User Manual
Contents
1. QC Hi A B__ Standard 10 QC Low Sample8 Sample 16 Sample 24 Sample 32 S A S Standard 5 Sample 1 Sample 9 Sample 17 Sample 25 Sample 33 S D__ Standard 2 5 Sample 2_ Sample 10 Sample 18 Sample 26 Sample 34 __ E Standard1 Sam F Standard 0 5 Sam _G __ Standard 0 25 Sample5 Sample 13 Sample 21 Sample 29 Sample 37 H Blank Sample 6 Sample 14 Sample 22 Sample 30 Sample 38 Figure 1 Example of a work sheet Page 11 of 24 VERSION 17 180908 03 12 CALCULATIONS NG Most microplate readers perform automatic calculations of analyte concentration The standard curve is constructed by plotting the mean absorbance at 450 nm Y of Standards against log of the known concentration X of Standards using the four parameter algorithm Results are reported as concentration of rat resistin ng ml in samples Alternatively the logit log function can be used to linearize the standard curve i e logit of the mean absorbance Y is plotted against log of the known concentration X of Standards The measured concentration of samples calculated from the standard curve must be multiplied by their respective dilution factor because samples have been diluted prior to the assay e g 0 73 ng ml from standard curve x 20 dilution factor 14 6 ng ml Rat Resistin ELISA Standard Curve 3 5 3 0 4 2 5 4 2 0 4 Absorbance 450 nm 0 5 0 0 a9 oot mm 0 1 10 100 Concentration of2. NN Kit Components State Quantity Antibody Coated Microtiter Strips ready to use 96 wells Biotin Labelled Antibody ready to use 13 ml Streptavidin HRP Conjugate ready to use 13 ml Master Standard lyophilized 1 vial Quality Control High lyophilized 1 vial Quality Control Low lyophilized 1 vial Dilution Buffer ready to use 20 ml Wash Solution Concentrate 10x concentrated 100 ml Substrate Solution ready to use 13 ml Stop Solution ready to use 13 ml Product Data Sheet Certificate of Analysis 1 pc 8 MATERIAL REQUIRED BUT NOT SUPPLIED TN Deionized distilled water Test tubes for diluting samples Glassware graduated cylinder and bottle for Wash Solution Dilution Buffer Precision pipettes to deliver 10 1000 ul with disposable tips Multichannel pipette to deliver 100 ul with disposable tips Absorbent material e g paper towels for blotting the microtitrate plate after washing Vortex mixer Orbital microplate shaker capable of approximately 300 rpm Microplate washer optional Manual washing is possible but not preferable Microplate reader with 450 10 nm filter Software package facilitating data generation and analysis optional Page 6 of 24 VERSION 17 180908 03 9 PREPARATION OF REAGENTS eee gt gt All reagents need to be brought to room temperature prior to use gt gt Always prepare only the appropriate quantity of reagents for your test gt gt Do not use components after the expiration date marked on t
3. Rat Resistin ng ml Figure 2 Standard Curve for rat resistin is plotted using the four parameter function as a proportion of rat resistin concentration and absorbance at 450 nm Page 12 of 24 VERSION 17 180908 03 13 PERFORMANCE CHARACTERISTICS NN gt gt Typical analytical data of Rat Resistin ELISA are presented in this chapter Sensitivity Limit of Detection LOD defined as concentration of analyte giving absorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank Abiank 3xSDpiank iS Calculated from the real resistin values in wells and is 0 05 ng ml Dilution Buffer is pipetted into blank wells e Limit of assay Results exceeding resistin level of 20 ng ml should be repeated with more diluted samples Dilution factor needs to be taken into consideration in calculating the resistin concentration Specificity The antibodies used in this ELISA are specific for rat resistin with no detectable crossreactivities to human and mouse recombinant resistin at 50 ng ml Sera of several mammalian species were measured in the assay See results below Mammalian serum Observed sample crossreactivity Bovine no Cat no Dog no Goat yes Hamster yes Horse no Monkey no Mouse no Pig no Rabbit no Rat yes Sheep no Page 13 of 24 VERSION 17 180908 03 Precision Intra assay Within Run n 8 results are presented after
4. calculation Sample Mean SD CV ng ml ng ml 1 14 07 0 73 5 2 2 21 12 1 03 49 Inter assay Run to Run n 5 results are presented after calculation Sample Mean SD CV ng ml ng ml 1 18 2 0 91 49 2 19 3 1 79 9 3 Spiking Recovery Serum samples were spiked with different amounts of recombinant rat resistin and assayed Results are presented after calculation Linearity Serum samples were serially diluted with Dilution Buffer and assayed at a further dilution of 3x Results are presented after calculation Sample Observed Expected Recovery O E ng ml ng ml 1 14 45 24 03 24 45 98 3 30 83 34 45 89 5 40 76 44 45 91 7 2 21 12 28 00 31 12 90 0 38 04 41 12 92 5 64 50 71 12 90 7 Sample Dilution Observed Expected Recovery ng ml ng ml O E 1 28 25 2x 15 89 14 12 112 5 4x 8 03 7 06 113 7 2 26 32 2x 15 13 13 16 114 9 4x 7 21 6 58 109 5 Page 14 of 24 VERSION 17 180908 03 Effect of freezing thawing No decline was observed in concentration of rat resistin in serum samples after repeated 3x freezing thawing cycles e Stability of samples at 4 C Samples should be stored at 20 C However no decline was observed in concentration of rat resistin in serum samples when stored at 4 C for 8 days To avoid microbial contamination add NaN3 to a final c
5. of 24 VERSION 17 180908 03 Reconstitute Master Standard and QCs prepare set of Standards Prepare Wash Solution d b Page 19 of 24 Assay Procedure Summary Antibody Coated Microtiter Strips Dilute Samples 20x Add Standards QCs and Samples 100 ul Incubate at RT for 1 hour 300 rpm Wash 3x Add Biotin Labelled Antibody 100 ul Incubate at RT for 1 hour 300 rpm lt a Wash 3x Add Streptavidin HRP Conjugate 100 pl i Incubate at RT for 30 min 300 rpm Was Add Substrate Solution 100 ul H Incubate at RT for 10 min Ss Add Stop Solution 100 ul Read absorbance and calculate results VERSION 17 180908 03 12 11 10 Page 20 of 24 VERSION 17 180908 03 NOTES Page 21 of 24 VERSION 17 180908 03 Page 22 of 24 VERSION 17 180908 03 Page 23 of 24 VERSION 17 180908 03 BIO ae BioCat GmbH Im Neuenheimer Feld 584 D 69120 Heidelberg Tel 49 6221 7141516 Fax 49 6221 7141529 E Mail info biocat com www biocat com Page 24 of 24 VERSION 17 180908 03
6. 5 30 2003 e Steppan C M Lazar M A Resistin and obesity associated insulin resistance TRENDS in Endocrinology and Metabolism 13 1 18 23 2002 e 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 e Kim K H et al A Cysteine rich Adipose Tissue specific Secretory Factors Inhibits Adipocte Differentiation The Journal of Biological Chemistry 276 14 11252 11256 2001 e 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 e 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 e Steppan C M at al A Family of Tissue specific Resistin like Molecules Proc Natl Acad Sci USA 98 2 502 506 2001 e Steppan C M et al The Hormone Links Obesity to Diabetes Nature 409 307 312 2001 Page 17 of 24 VERSION 17 180908 03 18 EXPLANATION OF SYMBOLS Catalogue number Content Lot number See instructions for use Biological hazard Expiry date Storage conditions Identification of packaging materials PAn PP BEE Page 18
7. Bio Cat No K1014 1 BA For Research Use Onl Page 1 of 24 VERSION 17 180908 03 CONTENTS ee eee 1 INTENDED USE 3 2 STORAGE EXPIRATION 3 3 INTRODUCTION 4 4 TEST PRINCIPLE 4 5 PRECAUTIONS 5 6 TECHNICAL HINTS 5 7 REAGENT SUPPLIED 6 8 MATERIAL REQUIRED BUT NOT SUPPLIED 6 9 PREPARATION OF REAGENTS 7 10 PREPARATION OF SAMPLES 9 11 ASSAY PROCEDURE 10 12 CALCULATIONS 12 13 PERFORMANCE CHARACTERISTICS 13 14 DEFINITION OF THE MASTER STANDARD 15 15 METHOD COMPARISON 15 16 TROUBLESHOOTING AND FAQS 16 17 REFERENCES 17 18 EXPLANATION OF SYMBOLS 18 gt gt This kit is manufactured by BioVendor Laboratorn medic na a s gt gt Use only the current version of Product Data Sheet enclosed with the kit Page 2 of 24 VERSION 17 180908 03 1 INTENDED USE EN The Rat Resistin ELISA is a sandwich enzyme immunoassay for the quantitative measurement of rat resistin gt gt Features e tis intended for research use only e The total assay time is less than 4 hours e The kit measures total serum homodimeric resistin e Assay format is 96 wells e Quality Controls are recombinant rat resistin based No animal sera are used e Standard is recombinant protein based e Components of the kit are provided ready to use concentrated or lyophilized 2 STORAGE EXPIRATION MN Store the complete kit at 2 8 C Under these conditions the kit is stable until the expiration date see label
8. also seems to inhibit adipogenesis In contrast the human adipogenic differentiation is likely to be associated with a down regulation of resistin gene expression Areas of investigation Insulin resistance Obesity 4 TEST PRINCIPLE MN In the Rat Resistin ELISA Standards Quality Controls and samples are incubated in microplate wells pre coated with polyclonal anti rat resistin antibody After 60 minutes incubation and washing biotin labelled polyclonal anti rat resistin antibody is added and incubated for 60 minutes with captured resistin After another washing streptavidin HRP conjugate is added After 30 minutes incubation and the last washing step the remaining conjugate is allowed to react with the substrate solution TMB The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured spectrophotometrically at 450 nm The absorbance is proportional to the concentration of resistin A standard curve is constructed by plotting absorbance values against concentrations of Standards and concentrations of unknown samples are determined using this standard curve Page 4 of 24 VERSION 17 180908 03 5 PRECAUTIONS eee For professional use only e Wear gloves and laboratory coats when handling immunodiagnostic materials e Do not drink eat or smoke in the areas where immunodiagnostic materials are being handled e This kit contains components of animal origin However these materials
9. heir label e Assay reagents supplied ready to use Antibody Coated Microtiter Strips Stability and storage Return the unused strips to the provided aluminium zip sealed bag with desicant and seal carefully Remaining Microtiter Strips are stable 3 months stored at 2 8 C and protected from the moisture Biotin Labelled Antibody Streptavidin HRP Conjugate Dilution Buffer Substrate Solution Stop Solution Stability and storage Opened reagents are stable 3 months when stored at 2 8 C e Assay reagents supplied concentrated or lyophilized Wash Solution Concentrate 10x Dilute Wash Solution Concentrate 10x ten fold in distilled water to prepare a 1x working solution Example 100 ml of Wash Solution Concentrate 10x 900 ml of distilled water for use of all 96 wells Stability and storage The diluted Wash Solution is stable 1 month when stored at 2 8 C Opened Wash Solution Concentrate 10x is stable 3 months when stored at 2 8 C Rat Resistin Master Standard Refer to the Cetrificate of Analysis for actual volume of Dilution Buffer needed for reconstitution of standard Reconstitute the lyophilized Master Standard with Dilution Buffer just prior to the assay Let it dissolve at least 15 minutes with occasionally gently shaking not to foam The resulting concentration of the rat resistin in the stock solution is 20 ng ml Page 7 of 24 VERSION 17 180908 03 Prepare set of standards using Dilution Buffer as fol
10. lows Volume of Standard Dilution Buffer Concentration Stock 20 ng ml 500 ul of stock 500 ul 10 ng ml 500 ul of 10 ng ml 500 ul 5 ng ml 500 ul of 5 ng ml 500 ul 2 5 ng ml 500 ul of 2 5 ng ml 750 ul 1 ng ml 500 ul of 1ng ml 500 ul 0 5 ng ml 500 ul of 0 5 ng ml 500 ul 0 25 ng ml Prepared Standards are ready to use do not dilute them Stability and storage Standard stock solution 20 ng ml should be aliquoted and frozen at 20 C for 3 months Avoid repeated freeze thaw cycles Quality Controls High Low Refer to the Certificate of Analysis for actual volume of Dilution Buffer needed for reconstitution and for current Quality Controls concentrations Reconstitute each Quality Control High and Low with Dilution Buffer just prior to the assay Let it dissolve at least 15 minutes with occasional gentle shaking not to foam Reconstituted Quality Controls are ready to use do not dilute them Stability and storage The reconstituted Quality Controls must be used immediately or aliquoted and frozen at 20 C for 3 month Avoid repeated freeze thaw cycles Page 8 of 24 VERSION 17 180908 03 10 PREPARATION OF SAMPLES Ne The kit measures rat resistin in serum Samples should be assayed immediately after collection or should be stored at 20 C Mix thoroughly thawed samples just prior to the assay and avoid repeated freeze thaw cycles which may cause erroneous results Avoid using hemolyzed o
11. of 24 VERSION 17 180908 03 17 REFERENCES ee GG gt gt References to Rat Resistin ELISA e Steppan C M Lazar M A The current biology of resistin Journal of Internal Medicine 255 439 447 2004 e Gong H Ni Y Guo X Fei L Pan X Guo M Chen R Resistin promotes 3T3 L1 preadipocyte differentiation Eur J Endocrinol 150 6 885 92 2004 e Nogueiras R Gallego R Gualillo O Caminos J E Garcia Caballero T Casanueva F F Dieguez C Resistin is expressed in different rat tissues and is regulated in a tissue and gender specific manner FEBS Lett 31 548 1 3 21 7 2003 e Satoh H Nguyen M T Miles P D Imamura T Usui I Olefsky J M Adenovirus mediated chronic hyper resistinemia leads to in vivo insulin resistance in normal rats J Clin Invest 114 2 224 31 2004 e Pravenec M Kazdova L Landa V Zidek V Mlejnek P Jansa P Wang J Qi N Kurtz TW Transgenic and recombinant resistin impair skeletal muscle glucose metabolism in the spontaneously hypertensive rat J Biol Chem 278 45209 45215 2003 e Milan G Granzotto M Scarda A Calcagno A Pagano C Federspil G Vettor R Resistin and adiponectin expression in visceral fat of obese rats effect of weight loss Obes Res 10 11 1095 103 2002 e Rajala M W Obici S Scherer P E Rossetti L Adipose derived resistin and gut derived resistin like molecule beta selectively impair insulin action on glucose production J Clin Invest 111 2 22
12. on 0 35 ml per well After final wash invert and tap the plate strongly against paper towel Add 100 ul of Streptavidin HRP Conjugate into each well Incubate the plate at room temperature ca 25 C for 30 minutes shaking at ca 300 rpm on an orbital microplate shaker Wash the wells 3 times with Wash Solution 0 35 ml per well After final wash invert and tap the plate strongly against paper towel Add 100 pl of Substrate Solution into each well Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminium foil is recommended Incubate the plate for 10 minutes at room temperature The incubation time may be extended up to 20 minutes if the reaction temperature is below than 20 C Do not shake the plate during the incubation Stop the colour development by adding 100 ul of Stop Solution Determine the absorbance by reading the plate at 450 nm The absorbance should be read within 5 minutes following step 12 Note If the microplate reader is not capable of reading absorbance greater than the absorbance of the highest standard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determinerat resistin concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Page 10 of 24 VERSION 17 180908 03 strip1 2 strip 3 4 strip 5 6 strip 7 8 strip 9 10 strip 11412 A __ Standard 20
13. on the box For stability of opened reagents see Chapter 9 Page 3 of 24 VERSION 17 180908 03 3 INTRODUCTION eee Resistin a product of the RSTN gene is a peptide hormone belonging to the class of cysteine rich secreted proteins which is termed the RELM family and is also described as ADSF Adipose Tissue Specific Secretory Factor and FIZZ3 Found in Inflammatory Zone Rat resistin contains 114 amino acids as a prepeptide and its hydrofobic signal peptide is cleaved before its secretion Resistin circulates in rat blood as a dimeric protein consisting of two 96 amino acid polypeptides which are disulfide linked Resistin may be an important link between obesity and insulin resistance Mouse resistin Specifically produced and secreted by adipocyte 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 They have also suggested that resistin is present at elevated levels in blood of obese mice and is down regulated by fasting and antidiabetic drugs TZDs On the other hand several Studies demonstrated reduced resistin expression in adipose tissue of obese mice and increased levels in leptin deficient ob ob mice and Zucker diabetic fatty rats in response to TZDs Other studies have shown that mouse resistin increases during the differentiation of adipocytes but it
14. oncentration 0 1 to the samples Reference ranges It is recommended that each laboratory include its own panel of control sample in the assay Each laboratory should establish its own normal and pathological reference ranges for resistin levels with the assay 14 DEFINITION OF THE MASTER STANDARD nn ee A recombinant protein is used as the Master Standard Resistin concentration depends on the method used for total protein determination Master Standard in the kit contains 20 ng of resistin measured by Bradford method Sigma Aldrich 15 METHOD COMPARISON D NGG Since no competitive commercial test is available Rat Resistin ELISA has not been compared to any other immunoassay Page 15 of 24 VERSION 17 180908 03 16 TROUBLESHOOTING AND FAQS Ne gt gt 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 room temperature e Improper wavelength when reading absorbance gt gt High signal and background in all wells Possible explanations e Improper or inadequate washing e Overdeveloping incubation time with Substrate Solution should be decreased before addition of Stop Solution e Incubation temperature over 30 C gt gt High coefficient of variation CV Possible explanation e Improper or inadequate washing e Improper mixing Standards Quality Controls or samples Page 16
15. r lipemic samples Dilute samples 20x with Dilution Buffer just prior to the assay e g 10 ul of sample 190 ul of Dilution Buffer when assaying samples in singlets or preferably 15 ul of sample 285 ul of Dilution Buffer for duplicates Mix well not to foam Vortex is recommended Stability and storage Samples should be stored at 20 or preferably at 70 C for long term storage Avoid repeated freeze thaw cycles Do not store the diluted samples See Chapter 13 for stability of serum samples if stored at 2 8 C effect of freezing thawing on the concentration of rat resistin Note It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results Page 9 of 24 VERSION 17 180908 03 11 ASSAY PROCEDURE eee 12 13 Pipet 100 ul of prepared Standards Quality Controls Dilution Buffer Blank and samples preferably in duplicates into the appropriate wells See Figure 1 for example of work sheet Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker Wash the wells 3 times with Wash Solution 0 35 ml per well After final wash invert and tap the plate strongly against paper towel Add 100 ul of Biotin Labeled Antibody into each well Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker Wash the wells 3 times with Wash Soluti
16. should be handled as potentially infectious e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide and tetramethylbenzidine TMB Wear gloves and eye and clothing protection when handling these reagents Stop and or Substrate Solutions may cause skin eyes irritation In case of contact with the Stop Solution and the Substrate Solution wash skin eyes thoroughly with water and seek medical attention when necessary e The materials must not be pipetted by mouth 6 TECHNICAL HINTS TN Reagents with different lot numbers should not be mixed Use thoroughly clean glassware Use deionized distilled water stored in clean containers Avoid any contamination among samples and reagents For this purpose disposable tips should be used for each sample and reagent e Substrate Solution should remain colourless until added to the plate Keep Substrate Solution protected from light e Stop Solution should remain colourless until added to the plate The colour developed in the wells will turn from blue to yellow immediately after the addition of the Stop Solution Wells that are green in colour indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution e Dispose of consumable materials and unused contents in accordance with applicable national regulatory requirements Page 5 of 24 VERSION 17 180908 03 7 REAGENT SUPPLIED
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