Human Insulin ELISA Kit User Manual Catalog # D
Contents
1. 13 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES 1 INTRODUCTION Insulin is a secreted peptide hormone that elicitsmetabolic effectssuch as increasesin glucose uptake and glycogen synthesisleading to a decrease in blood glucose concentration Insulin isfirst formed as a precursormolecule preproinsulin which islatercleaved to proinsulin and finallyto the mature Insulin hormone Mature Insulin consists of 51 amino acids containedwithin an A chain and a B chain that are connected by 2 disulfide bridges It increases cell permeabilityto monosaccharides amino acids and fatty acids Insulin is secreted bythe pancreas at basal levelsin the absence of exogenousstimuli with secretion increasing in response to glucose Insulin action is effected by the binding of Insulin to cell surface receptors on the targetcell membrane Defects of Insulin are the cause of hyperproinsulinemia and of type II diabetes mellitus FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Il ASSAY PRINCIPLES The Human Insulin ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of Human Insulin in Cell Culture Supernatants Serum Plasma Tissue Homogenates This assay employs an antibody specific for Human Insulin coated on 96 well plate Standards and samples are pipetted into the wells and Insulin present in a sample2. ASSAY PROCEDURE SUMMARY Prepare all reagents samples and standards Add 50 ul Standard or Sample Add 50 ul Biotin Labeled Detection Antibody Working Solution Wash plate times with Wash Buffer Working Solution e Add 100 ul Streptavidin HRP Working Solution Wash plate 5 times with Wash Buffer Working Solution Add 100 uI TMB Substrate Solution Add 100 ul Stop Solution Read the plate at 450nm FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES X TYPICAL DATA The standard curve is for demonstration only A standard curve must be run with each assay 10 g 1 1 2 1 10 100 Human Insulin Concentration mU L XI SENSITIVITY The minimum detectable dose of Human Insulin is typically less than 0 5 mU L XII SPECIFICITY The Human Insulin ELISA Kit allows for the detection and quantification of endogenous levels of natural and or recombinant Human Insulin proteins within the range of 3 13 mU L 100 mU L FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES XIII CROSS REACTIVITY No detectable cross reactivity with other relevant proteins REFERENCES FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES XV TROUBLESHOOTING GUIDE Problem High signal and background in all wells No signal Too much signal whole plate turned uniformly blu
3. is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti Human Insulin antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Insulin bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES COMPONENTS Human Insulin Standard 100 mU L 500 ul Human Insulin Standard 50 mU L 500 ul Human Insulin Standard 25 mU L Human Insulin Standard 12 5 mU L Human Insulin Standard 6 25 mU L 500 ul Human Insulin Standard 3 13 mU L 500 ul Biotin Labeled Detection Antibody 100X Streptavidin HRP 100X Detection Antibody Diluent 12 ml Streptavidin HRP Diluent Wash Buffer 20X 30 ml TMB Substrate Solution Plate Adhesive Strips Technical Manual IV STORAGE AND STABILITY All kit components are stable at 2 to 8 C Standard recombinant protein should be stored at 20 C or 80 C recommended at 80 C after reconstitution Opened Microplate Wells or reagents may be store for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant pack reseal along entire edge Note the kit can be used within one year if the wh
4. Human Insulin ELISA Kit User Manual Catalog Sandwich Enzyme Linked Immunosorbent Assay for Quantitative Detection of Human Insulin Concentrations in Cell Culture Supernatants Serum Plasma Tissue Homogenates For research use only Not for diagnostic or therapeutic procedures FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES l INTRODUCTION tinea satar dhabeestatieets desde a E cosa tastes oda diesel bry 2 ASSAY PRINCIPLES oa aar aana AE 3 II KIT COMPONENTS rise AE ETE EEEE E AAEE 4 IV STORAGEAND STABILITY oiner ie E A EE EA T a 4 V MATERIALS REQUIRED BUT NOT 5 VI HEALTH AND SAFETY PRECAUTIONS cee inne iritatia anaa aias 5 VIN REAGENT PREPARATION Fenisiese 6 VIII ASSAY PROCEDURE 5 Bedceestdedanesashouatadzasaandae 8 ASSAY PROCEDURE 5 8 10 KY RIGALADATA sea n a a a r aaa 11 Xl SENSITIMTY de A EAEE AE A a 11 Xl SPECIBICI TY nnana Ahern oda AAO 11 XII CROSS REACTIVITY E aoei ie i a 12 XIV 12 XV TROUBLESHOOTING
5. R RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES The Biotin Labeled Detection Antibody should be diluted in 1 100 with the Detection Antibody Diluent and mixed thoroughly The solution should be prepared no more than 2 hours prior to the experiment 3 Streptavidin HRP Working Solution Preparation The Streptavidin HRP should be diluted in 1 100 with the Streptavidin HRP Diluent and mixed thoroughly The solution should be prepared no more than 1 hour prior to the experiment 4 Wash Buffer Working Solution Preparation Pour entire contents 30 ml of the Wash Buffer Concentrate into a clean 1 000 ml graduated cylinder Bring final volume to 600 ml with glass distilled or deionized water 1 20 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Vill ASSAY PROCEDURE The Streptavidin HRP Working Solution and TMB Substrate Solution must be kept warm at 37 C for 30 minutes before use When diluting samples and reagents they must be mixed completely and evenly Standard detection curve should be prepared for each experiment The user will decide sample dilution fold by crude estimation of protein amount in samples 1 Add 50 ul of each standard and sample into appropriate wells 2 Add 50 ul of Biotin Labeled Detection Antibody Working Solution into each well and incubate the plate at 37 C for 60 minutes 3 Wash plate 3 times with Wash Buffer Working Solution and each time let W
6. ash Buffer Working Solution stay in the wells for 1 2 minutes Discard the Wash Buffer Working Solution and blot the plate onto paper towels or other absorbent material 4 Add 100 ul of Streptavidin HRP Working Solution into each well and incubate the plate at 37 C for 45 minutes 5 Wash plate 5 times with Wash Buffer Working Solution and each time let wash buffer stay in the wells for 1 2 minutes Discard the wash buffer and blot the plate onto paper towels or other absorbent material 6 Add 100 ul of TMB Substrate Solution into each well and incubate plate at 37 C in dark for 30 minutes 7 Add 100 ul of Stop Solution into each well The color changes into yellow immediately 8 Read the O D absorbance at 450nm in a microplate reader within 30 minutes after adding the Stop Solution For calculation the relative O D 450 the O D 450 of each well the 450 of Zero well The standard curve can be plotted as the relative O D 450 of each FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES standard solution Y vs the respective concentration of the standard solution X The concentration of the samples can be interpolated from the standard curve Note If the samples measured were diluted multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES IX
7. ation assay immediately or aliquot and store samples at 20 C Serum Allow the serum to clot in a serum separator tube about 4 hours at room temperature Centrifuge at approximately 1000 X g for 15 minutes Analyze the serum immediately or aliquot and store samples at 20 C Plasma Collect plasma using heparin or EDTA as an anticoagulant Centrifuge for 15 minutes at 1500 X g within 30 minutes of collection Assay immediately or aliquot and store samples at 20 C Cell Lysates Collect cells and rinse cells with PBS Homogenize and lyse cells throughly in lysate solution Centrifuge cell lysates at approximately 10000 X g for 5 minutes to remove debris Aliquots of the cell lysates were removed and assayed Bone Tissue Extract demineralized bone samples in 4 M Guanidine HCl and protease inhibitors Dissolve the final sample in 2 M Guanidine HCl Tissue Homogenates Rinse tissue with PBS to remove excess blood chopped into 1 2 mm pieces and homogenize with a tissue homogenizer in PBS or in lysate solution lysate solution tissue net weight 10ml 1g i e Add 10ml lysate solution to 1g tissue Centrifuge at approximately 5000 X g for 5 minutes Assay immediately or aliquot and store homogenates at 20 C Avoid repeated freeze thaw cycles Urine Urinary samples should be cleared by centrifugation and then can be used directly without dilution Storage at 20 C 2 Biotin Labeled Detection Antibody Working Solution Preparation FO
8. e Standard curve achieved but poor discrimination between point No signal when a signal is expected but standard curve looks fine Samples are reading too high but standard curve is fine Edge effect Possible Cause e Insufficient washing e Too much Streptavidin HRP e Incubation time too long Development time too long e Reagent added in incorrect order or incorrectly prepared e Standard has gone bad If there is a signal in the sample wells e Assay was conducted from an incorrect starting point e Insufficient washing unbound Streptavidin HRP remaining Too much Streptavidin HRP e Plate sealer or reservoir reused resulting in presence of residual Streptavidin HRP e Plate not developed long enough e Improper calculation of standard curve dilution e Sample matrix is masking detection e Samples contain protein levels above assay range e Uneven temperature around work surface Solution Increase number of washes e Increase time of soaking between in wash e Check dilution titration e Reduce incubation time e Decrease the incubation time before the stop solution is added e Review protocol e Check the condition of stored standard e Reagents allows to come 20 30 C before performing assay Increase number of washes Carefully e Check dilution e Use fresh plate sealer and reagent reservoir for each step Increase substrate solution incubation time e Check d
9. ilution make new standard curve e More diluted sample Recommended e Dilute samples and run Again e Avoid incubating plate in areas where environmental conditions vary e Use plate sealer FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
10. ole kit is stored at 20 C Avoid repeated freeze thaw cycles FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES V MATERIALS REQUIRED BUT NOT PROVIDED 1 Microplate reader capable of measuring absorbance at 450 nm 2 Adjustable pipettes and pipette tips to deliver 2 ul to 1 volumes 3 Adjustable 1 25 ml pipettes for reagent preparation 4 100 ml and 1 liter graduated cylinders 5 Absorbent paper 6 Distilled or deionized water 7 Computer and software for ELISA data analysis 8 Tubes to prepare standard or sample dilutions VI HEALTH AND SAFETY PRECAUTIONS 1 Reagents provided in this kit may be harmful if ingested inhaled or absorbed through the skin Please carefully review the MSDS for each reagent before conducting the experiment 2 Stop Solution contains 2 N Sulfuric Acid H2SO and is an extremely corrosive agent Please wear proper eye hand and face protection when handling this material When the experiment is finished be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Vil REAGENT PREPARATION 1 Sample Preparation Store samples to be assayed within 24 hours at 2 8 C For long term storage aliquot and freeze samples at 20 C Avoid repeated freeze thaw cycles Cell culture supernates Remove particulates by centrifug
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