Percentile (µg/l) Table 6: Girls Tanner stages 5 BMI (kg/m 2 ) Percentile (µg/l) Table 7: Boys Tanner stages 5 Figure 5: Observed ranges of human serum levels referring to BMI: Girls Tanner stage 5 (see text for details). Figure 6: Observed ranges of human serum levels referring to BMI: Boys Tanner stage 5 (see text for details). 20

21 BMI (kg/m 2 ) Percentile (µg/l) Table 8: Adult women BMI (kg/m 2 ) Table 9: Adult men Percentile (µg/l) Figure 7: Observed ranges of human serum levels referring to BMI: Adult women (see text for details). Figure 8: Observed ranges of human serum levels referring to BMI: Adult men (see text for details). 21

22 Test Performance Calibration The Standards from the kit are prepared from recombinant Leptin, calibrated against the WHO international standard NIBSC 97/594. Precision (Inter assay) Sample Mean value Standard deviation CV (%) Sample Sample Sample (Intra assay) Sample N Mean value (ng/ml) Standard deviation (ng/ml) CV (%) Sample Sample Detection Limit The analytical sensitivity of the assay yields 0.2 ng/ml (2 x SD of zero standards). Recovery Test The recovery of recombinant hleptin in different human sera yielded on average 102 % of the theoretical expected value. Parallelism Dilution Sample 1 (ng/ml) Dilution Sample 2 (ng/ml) 1: : : : : : : : : : Mean/1 SD/CV % 36.96/2.5/6.8 Mean/1 SD/CV % 17.4/0.86/4.97 Mean = Average Value, SD = Standard Deviation, CV = Variation coefficient 22

23 Interference Serum samples have been spiked with different concentrations of possibly interfering substances and the amount of leptin was measured and compared with the leptin concentration in the same sample without any spiking. None of the tested substances interfered significantly with leptin measurement. % Triglyceride 100 mg/ml Bilirubin 100 µg/ml Hemoglobin 100 µg/ml Sample Sample Sample Cross-reactivity This assay is specific for human leptin. There is a low cross-reaction with mouse, rat, horse, sheep and chicken-leptin. No cross-reactivity was found with other proteins such as insulin or IGF-I. Remark The data quoted in this instruction should be used for guidance only. It is recommended that each laboratory includes its own panel of control samples in the assay. In order to follow GLP guidelines, each laboratory should establish its own ranges for leptin levels. 23

24 TECO Human Leptin Assay Procedure Quick Guide Bring samples and reagents to room temperature. Mix the samples well. Standards thru : Reconstitute each vial with 750 µl Dilution Buffer. Controls and : Reconstitute each vial with 500 µl Dilution Buffer. Washing Buffer : Dilute 1:20 with Distilled water. Prepare the required number of Assay Strips Pipette 100 µl Dilution Buffer in all wells Add 20 µl Dilution Buffer into wells (Blank) Pipette 20 µl Standards thru, Controls and and Samples After sealing the plate, incubate 60 min at C on a plate shaker 350 rpm Aspirate and wash 3 x with 350 µl Wash Buffer, aspirate and tap the inverted wells gently on a clean dry absorbent surface Add 100 µl Antibody HRP Conjugate into each well After sealing the plate, incubate 30 min at C on a plate shaker 350 rpm Aspirate and wash 3 x with 350 µl Wash Buffer, aspirate and tap the inverted wells gently on a clean dry absorbent surface Add 100 µl TMB Substrate into each well Incubate 15 min at C in the dark Add 100 µl Stop Solution into each well Measure the absorbance at 450 nm within 30 minutes Quantification software, 4-parameter fit: y = (A-D)/(1+(x/C)^B)+D Reference measurement should be performed at nm Please read Kit instruction before using the Quick Guide.