IFU - DRG International
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1. COUNTRIES 8 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 9 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 10 Add 100 uL of Substrate Solution into each well Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminium foil is recommended 11 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 12 Stop the colour development by adding 100 uL of Stop Solution 13 Determine the absorbance of each well using a microplate reader set to 450 nm preferably with the reference wavelength set to 630 nm acceptable range 550 650 nm Subtract readings at 630 nm 550 650 nm from the readings at 450 nm The absorbance should be read within 5 minutes following step 12 Note If some samples and standard s have absorbances above the upper limit of your microplate reader perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine resistin concentration of off scale standards and samples The readings at 405 nm should not replace the readings for samples that were in range at 450 nm Note 2 Manual washing Aspirate wells and p2. DREG is DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES This kit is intended for Research Use Only Not intended for diagnostic purposes Please use only the valid version of the package insert provided with the kit 1 INTENDED USE The Resistin human ELISA is a sandwich enzyme immunoassay for measurement of human resistin Features The total assay time is less than 4 hours The kit measures total resistin in serum and plasma EDTA citrate heparin Assay format is 96 wells Quality Controls are human serum based Standard is recombinant protein based Components of the kit are provided ready to use concentrated or lyophilized 2 STORAGE EXPIRATION Store the complete kit at 2 C 8 C Under these conditions the kit is stable until the expiration date see label on the box 3 INTRODUCTION 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 or FIZZ3 Found in Inflammatory Zone Human resistin contains 108 amino acids as a prepeptide and its hydrophobic signal peptide is cleaved before its secretion 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 invest
3. ICAL HINTS 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 Substrate Solution should remain colourless until added to the plate Keep Substrate Solution protected from light 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 Dispose of consumable materials and unused contents in accordance with applicable national regulatory requirements DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 2 DREG is DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES 7 REAGENT SUPPLIED 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 Conc 10x concentrated 100 mL Subst
4. absorbance 2 High signal and background in all wells Possible explanations Improper or inadequate washing Overdeveloping incubation time with Substrate Solution should be decreased before addition of Stop Solution Incubation temperature over 30 C DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 13 DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES 3 High coefficient of variation CV Possible explanation Improper or inadequate washing Improper mixing Standards Quality Controls or samples 16 REFERENCES 1 Sadashiv Tiwari S Paul BN Kumar S Chandra A Dhananjai S Negi MP Over expression of resistin in adipose tissue of the obese induces insulin resistance World J Diabetes 3 7 135 141 2012 2 Malo E Ukkola O Jokela M Moilanen L K h nen M Nieminen MS Salomaa V Jula A Kes niemi YA Resistin is an indicator of the metabolic syndrome according to five different definitions in the Finnish Health 2000 survey Metab Syndr Relat Disord 9 3 203 210 2011 Lazar MA Resistin and Obesity associated metabolic diseases Horm Metab Res 39 10 710 716 2007 Meier U Gressner AM Endocrine regulation of energy metabolism review of pathobiochemical and clinical chemical aspects of leptin ghrelin adiponectin and resistin Clin Chem 50 9 1511 1525 2004 5 Kershaw EE Flier JS Adipose tiss
5. arin Plasma Concentration of Human Resistin ng ml Volunteers DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 10 DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES Figure 3 Resistin levels measured using Human Resistin ELISA from10 individuals using serum heparin citrate and EDTA plasma respectively 12 2 Stability of samples stored at 2 C 8 C Samples should be stored at 80 C However no decline in concentration of resistin was observed in serum and plasma samples after 7 days when stored at 2 C 8 C To avoid microbial contamination samples were treated with aminocaproic acid and sodium azide resulting in the final concentration of 0 03 and 0 1 respectively Incubation Temp Plasma ng ml Period Citrate 2 C 8 C 7 days Sample Heparin 12 23 12 18 12 3 Effect of Freezing Thawing No decline was observed in concentration of human resistin in serum and plasma samples after repeated 3x freeze thaw cycles However it is recommended to avoid unnecessary repeated freezing thawing of the samples DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 11 DREG is DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES Volunte
6. er Number of f t Serum Plasma ng mL No cycles ng mL Heparin Citrate EDTA 12 4 Reference range 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 references ranges for resistin levels with the assay 13 DEFINITION OF THE STANDARD A recombinant protein is used as the standard The recombinant resistin is a 19 5 kDa dimeric protein consisting of two 92 amino acid polypeptide chains which are disulfide linked 14 METHOD COMPARISON Human Resistin ELISA was compared to the other commercial ELISA assay by measuring 78 serum samples The following correlation graph was obtained DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 12 DRE DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES Method Com parison 13 i y 0 9236x 0 2686 a 11 RF 0 8363 10 n 7s 3 9 D 8 B 7 6 amp 5 I 4 3 2 1 0 0 1 2 3 4 5 6 7 8 g 10 11 12 413 Competitor Human Resistin ELISA tigi Figure 4 Method comparison 15 TROUBLESHOOTING AND FAQS 1 Weak signal in all wells Possible explanations Omission of a reagent or a step Improper preparation or storage of a reagent Assay performed before reagents were allowed to come to room temperature Improper wavelength when reading
7. igations 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 DRG International Inc USA Fax 973 564 7556 e mail corp drg international com l DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES 4 TEST PRINCIPLE In the DRG Resistin human ELISA standards quality controls and samples are incubated in microplate wells pre coated with polyclonal anti human resistin antibody After 60 minutes incubation and washing biotin labelled second polyclonal anti human resistin antibody is added and incubated with captured resistin for 60 minutes After another washing streptavidin HRP conjugate is added After 60 minutes incubation and the last washing step the remaining conjugate is allowed to react
8. ipet 0 35 mL Wash Solution into each well Aspirate wells and repeat twice After final wash invert and tap the plate strongly against paper towel Make certain that Wash Solution has been removed entirely DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 7 DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES strip 1 2 strip 3 4 strip 5 6 strip 7 8 strip 9 10 strip 11 12 A Standard 50 QC Low Sample 8 Sample 16 Sample 24 Sample 32 B Standard 20 Sample 1 Sample 9 Sample 17 Sample 25 Sample 33 C Standard 10 Sample 2 Sample 10 Sample 18 Sample 26 Sample 34 D Standard 5 Sample 3 Sample 11 Sample 19 Sample 27 Sample 35 E Standard 2 Sample 4 Sample 12 Sample 20 Sample 28 Sample 36 F Standard 1 Sample 5 Sample 13 Sample 21 Sample 29 Sample 37 G Blank Sample 6 Sample 14 Sample 22 Sample 30 Sample 38 H OC High Sample 7 Sample 15 Sample 23 Sample 31 Sample 39 Figure 1 Example of a work sheet 12 CALCULATIONS Most microtiter plate readers perform automatic calculations of analyte concentration The standard curve is constructed by plotting the mean absorbance Y of Standards against the known concentration X of Standards in logarithmic scale using the four parameter algorithm Results are reported as concentration of resistin in ng mL in samples Alternatively the logit log functio
9. ma samples when stored at 2 C 8 C effect of freezing thawing and effect of sample matrix serum plasma on the concentration of human resistin Note It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results Ask for protocol at drg drg diagnostics de if assaying tissue culture medium synovial fluid and cerebrospinal fluid CSF 11 ASSAY PROCEDURE 1 Pipet 100 uL of diluted Standards Quality Controls Dilution Buffer Blank and samples preferably in duplicates into the appropriate wells See Figure 1 for example of work sheet 2 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 3 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 4 Add 100 uL of Biotin Labelled Antibody solution into each well 5 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 6 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 7 Add 100 uL of Streptavidin HRP Conjugate into each well DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 6 DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU
10. n 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 Samples Quality Controls and Standards are all diluted 3x prior to analysis so there is no need to take this dilution factor into account DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 8 DREG is DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES Human Resistin ELISA Standard Curve 3 0 25 z g 2 0 z 3 1 5 F 1 0 2 0 5 0 0 1 10 Concentration of Human Resistin ng ml Figure 2 Typical Standard Curve for Human Resistin ELISA 12 1 Effect of sample matrix Heparin citrate and EDTA plasmas were compared to respective serum samples from the same 10 individuals Results are shown below DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 9 DREG is DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES Volunteer Serum ng mL Plasma ng mL No EDTA 6 60 2 ow 1007 s9 11 69 933 4 336 5 278 6 2 29 14 62 5 709 o Zi i 580 Mean ng mL 609 651 565 808 Mean Plasma Serum Ff 1069 92 7 132 6 Coefficient of determination R P oo 0 89 Effect of Sample Matrix E Serum m EDTA Plasma O Citrate Plasma O Hep
11. rate Solution ready to use 13 mL Stop Solution ready to use 13 mL Instructions for Use Certificate of Analysis 1 pe 8 MATERIAL REQUIRED BUT NOT SUPPLIED 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 preferably with reference wavelength 630 nm alternatively another one from the interval 550 650 nm Software package facilitating data generation and analysis optional 9 PREPARATION OF REAGENTS All reagents need to be brought to room temperature prior to use Always prepare only the appropriate quantity of reagents for your test Do not use components after the expiration date marked on their label DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 3 DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES 9 1 Assay reagents supplied ready to use 1 Antibody Coated Microtiter Strip
12. rly 3 Wash Solution Conc 10x Dilute Wash Solution Concentrate 10x ten fold in distilled water to prepare a 1x working solution DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 5 DREG is DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES 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 C 8 C Opened Wash Solution Concentrate 10x is stable 3 months when stored at 2 C 8 C 10 PREPARATION OF SAMPLES The kit measures human resistin homodimeric in serum and plasma EDTA citrate heparin 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 or lipemic samples Dilute samples 3x with Dilution Buffer just prior to the assay e g 50 uL of sample 100 uL of Dilution Buffer for singlets or preferably 100 uL of sample 200 uL of Dilution Buffer for duplicates Mix well not to foam Vortex is recommended Stability and storage Samples should be stored at 20 C or preferably at 70 C for long term storage Do not store the diluted samples See Chapter 13 for stability of serum and plas
13. s Stability and storage Return the unused strips to the provided aluminium zip sealed bag with desiccant and seal carefully Remaining Microtiter Strips are stable 3 months stored at 2 C 8 C and protected from the moisture Biotin Labelled Antibody Streptavidin HRP Conjugate Dilution Buffer Substrate Solution ONT oe Ge D Stop Solution Stability and storage Opened reagents are stable 3 months when stored at 2 C 8 C 9 2 Assay reagents supplied concentrated or lyophilized 1 Human Resistin Master Standard Refer to the Certificate of Analysis for current 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 occasional gentle shaking not to foam The resulting concentration of the resistin in the stock solution is 50 ng mL Prepare set of standards using Dilution Buffer as follows DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 4 DREG GC DRG Resistin human ELISA EIA 4572 REVISED 7 APRIL 2014 RM VERS 7 1 IN THE USA AND NON EU COUNTRIES Volume of Standard Dilution Buffer Concentration stock 50 ng mL 500 uL of stock 20 ng mL 500 uL of std 20 ng mL 10 ng mL 500 uL of std 10 ng mL 5 ng mL 500 uL of std 5 ng mL 2 ng mL 500 uL of std 2 ng mL 500 uL 1 ng mL Dilute each concentration of standard 3x
14. ue as an endocrine organ J Clin Endocrinol Metab 89 6 2548 2556 2004 6 Hartman HB Hu X Tyler KX Dalal CK Lazar MA Mechanisms Regulating Adipocyte Expression of Resistin J Biol Chem 277 22 19754 19761 2002 7 Steppan CM Bailey ST Bhat S Brown EJ Banerjee RR Wright CM Patel HR Ahima RS Lazar MA The Hormone Links Obesity to Diabetes Nature 409 6818 307 312 2001 Rev 2014 04 04 ENG 001 A cc DRG International Inc USA Fax 973 564 7556 e mail corp drg international com 14
15. with Dilution Buffer prior to the assay e g 50 uL of standard 100 uL of Dilution Buffer for singlets or preferably 100 uL of standard 200 uL of Dilution Buffer for duplicates Mix well not to foam Stability and storage Set of Standards 50 1 ng mL should be aliquoted and frozen at 20 C for 3 months Avoid repeated freeze thaw cycles Do not store the diluted Standard solutions 2 Quality Controls High Low Refer to the Certificate of Analysis for current Quality Control concentration Reconstitute each Quality Control High and Low with 350 uL of Dilution Buffer just prior to the assay Let it dissolve at least 30 minutes with occasional gentle shaking not to foam Dilute Quality Controls prior to the assay 3x with Dilution Buffer e g 50 uL of Quality Control 100 uL of Dilution Buffer for singlets or preferably 100 uL of Quality Control 200 uL of Dilution Buffer for duplicates Stability and storage The reconstituted Quality Controls must be used immediately or aliquoted and frozen at 20 C for 3 months Avoid repeated freeze thaw cycles Do not store the diluted Quality Controls Note Concentration of analyte in Quality Controls need not be anyhow associated with normal and or pathological concentrations in serum or another body fluid Quality Controls serve just for control that the kit works in accordance the with user s manual and Certificate of Analysis and that ELISA test was carried out prope
16. with the substrate solution TMB The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured 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 5 PRECAUTIONS For professional use only Wear gloves and laboratory coats when handling kit materials Do not drink eat or smoke in the areas where kit materials are being handled This kit contains components of human origin These materials were found nonreactive for HBsAg HCV antibody and for HIV 1 2 antigen and antibody However these materials should be handled as potentially infectious as no test can guarantee the complete absence of infectious agents This kit contains components of animal origin These materials should be handled as potentially infectious 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 The materials must not be pipetted by mouth 6 TECHN
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