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1. 1 10 100 Concentration of Human Resistin ng ml B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual Vill Troubleshooting Guide 1 Lack of signal or weak signal in all wells Possible explanations e Omission of a reagent or a step e Improper preparation or storage of a reagent e Assay performed before reagents were allowed to come to 25 C e Plate reader did not perform well 2 High signal and background in all wells Possible explanations e Improper or inadequate washing be certain that all wash volumes and repetitions were correct e Decrease color development time e The plate should be read within 5 minutes of stopping the color development 3 High background in sample wells only Possible explanations e Sample concentration was too high 4 Weak signal in sample wells only Possible explanations e Sample concentration was too low B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual IX References Banerjee R R Lazar M A Dimerization of resistin and resistin like moleules is determined by a single cystein The Journal of Biological Chemistry 276 25970 25973 2001 Fasshauer M at al Tumor Necrosis Factor a Is a Negative Regulator of Resistin Gene Expression and Secretion in 3T3 L1 Adipocytes Biochemical and Biophysical Research Communications 208 1027 1031 2001 Hartman H B La2. 2 80 Do not store the 1 3 diluted Qualit y Controls Refer to the Certificate of Analysis for Quality Control concentrations The Quality Controls serve to confirm that the kit works in accordance with Certificate of Analysis and the ELISA test was carried out properly B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual V Sample Preparation Allow all the reagents to equilibrate at room temperature 25T prior to the start of the sample preparation Samples should be assayed immediately after collection or stored at 20 or 70 for long term Thoroughly mix thawed samples prior to assaying Aliquot samples to avoid repeat freeze thaw cycles Avoid using hemolyzed or lipemic samples Serum and plasma samples 1 For Assaying Samples in singlets Mix 50 ul of samples with 100 ul of 1X Sample Diluent 2 For Assaying Samples in duplicates Mix 100 ul of samples with 200 ul of 1X Sample Diluent Do not store 1 3 diluted samples Tissue culture supernatant Quantity of protein in cell culture supernatant may vary by cell line e g HIB 1B LS14 etc we recommend running several dilutions to determine optimal dilution for the assay Undiluted culture supernatant can also be assayed Select a few samples to determine optimal dilution for assaying 1 Undiluted tissue culture supernatants 2 Dilute 1 3 with 1X Sample Diluent 50ul of sample 100 ul of Sample Diluent 3 Dilute 1 6
3. with 1X Sample Diluent 25ul of sample 125 ul of Sample Diluent Choose suitable dilution and repeat the assay with all samples B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual VI Human Resistin ELISA Protocol Note Allow all reagents to equilibrate at room temperature 25 prior to the start of the assay and prepare 1X Wash Solution Quality Controls Resistin Standards and samples as described in the previous sections di 10 11 12 13 Remove Primary Antibody Coated Plate from its foil pouch Remove any unneeded strips from the plate frame reseal them in the foil pouch and return the foil pouch to 2 8T If a 96 well plate washer is used the plate frame should be completely filled with wells by adding as many null strips as necessary Identify well position s for each sample on a data sheet or plate map Add 100 ul of diluted Standards Quality Controls Sample Diluent blank and samples to the appropriate number of antibody coated wells Every plate must include the standard series to properly correlate the sample readings Cover plate s securely and incubate at room temperature 25C for 60 minutes on an orbital microplate shaker at ca 300 rpm After incubation wash the plate s 3 times with Wash Solution as follows a Completely aspirate the liquid from the wells using a plate washer b Fill each well with 1X Wash Solution 350 ul well and immediately
4. A Kit User Manual l Introduction and Protocol Overview Resistin a product of the RSTN gene is a peptide hormone belonging to the class of cysteine rich secreted proteins which are termed the RELM family and is also described as ADSF Adipose Tissue Specific Secretory Factor and FIZZ3 Found in Inflammatory Zone Human resistin contains 108 amino acids as a pre peptide Its hydrophobic signal peptide is cleaved before it is secreted Resistin circulates in human blood as a dimeric protein consisting of two 92 amino acid polypeptides which are disulfide linked via Cys26 Much of the early investigations about the resistin molecule are based on the mouse model Resistin produced and secreted primarily by adipocytes in mice acts on skeletal muscle myocytes hepatocytes and adipocytes themselves so that it reduces their sensitivity to insulin Steppan et al have suggested that resistin suppresses the ability of insulin to stimulate glucose uptake Other studies have shown that mouse resistin increases during the differentiation of adipocytes but it also seems to inhibit adipogenesis Compared to the mouse model human adipogenic differentiation is likely to be associated with a down regulation of resistin gene expression On the other hand resistin was found to be expressed at high levels in human monocytes macrophages and bone marrow Recent investigations have shown that human resistin is correlated with metabolic syndrome and obesity rel
5. B Bridge International Inc kA Human Resistin ELISA Kit User Manual UM 100301 Published 01 August 2004 Catalog K1003 1 See List of Components for Storage Conditions FOR RESEARCH USE ONLY Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual Table of Contents l Introduction and Protocol Overview 3 I List of Components 5 IIl Additional Materials Required 6 IV Reagent Preparation and Storage 7 V Sample Preparation 8 VI Human Resistin ELISA Protocol 9 VII Calculation of Results 11 VIII Troubleshooting Guide 12 IX References 13 Notice to Purchaser This product is to be used for Research Purposes Only It is not to be used for Drug or Diagnostic Purposes nor is it intended for Human Use B Bridge products may not be resold modified for resale or used to manufacture commercial products without the express written consent of B Bridge International Inc EXCEPT AS OTHERWISE EXPRESSLY SET FORTH IN THIS USER MANUAL B BRIDGE DOES NOT MAKE ANY REPRESENTATION OR WARRANTIES OR CONDITIONS OF ANY KIND EITHER EXPRESSED OR IMPLIED WITH RESPECT TO THE PRODUCTS OR INFORMATION DISCLOSED HEREUNDER INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FIT FOR A PARTICULAR PURPOSE OR NONINFRINGEMENT OF THE INTELLECTUAL PROPERTY RIGHTS OF THIRD PARTIES 2004 B Bridge International Inc All Rights Reserved B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELIS
6. TMB The reaction is quenched by addition of acidic solution and absorbance of the resulting product is measured at 450 nm The absorbance is proportional to the concentration of resistin A standard curve is constructed by plotting absorbance values versus resistin concentrations of calibrators and concentrations of unknown samples are determined using this standard curve Figure 1 Assay Principle Biotin labeled rabbit ma polyclonal anti human sake pt Substrate Resistin isti i onjugate resistin antibody H 0 TMB ae i OPH To 1st Reaction 2nd Reaction 3rd Reaction Color Development IS pile a RRR A goat polyclonal anti human resistin antibody coated plate gt B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual ll List of Components e Store all components at 2 8C DO NOT FREEZE 1 10X WASH SOLUTION 1 Bottle 100 ml 2 1X SAMPLE DILUENT 1 Bottle 20 ml 3 PRIMARY ANTIBODY COATED PLATE 1 Plate One plate holds 12x8 well strips 96 wells with adsorbed Goat Polyclonal Anti Human Resistin Antibody Plate is provided in a resealable foil pouch with desiccant 4 HUMAN RESISTIN MASTER STANDARD 1 Vial Lyophilized 5 QUALITY CONTROL 2 Vials High and Low lyophilized 6 BIOTINYLATED SECONDARY ANTIBODY SOLUTION 1 Bottle 13 ml Rabbit Polyclonal Anti Human Resistin Antibody Biotin Labeled 7 CONJUGATE SOLUTION 1 Bottle 13 ml Streptavidin Horseradish Peroxidase Conjugate 8 S
7. UBSTRATE SOLUTION 1 Bottle 13 ml H O2Tetramethylbenzidine 9 STOP SOLUTION 1 Bottle 13 ml 0 2M H2SO4 MSDS forms are available on our website please visit www b bridge com B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual Additional Materials Required The following materials are required but not supplied Graduated cylinder Micropipettor s and disposable pipette tips Null strips for 96 well plate 96 well plate washer Paper towels or absorbent paper Plate reader capable of measuring absorbance at a wavelength of 450 nm Orbital microplate shaker capable of approximately 300 rom Tubes for sample dilutions Deionized water B Bridge International Inc Human Resistin ELISA 04152013 ih 2 3 Human Resistin ELISA Kit User Manual IV Reagent Preparation and Storage Allow all the reagents to equilibrate at room temperature 25T prior to the start of the reagent preparation 1X Wash Solution Prepare 1X Wash Solution by mixing 100 ml of 10X Wash Solution with 900 ml of deionized water or equivalent After preparation store 1X Wash Solution at 2 8 The diluted Wash Solution is stable for 1 month at 2 8 Human Resistin Standard Solution Reconstitute the Resistin Master Standard according to the Certificate of Analysis Let the lyophilized standard dissolve in 1X Sample Diluent for at least 15 minutes prior to serial dilutions for the assay Prepare each R
8. aspirate Avoid overflow c Repeat Step 4b two more times for a total of three washes d Invert the plate s and gently tap on a clean absorbent towel Dispense 100 ul of the Biotinylated Secondary Antibody Solution into each well Cover plate s securely and incubate at room temperature 25C for 60 minutes on an orbital microplate shaker at ca 300 rpm Repeat the wash procedure described in step 4 Dispense 100 ul of Conjugate Solution into each well Cover plate s securely and incubate at room temperature 25C for 60 minutes on an orbital microplate shaker at ca 300 rpm Repeat the wash procedure described in step 4 Dispense 100 ul of Substrate Solution into each well Avoid exposing the microtiter plate to direct sunlight Covering the plate with aluminum foil is recommended Cover plate s securely and incubate at room temperature 25 for 10 minutes with no shaking The incubation time may be extended up to 20 minutes if the reaction temperature is below 20T Dispense 100 ul of Stop Solution into each well The plate should be read immediately Read the plate at 450 nm using a plate reader with reference wavelength 630 nm Subtract the readings at 630 nm from the readings at 450 nm B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual Note If some samples and standards have absorbances above the upper limit of your microplate reader perform a second reading at 405 nm A new s
9. ated disorders Malo et al have reported that resistin levels are positively associated with waist circumference tumor necrosis factor a and insulin resistance assessed by the homeostasis model and inversely correlated with total cholesterol HDL cholesterol and LDL cholesterol Moreover Sadhasiv et al found a positive correlation of SAT subcutaneous adipose tissue resistin MRNA expression with serum resistin BMI and insulin resistance HOMA index Based on the above reports human resistin might be an important marker that acts as the link between obesity and insulin resistance Resistin can play a role also in inflammation processes and in atherosclerosis The B Bridge Human Resistin ELISA Kit is designed to measure the concentration of human resistin homodimeric in human serum plasma EDTA citrate heparin or tissue culture medium B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual The principle of the assay is shown in Figure 1 Standards or samples are incubated in microtiter wells coated with a goat polyclonal anti human resistin antibody The wells are washed and a biotin labeled rabbit polyclonal anti human resistin antibody is added and incubated with the captured resistin After a thorough wash streptavidin horseradish peroxidase conjugate is added Following subsequent incubation and washing the bound conjugate is allowed to react with the substrate H2O tetramethylbenzidine
10. esistin Standard 20 ng ml 10 ng ml 5 ng ml 2 ng ml and 1 ng ml by serially diluting the stock Resistin Master Standard Solution 50 ng ml with 1X Sample Diluent as shown in the table Use the reconstituted Resistin Standard 50 ng ml and 1X Sample Diluent for the 50 0 ng ml and 0 ng ml standard solutions respectively Table 1 Serial Dilution of Resistin Standards Master Standard Vol 1X Diluent Concentration Master Std Stock 50 ng ml 500 ul of Master Std Stock 750 ul 20 ng ml 500 ul of 20 ng ml Std 500 ul 10 ng ml 500 ul of 10 ng ml Std 500 ul 5 ng ml 500 ul of 5 ng ml Std 750 ul 2 ng ml 500 ul of 2 ng ml Std 500 ul 1 ng ml Further dilute the serial diluted Standard Solutions 1 3 prior to use by mixing 100 ul of Standard Solutions with 200 ul of Sample Diluent for duplicates Do not store the diluted standard solutions Reconstituted and stock Resistin Standard Solutions 50 1 ng ml should be aliquoted and frozen at 20 for 3 months Do not store the dilut ed 1 3 standard solutions Quality Controls Reconstitute the Quality Controls with 350 ul Sample Diluent mix and let sit for 30 minutes at room temperature Further dilute the reconstituted Quality Controls 1 3 with Sample Diluent prior to use 50 ul control 100 ul Sample Diluent for singlets or 100 ul Quality Control 200 ul Sample Diluent for duplicates Reconstituted Quality Controls are stable until the expiration date at
11. tandard curve constructed using the values measured at 405 nm is used to determine resistin concentrations of off scale standards and samples Figure 2 Flow Chart of Assay Procedure Goat Polyclonal Anti Human Resistin Antibody Coated Plate Diluted Standards Quality Controls Sample Diluent and Samples 100 uL 1st Reaction 25 60 min 300 rom Prepare Wash Solution Wash x 3 350 uL x 3 Biotinylated Rabbit Polyclonal Anti Human Resistin Antibody Solution 100 uL 2nd Reaction 25 60 min 300 rom Wash x 3 350 uL x 3 Streptavidin HRP Conjugate Solution 100 uL 3rd Reaction 25 60 min 300 rom Wash x 3 350 uL x 3 Substrate Solution 100 uL Color Development 25 10 min no shaking Stop Solution 100 uL Read Absorbance at 450 nm B Bridge International Inc Human Resistin ELISA 04152013 Human Resistin ELISA Kit User Manual Vil Calculation of Results 1 Subtract the mean absorbance value of the 0 ng ml blank from each mean absorbance value of the standard series and samples tested Net Absorbance 2 Plot the log of known concentrations of each standard and the calculated Net Absorbances on the X axis and Y axis respectively Fit an appropriate regression curve to the plotted points 3 Determine the resistin concentrations of the samples by interpolation of the regression curve formula Figure 3 Typical Standard Curve Human Resistin ELISA ae Standard Curve Absorbance 450 nm
12. zar M A at al Mechanisms Regulating Adipocyte Expression of Resistin The Journal of Biological Chemistry 277 22 19754 19761 2002 Juan CC at al Suppressed Gene Expression of Adipocyte Resistin in an Insulin resistant Rat Model Probably by Elevated Free Fatty Acids Biochemical and Biophysical Research Communications 289 1328 1333 2001 Kim KH et al A Cysteine rich Adipose Tissue specific Secretory Factors Inhibits Adipocte Differentiation The Journal of Biological Chemistry 276 14 11252 11256 2001 Steppan C M Lazar M A Resistin and obesity associated insulin resistance TRENDS in Endocrinology and Metabolism 13 1 18 23 2002 Steppan C M et al The Hormone Links Obesity to Diabetes Nature 409 307 312 2001 Steppan C M at al A Family of Tissue specific Resistin like Molecules Proc Natl Acad Sci USA 98 2 502 506 2001 Way J M et al Adipose Tissue Resistin Expression Is Severly Suppressed in Obesity and Stimulated by Peroxisome Proliferator activated Receptor y Agonists The Journal of Biological Chemistry 276 28 25651 25653 2001 B Bridge International Inc Human Resistin ELISA 04152013
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