Leptin ELISA (Ultrasensitive)

Transcription

1 Leptin ELISA (Ultrasensitive) For quantitative determination of Leptin in serum, plasma, urine, saliva and breast milk. For Research Use Only. Not For Use In Diagnostic Procedures. Catalog Number: 22-LEPHUU-E01 Size: 96 wells Version: ALPCO February 25, G Keewaydin Drive, Salem, NH P: (800) F: (603) ts@alpco.com 1

2 INTENDED USE Measurement of human Leptin in human serum, plasma, urine, saliva, and breast milk. For research use only. Not for use in diagnostic procedures. INTRODUCTION Leptin, the product of the OB gene (1,2), is a recently discovered 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). 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. This ELISA kit is suited for measuring human leptin in serum or plasma, other biological fluids (e.g. urine or cerebrospinal fluid), and conditioned adipocyte culture media for scientific purposes. 2

3 REAGENTS PROVIDED 1) MTP 2) STD 3) BUF 4) Control 5) Ab CONJ 6) WASHBUF 20x Microtiter plate, ready for use: Microtiter plate with 96 wells, 12 x 8 well strips (separately breakable), coated with antibody against leptin, packed in a laminate bag. Standards A-E, 1 ml lyophilized: Contain recombinant Leptin and have to be reconstituted with 1 ml Dilution Buffer VP each. After using store the reconstituted standards in the original flasks as soon as possible at 20 C. When using the standards anew, please thaw them rapidly but gently (no temperature rise over the room temperature and no powerful vortexing), 3 of these freezingthawing cycles showed no influence on the assay. Standard values are between ng/ml (0.05; 0.5; 1.5; 3.5; 5 ng/ml) recombinant human leptin Dilution Buffer VP, 60 ml, ready for use, please use for the reconstitution of Standards A-E, Control Sera KS1 & KS2. This buffer is also used to dilute the controls and sample(s). Control Sera KS1 & KS2, each 250 µl lyophilized: Contains human serum. Reconstitute each with 250 µl Dilution Buffer VP. Exact human leptin concentration and the acceptable range are given on the certificate. The reconstituted Control Sera must be stored in the original flask as soon as possible at 20 C after use. When using anew, please thaw it rapidly but gently (no temperature rise over the room temperature and no powerful vortexing), 3 of these freezing-thawing cycles showed no influence on the assay. anti-leptin Antibody POD-Conjugate AK, 12 ml, ready for use: Contains a mix of biotinylated anti-human leptin antibody and Horseradish peroxides conjugated streptavidin. Use 100 µl per well in the assay Washing Buffer (WP), 50 ml, 20 X concentrated solution. Washing Buffer (WP) has to be diluted 1:20 with distilled or demineralized water before use (e.g. add the complete contents of the flask (50 ml) into a graduated flask and fill up with A.dest. to 1000 ml). Attention: After dilution the Washing Buffer is only 4 weeks stable, dilute only according to requirements. 7) SUBST Substrate (S), 12 ml, ready for use, horseradish-peroxidase-(hrp)-substrate, stabilized H 2O 2 Tetramethylbencidine. 8) H2SO4 Stopping Solution (SL), 12 ml, ready for use, 0.2 M sulphuric acid, Caution acid. 9) Sealing tape for covering of the microtiter plate, 2 x, adhesive. MATERIALS REQUIRED BUT NOT PROVIDED Precision pipettes, Micropipettes and multichannel pipettes with disposable plastic tips Distilled or deionized water for dilution of the Washing Buffer (WP) Vortex-mixer Microtiter plate shaker (350 rpm) Microtiter plate washer (recommended) Microplate reader ("ELISA-Reader") with filter for 450 and 590 nm Polyethylene PE/Polypropylene PP tubes for dilution of samples WARNINGS AND PRECAUTIONS For Research Use Only. Not for Use in Diagnostic Procedures. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. Before use, all kit components should be brought to room temperature at C. Precipitates in buffers should be dissolved before use by thorough mixing and warming. Do not mix reagents of different lots. Do not use expired reagents. The microplate contains snap-off strips. Unused wells must be stored at 2-8 C in the sealed foil pouch and used in the frame provided. 3

4 Caution: This kit contains material of human and/or animal origin. Source human serum for the Control Sera provided in this kit was tested and found non-reactive for Hepatitis-B surface antigen (HBsAg), Hepatitis C virus (HCV), and Human Immunodeficiency Virus 1 and 2 (HIV) antibody. No known test methods can offer total assurance of the absence of infectious agents; therefore all components and specimens should be treated as potentially infectious. Following components contain < 0.01% 2-Methyl-4-isothiazolin-3-one solution as preservative: AK, VP R34 R43 S26 Irritating to eyes and skin Sensibilization through skin contact possible In case of contact with eyes, rinse immediately with plenty of water and seek medical advice S36/37 Wear suitable protective clothing and gloves S45 In case of accident or if someone feels unwell, seek medical advice Following components contain < 0.01%(w/w) 5-chloro-2-methyl 2H isothiazol-3-one and 2-methyl-2Hisothiazol-3-one as preservative: AK, VP, WP R36/38 Irritating to eyes and skin R43 Sensibilization through skin contact possible S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice S28.1 S28.1 After contact with skin, wash immediately with plenty of water Stop solution contains 0.2 M Sulfuric Acid (H 2SO 4) R36/38 Irritating to eyes and skin S26 In case of contact with eyes, rinse immediately with plenty of water and seek medical advice S28.1 After contact with skin, wash immediately with plenty of water S36/37 Wear suitable protective clothing and gloves. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for each step. Use separate pipette tips for each sample, control and reagent to avoid cross contamination. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir for dispensing a substrate solution that had previously been used for the conjugate solution may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps. TMB-Substrate (S) contains 3,3,5,5 Tetramethylbenzidine. Store and Incubate in the dark. R20/21/R22 R36/37/38 S26 Harmful by inhalation, in contact with skin and if swallowed Irritating to eyes, respiratory system and skin In case of contact with eyes, rinse immediately with plenty of water and seek medical advice S28.1 After contact with skin, wash immediately with plenty of water S36/37 Wear suitable protective clothing and gloves General first aid procedures: Skin contact: Wash affected area thoroughly with water. Discard contaminated cloths and shoes. Eye contact: In case of contact with eyes, rinse immediately with plenty of water at least 15 minutes. In order to assure an effectual rinsing spread the eyelids. Ingestion: If swallowed, wash out mouth thoroughly with water. Immediately see a physician. Do not eat, drink or smoke in these areas. Never pipette the materials with the mouth. Spilled material must be wiped off immediately and should become disinfected. Clean contaminated areas and equipment with a suitable detergent. 4

5 PRINCIPLE The ELISA for Leptin E077 is a so-called Sandwich-Assay using two specific and high affinity antibodies. The Leptin in the samples binds to the first antibody coated on the microtiter plate. In the following step the second specific anti-leptin-antibody binds in turn to the immobilised Leptin. The second antibody is biotinylated and will be applied in a mixture with a Streptavidin-Peroxidase-Enzyme Conjugate. In the closing substrate reaction the turn of the colour will be catalysed quantitatively depending on the Leptin-level of the samples. SPECIMEN Serum and plasma samples, urine, saliva and breast milk are applicable. Significant deviation of Leptin levels in corresponding Serum- or EDTA-Plasma samples were not found. If commercially available tubes for Citrat-plasma are used for preparation, samples will be diluted, resulting Leptin values will be therefore reduced. The blood sample for serum preparation should be gained according to standardized venipucture procedure. Hemolytic reactions have to be avoided. The blood has to be allowed to clot min at RT. After complete clotting, serum is separated by centrifugation (3000g, 10 min). In case of normal nutritional status serum and plasma samples should be gained in the morning or the early afternoon, because leptin undergoes circadian changes with a maximal serum value at 2 am. Storage of the samples Storage at RT max. 2 days Storage at 20 C max. 2 years No more than five freeze/thaw cycles recommended. Sample Preparation Samples have to be diluted in Dilution Buffer (VP). A standard dilution of 1:10 is recommended. Suggestion for dilution protocol: Dilute e.g. 225 µl Dilution Buffer VP in PE-/PP-Tubes (application of a multi-stepper is recommended in larger series), add 25 µl Serum-or Plasma (dilution 1:10). After mixing use 2 x 100 µl from the dilution in the assay. If Leptin levels over 50 ng/ml are expected, the sample should be diluted higher in Dilution Buffer VP, e.g. 1:20. The Leptin concentrations may be completely different in body fluids of human origin other than serum or cell culture supernatants. In samples of urine or breast milk very low levels were determined. Furthermore this kind of samples must be diluted at least 1:5. If samples are used undiluted, the reaction will be interfered and the values are reduced (s. Assay Characteristics and Validation). TECHNICAL NOTES The assay has to be conducted strictly according the test protocol herein. Reagents with different lot numbers cannot be mixed. The microtiter plate and reagents are stable until the indicated expiry if stored unopened and protected from sunlight at 2 8 C. The shelf life of the components after opening is not affected, if used appropriately. 5

6 Bring all reagents to room temperature (20-25 C) before use. Possible precipitations in the buffers have to be resolved before usage by mixing and / or warming. Incubation at room temperature means: C The incubation steps should be performed at mean rotation frequency of a particularly suitable microtiter plate shaker. It is recommended to use 350 rpm. Due to certain technical differences deviations may occur, in case the rotation frequency must become adjusted. Insufficient shaking may lead to inadequate mixing of the solutions and thereby to low optical densities, high variations and/or false values, excessive shaking may result in high optical densities and/or false values. Proper washing is of basic importance for a secure, reliable and precise performance of the test. Incomplete washing is common and will adversely affect the test outcome. Possible consequences may be uncontrolled unspecific variations of measured optical densities, potentially leading to false results calculations of the examined samples. Effects like high background values or high variations may indicate washing problems. All washing must be performed with the provided washing buffer diluted to usage concentration. Washing volume per washing cycle and well must be 300 µl at least. The danger of handling with potentially infectious material must be taken into account. When using an automatic microtiter plate washer, the respective instructions for use must be carefully followed. Device adjustments, e.g. for plate geometry and the provided washing parameters, must be performed. Dispensing and aspirating manifold must not scratch the inside well surface. Provisions must be made that the remaining fluid volume of every aspiration step is minimized. Following the last aspiration step of each washing cycle, this could be controlled, and possible remaining fluid could then be removed, by inverting the plate and repeatedly tapping it dry on non fuzzy absorbent tissue. Manual washing is an adequate alternative option. Washing Buffer may be dispensed via a multistepper device, a multichannel pipette, or a squirt bottle. The fluid may be removed by dynamically swinging out the microtiter plate over a basin. If aspirating devices are used, care has to be taken that the inside well surface is not scratched. Subsequent to every single washing step, the remaining fluid should be removed by inverting the plate and repeatedly tapping it dry on non fuzzy absorbent tissue. Standards and Controls For the reconstitution of the lyophilized components (Standards A - E and Control Sera KS1 &KS2) the kit Dilution Buffer VP has to be used. It is recommended to keep reconstituted reagents at room temperature for 15 minutes and then to mix them thoroughly but gently (no foam) with a Vortex mixer. The reconstituted standard and controls can be stored for 2 month at 20 C. Repeated freeze/thaw cycles have to be avoided. Controls KS1 and KS2 kit controls will be diluted 1:10 in Dilution Buffer by adding 25uL of KS1 or KS2 to 225uL Dilution Buffer VP in PE-/PP-Tubes or microtiter plates. After mixing, use 2 x 100 L from the dilution in the assay. Washing Buffer The required volume of washing buffer is prepared by 1:20 dilution of the provided 20fold concentrate with deionised water. The diluted washing buffer is stable for 4 weeks at 2-8 C. It has to be at room temperature for usage. Microtiter plate Unused microtiter plate stripes have to be stored airtight together with the desiccant bag at 2-8 C. The labelled expiry is not influenced in case of proper storage. ASSAY PROCEDURE All determinations (Standards, Control Sera KS1 & KS2 and samples) should be assayed in duplicate. For optimal results, accurate pipetting and adherence to the protocol are recommended. 6

7 When performing the assay, the Standards, Control Sera and the samples should be pipetted as fast as possible (e.g., <15 minutes). All incubations have to be conducted at room temperature (20-25 C) To avoid distortions due to differences in incubation times, Antibody-POD-Conjugate AK as well as the following Substrate Solution S should be added to the plate in the same order and in the same time interval as the samples. Stop Solution SL should be added to the plate in the same order as the Substrate Solution. 1) Add 100 µl Dilution Buffer VP to the first wells (blank). Subsequently, add 100µL of each standard or diluted Control (KS1&KS2) or diluted Sample to the following wells. 2) Cover the wells with sealing tape and incubate the plate for 1 hour shaking with 350 rpm. 3) After incubation aspirate the contents of the wells into a disinfectant (possible risk of infection.) and wash the wells 3 times with 300 µl of Washing Buffer WP / well respectively. The washing buffer WP should incubate at least for 15 seconds/cycle 4) Pipette 100 µl of the Antibody-POD-Conjugate AK in each well and incubate 30 minutes shaking with 350 rpm. 5) After incubation wash the wells 3 times with Washing Buffer as described in step 3 6) Pipette 100 µl of the Substrate (S) in each well. 7) Incubate the plate for 15 minutes in the dark at room temperature (20-25 C). 8) Stop the reaction by adding 100 µl of Stopping Solution (SL). 9) Measure the color reaction within 30 minutes at 450 nm (reference filter 590 nm). CALCULATION OF RESULTS Establishing the Standard Curve For the evaluation of the assay it is preconditioned that the absorbance values of the blank should be below 0.25, these of standard E should be above 1.0. Samples, which yield higher absorbance values than Standard E are beyond the standard curve, for reliable determinations these samples should be tested anew with a higher dilution. Standards are provided in the following concentrations (use the concentration unit as preferred): Standard A B C D E ng/ml ) Calculate the mean absorbance value for the blank from the duplicated determination (well A1/A2). 2) Subtract the mean absorbance of the blank from the mean absorbance of all other values. 3) Plot the standard concentrations on the x-axis versus the mean value of the absorbance of the standards on the y-axis. 4) Recommendation: Calculation of the standard curve should be done by using a computer program. A higher-grade polynomial, or four parametric logistic (4-PL) curve fit or nonlinear regression are usually suitable for the evaluation (as might be spline or point-to-point alignment in individual cases). 7

8 5) The concentration in ng/ml of the samples can be calculated by multiplication with the respective dilution factor. The exemplary shown standard curve in Fig.1 cannot be used for calculation of the test results. Each research lab will have to establish a standard curve for each test conducted. Exemplary calculation of the leptin concentration of an undiluted sample: Measured extinction of the sample Measured extinction of the blank The measurement program will calculate the leptin concentration of the diluted sample automatically by using the difference of sample and blank (0.278) for the calculation. One only has to determine the most suitable curve fit (here: polynomial 2 nd degree). In this exemplary case the following equation is solved by the program to calculate the Leptin concentration in the sample: = ,64743x x² = x If the dilution factor (1:10) is taken into account the leptin concentration of the undiluted sample is x 10 ng/ml= ng/ml Fig. 1: Exemplary Standard Curve with a polynomial 2 rd degree as curve fit. PERFORMANCE CHARACTERISTICS Standards The Standards of the ELISA E077 are prepared from recombinant Leptin in concentrations of 0.05; 0.5; 1.5; 3.5; and 5 ng/ml. Sensitivity The analytical sensitivity of the assay yields 0.01 ng/ml (2x SD of zero standards in 12-fold determinations). 8

9 Specificity This assay is specific for human leptin. Sera of the cited species were used as sample in this assay system. No measurable cross reactivity was detected in serum of: Horse, Cow, Chicken, Rabbit, Dog, Guinea pig, Sheep, Mouse, Goat, Donkey, Rat, Cat, Pig Reproducibility and Precision Table 1 : Inter-Assay-Variation Mean value (ng/ml) Standard Deviation (ng/ml) VC (%) Sample Sample Sample Table 2: Intra-Assay-Variation Mean value (ng/ml) Standard Deviation (ng/ml) VC (%) Sample Sample

10 LITERATURE 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. 372: Halaas JL, Gajiwala KS, Maffei M, et al Weight-reducing effects of the plasma protein encoded by the obese gene. Science. 269: 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. 92: Rentsch J, Chiesi M Regulation of ob gene mrna levels in cultured adipocytes. FEBS Lett. 379: Wabitsch M, Jensen PB, Blum WF, et al Insulin and cortisol promote leptin production in cultured human fat cells. Diabetes. 45: Stephens TW, Basinski M, Bristow PK, et al The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature. 377: 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. 269: Pelleymounter MA, Cullen MJ, Baker MB, et al Effects of the obese gene product on body weight regulation in ob/ob mice. Science. 269: 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. 93: Tartaglia LA, Dembski M, Weng X, et al Identification and expression cloning of a leptin receptor, OB-R. Cell. 83: 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. 84: Lee GH, Proenca R, Montez JM, et al Abnormal splicing of the leptin receptor in diabetic mice. Nature. 379: 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. 219: 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. 45: Sinha MK, Opentanova I, Ohannesian JP, et al Evidence of free and bound leptin in human circulation. J Clin Invest. 98: Ahima RS, Prabakaran D, Mantzoros C, et al Role of leptin in the neuroendocrine response to fasting. Nature. 382: 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. 12: 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. 45: Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG Identification of targets of leptin action in rat hypothalamus. J Clin Invest. 98: Campfield LA, Smith FJ, Burn P The OB protein (leptin) pathway - a link between adipose tissue mass and central neural networks. Horm Metab Res. 28: 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. 28: Chan YY, Steiner RA, Clifton DK Regulation of hypothalamic neuropeptide-y neurons by growth hormone in the rat. Endocrinol. 137: 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 insulin-like growth factor I secretion in intact adult male rats. Endocrinol. 137: Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB, Flier JS Leptin levels reflect body lipid content in mice: evidence for diet-induced resistance to leptin action. Nat Med. 1:

11 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. 1: Considine RV, Sinha MK, Heiman ML, et al Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med. 334: 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. 45: 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. 97: 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. 91: Saladin R, De-Vos P, Guerre-Millo M, et al Transient increase in obese gene expression after food intake or insulin administration. Nature. 377: 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. 44: 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. 45: Malström R, Taskinen M-R, Karonen S-L, Yki-Järvinen H Insulin increases plasma leptin concentrations in normal subjects and patients with NIDDM. Diabetologia. 39: 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. 270: Slieker LJ, Sloop KW, Surface PL, et al Regulation of expression of ob mrna and protein by glucocorticoids and camp. J Biol Chem. 271: Miell JP, Englaro P, Blum WF Dexamethasone induces an acute and sustained rise in circulating leptin levels in normal human subjects. Horm Metab Res. 28: 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. 97: Havel PJ, Kasim-Karakas S, Dubuc GR, Mueller W, Phinney SD Gender difference in plasma leptin concentrations. Nature Med. 2: Rosenbaum M, Nicolson M, Hirsch J, et al Effects of gender, body composition, and menopause on plasma concentrations of leptin. J Clin Endocrinol Metab. 81: Hassink SG, Sheslow DV, de Lancey E, Opentanova I, Considine RV, Caro JF 1996 Serum leptin in children with obesity: relationship to gender and development. Pediatrics. 98: 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. 28: Caro JF, Kolaczynski JW, Nyce MR, et al Decreased cerebrospinal-fluid/serum leptin ratio in obesity: a possible mechanism for leptin resistance. Lancet. 348: 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. 27: Adresse NIBSC: Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, Great Britain. 45. Blum WF, Juul A; Reference ranges of serum leptin,s In:Leptin -the voice of adipose tissue, Blum WF et al, eds., Johann Ambrosius Verlag, Heidelberg,

12 SUMMARY - hleptin-sensitive ELISA Reconstitution/ Dilution of reagents Standards A-E reconstitute in Dilution Buffer VP (after using, store at 20 C) 1 ml Control Serum KS1&KS2 reconstitute in Dilution Buffer VP 250 µl (after using, store at 20 C) Washing Buffer WP dilute in A. dest. (e.g. add the complete contents of 1:20 the flask (50 ml) into a graduated flask and fill with A.dest. to 1000 ml). Sample dilution varies regarding the sample type, serum samples generally e.g. 1:10, use 100 µl per determination. Before assay procedure bring all reagents to the room temperature. Proposal of Assay Procedure for double determinations Pipette Reagents Well positions 100 µl Dilution Buffer VP as blank A1 and A2 100 µl Standard A (0.05 ng/ml) B1 and B2 100 µl Standard B (0.5 ng/ml) C1 and C2 100 µl Standard C (1.5 ng/ml) D1 and D2 100 µl Standard D (3.5 ng/ml) E1 and E2 100 µl Standard E (5 ng/ml) F1 and F2 100 µl Control Serum KS1 G1 and G2 100 µl Control Serum KS2 H1 and H2 100 µl Sample Cover the wells with the sealing tape Incubation: 1 h at RT (20-25 C), 350 rpm Pipette sample in the rest of the wells according to requirements 3x 300 µl Aspirate the contents of the wells and wash 3x with 300 µl Wash each well Buffer WP/well 100 µl Antibody-HRP-Conjugate AK each well Incubation: 30 min at RT (20-25 C), 350 rpm 3x 300 µl Aspirate the contents of the wells and wash 3x with 300 µl Wash each well Buffer WP 100 µl Substrate Solution S each well Incubation: 15 min in the dark at RT (20-25 C) 100 µl Stop Solution SL each well Measure the absorbance within 30 min at 450 nm ( 590 nm reference) 13