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E90190Hu 96 Tests Enzyme-linked Immunosorbent Assay Kit For Glycated Hemoglobin (HbA1C) Organism: Homo sapiens (Human) Instruction manual FOR IN VITRO USE AND RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
7th Edition (Revised in November, 2011)

The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of HbA1C in human plasma.

Reagents Pre-coated, ready to use 96-well strip plate Standard (lyophilized) Detection Reagent A (green) Detection Reagent B (red) TMB Substrate Wash Buffer (30 × concentrate) Quantity 1 2 1×120µL 1×120µL 1×9mL 1×20mL Reagents Plate sealer for 96 wells Standard Diluent Assay Diluent A (2 × concentrate) Assay Diluent B (2 × concentrate) Stop Solution Instruction manual Quantity 4 1×20mL 1×6mL 1×6mL 1×6mL 1

1. Microplate reader with 450 ± 10nm filter. 2. Precision single or multi-channel pipettes and disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution

1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20oC upon receipt while the others should be at 4 oC. 2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.

Therefore. When performing the assay. 3. Set up 7 points of diluted standard such as 200ng/mL. 3. Dilute to the working concentration with working Assay Diluent A or B. Wash Solution . Note: 1. 5.Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. shake gently(not to foam). [ REAGENT PREPARATION ] 1.Briefly spin or centrifuge the stock Detection A and Detection B before use. and the last EP tubes with Standard Diluent is the blank as 0ng/mL. more than 6mL Assay Diluent A and Assay Diluent B are contained in the bottles. 50ng/mL. Avoid repeated freeze/thaw cycles.5 6 6.Dilute 20mL of Wash Solution concentrate (30×) with 580mL of deionized or distilled water to prepare 600 mL of Wash Solution (1×).12ng/mL. Centrifuge samples for 15 minutes at 1000×g within 30 minutes of collection. Bring all kit components and samples to room temperature (18-25oC) before use. Standard . 100ng/mL. Detection Reagent A and Detection Reagent B . please precisely pipette required amount of Diluent and make double dilution in a new container.12 8 0 3. 2. Samples to be used within 5 days may be stored at 4oC.Reconstitute the Standard with 0.Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit. 2.Collect plasma using EDTA as an anticoagulant. TMB substrate . Please prepare 7 tubes containing 0. 25ng/mL.) 4. Remove plasma and assay immediately or store samples in aliquot at -20oC or -80oC. in every test. 6. so hemolytic specimen can not be detected. respectively (1:100).     . The prepared working dilution can't be frozen.25mL Standard Diluent and produce a double dilution series according to the picture shown below. Assay Diluent A and Assay Diluent B .Dilute 6mL of Assay Diluent A or B Concentrate(2×) with 6mL of deionized or distilled water to prepare 12 mL of Assay Diluent A or B.25 7 3. bring samples to room temperature.5 mL of Standard Diluent. (In fact. kept for 10 minutes at room temperature.25ng/mL. 12. Tube ng/mL 1 200 2 100 3 50 4 25 5 12. [ SAMPLE COLLECTION AND STORAGE ] Plasma . otherwise samples must be stored at -20oC ( 1 month) or -80oC ( 2 months) to avoid loss of bioactivity and contamination. The concentration of the standard in the stock solution is 200ng/mL. Sample hemolysis will influence the result. 6.5ng/mL. Mix each tube thoroughly before the next transfer.

a preliminary experiment to determine the validity of the kit is necessary. and avoid foaming and mix gently until the crystals are completely dissolved. Otherwise. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. cell number or sampling time. To minimize imprecision caused by pipetting. 4. Aspirate the solution and wash with 350µL of 1× Wash Solution to each well using a squirt bottle. If the samples are not indicated in the manual. 2. remove any remaining Wash Buffer by aspirating or decanting. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e. Fresh samples without long time storage is recommended for the test. Please reserve sufficient samples in advance. multi-channel pipette. Detection Reagent A and Detection Reagent B can be used only once. users must determine the optimal sample dilutions for their particular experiments. Inc. and let it sit for 1~2 minutes. 4. 2. is only responsible for the kit itself. Please do not dissolve the reagents at 37oC directly. Please predict the concentration before assaying. The reconstituted Standards. 3. 5. samples from cell culture supernatant may not be detected by the kit. use small volumes and ensure that pipettors are calibrated. don’t wash. Remove the liquid of each well. Prepare standard within 15 minutes before assay. protein degradation and denaturalization may occur in those samples and finally lead to wrong results.. Contaminated water or container for reagent preparation will influence the detection result. 5. warm to room temperature and mix gently until the crystals are completely dissolved. antibody targets conformational epitope rather than linear epitope). manifold dispenser or autowasher. [ ASSAY PROCEDURE ] 1. Add 100µL each of dilutions of standard (read Reagent Preparation).Note: 1. 4. Making serial dilution in the wells directly is not permitted. but not for the samples consumed during the assay. Cover with the Plate sealer. If crystals have formed in the Wash Solution concentrate (30×). Totally wash 3 times.g. Incubate for 2 hours at 37oC. Determine wells for diluted standard. 6. If values for these are not within the range of the standard curve. Invert the plate and blot it against absorbent paper. 2. 1 well for blank. Influenced by the factors including cell viability. some native or recombinant proteins from other manufacturers may not be recognized by our products. 3. 7. The user should calculate the possible amount of the samples used in the whole test. [ SAMPLE PREPARATION ] 1. 6. Uscn. . blank and sample. After the last wash. Prepare 7 wells for standard. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals. Add 100µL of Detection Reagent A working solution to each well. Incubate for 1 hour at 37oC after covering it with the Plate sealer. 3. blank and samples into the appropriate wells. It is recommended to suck more than 10µL for once pipetting. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction.

DO NOT let the strips DRY at any time during the assay. Once reagents are added to the well strips. 5. is recommended. If color change does not appear uniform. Do not touch the well wall. change pipette tips between additions of standards. a humidifier is recommended to be used at that condition. After the last wash. This will ensure equal elapsed time for each pipetting step. The environment humidity which is less than 60% might have some effects on the final performance. Add 50µL of Stop Solution to each well. Repeat the aspiration/wash process for total 5 times as conducted in step 4. 7. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. only those wells that contain HbA1C. if the color is too deep. Then. although not required. The liquid will turn blue by the addition of Substrate Solution. 2.5. Next. Add 100µL of Detection Reagent B working solution to each well. proper adhesion of plate sealers during incubation steps is necessary. Add 90µL of Substrate Solution to each well. Please protect it from light. without interruption. Complete removal of liquid at each step is essential for good performance. 6. Samples or reagents addition Please use the freshly prepared Standard. The liquid will turn yellow by the addition of Stop solution. Note: 1. and reagents. observation once every 10 minutes). 4. total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. 6. remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. 8. 3. Incubation time and temperature must be controlled. run the microplate reader and conduct measurement at 450nm immediately. Rest wells should be resealed and stored at -20oC. Cover with a new Plate sealer. Assay preparation: Keep appropriate numbers of wells for 1 experiment and remove extra wells from microplate. Mix the liquid by tapping the side of the plate. For each step in the procedure. Insufficient washing will result in poor precision and false elevated absorbance reading. Incubation: To ensure accurate results. To avoid cross-contamination. gently tap the plate to ensure thorough mixing. Incubate for 15 . 9. [ TEST PRINCIPLE ] The microtiter plate provided in this kit has been pre-coated with an antibody specific to HbA1C. Protect from light. Washing: The wash procedure is critical. use separated reservoirs for each reagent. add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at ¡ ¡ ¡ ¡ . Controlling of reaction time: Observe the change of color after adding TMB Substrate (e. 7. samples. Duplication of all standards and specimens. biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. Do not allow wells to sit uncovered for extended periods between incubation steps. TMB Substrate is easily contaminated. Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added. Incubate for 30 minutes at 37oC after covering it with the Plate sealer.25 minutes at 37oC (Don't exceed 30 minutes). therefore. Please carefully add samples to wells and mix gently to avoid foaming. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for HbA1C. Also.g.

curve expert 1. [ DETECTION RANGE ] 3. Using some plot software. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. plotting log of the data to establish standard curve for each test is recommended. [ TYPICAL DATA ] In order to make the calculation easier. the concentration read from the standard curve must be multiplied by the dilution factor. is also recommended. with HbA1C concentration on the y-axis and absorbance on the x-axis. value of the standard (X-axis) against the known concentration of the standard (Y-axis). If samples have been diluted. of the samples to the standard curve. although concentration is the independent variable and O. The sensitivity of this assay. Create a standard curve on log-log graph paper. 3.12ng/mL. 100ng/mL. and samples and subtract the average zero standard optical density. the O.g. washing technique or temperature effects). The concentration of HbA1C in the samples is then determined by comparing the O. for instance. However. pipetting technique. 6. value is the dependent variable.D. The standard curve concentrations used for the ELISA’s were 200ng/mL.30.25ng/mL. 50ng/mL. Draw the best fit straight line through the standard points and it can be determined by regression analysis.D.a wavelength of 450nm ± 10nm. 25ng/mL. [ CALCULATION OF RESULTS ] Average the duplicate readings for each standard. 12.24ng/mL. . Typical Standard Curve for Human HbA1C ELISA. Typical standard curve below is provided for reference only.12-200ng/mL. or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. control.5ng/mL. [ SENSITIVITY ] The minimum detectable dose of human HbA1C is typically less than 1. operator. values of the standard curve may vary according to the conditions of assay performance (e.D.D. we plot the O.

¢ ¢ ¢ ¢ Sample 1 2 1 4 1 8 1 16 . it is impossible for us to complete the cross. human EDTA plasma(n=5) 86-98% 93-101% 86-95% 84-99% [ PRECISION ] Intra-assay Precision (Precision within an assay): 3 samples with low. No significant cross-reactivity or interference between human HbA1C and analogues was observed.[ SPECIFICITY ] This assay has high sensitivity and excellent specificity for detection of human HbA1C. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end. especially room temperature. air humidity. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12% [ STABILITY ] The stability of ELISA kit is determined by the loss rate of activity. therefore. which means 3 days at 37oC equaling 6 months at 4oC). 8 replicates in each plate. [ RECOVERY ] Matrices listed below were spiked with certain level of recombinant human HbA1C and the recovery rates were calculated by comparing the measured value to the expected amount of HbA1C in samples. Matrix human EDTA plasma(n=5) Recovery range (%) 85-97 Average(%) 92 [ LINEARITY ] The linearity of the kit was assayed by testing samples spiked with appropriate concentration of human HbA1C and their serial dilutions. incubator temperature should be strictly controlled. respectively. cross reaction may still exist.values of the kit kept at 37oC with that of at recommended temperature. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. 1 day storage at 37oC can be considered as 2 months at 4oC. middle and high level human HbA1C were tested on 3 different plates. middle and high level human HbA1C were tested 20 times on one plate. Inter-assay Precision (Precision between assays): 3 samples with low.reactivity detection between human HbA1C and all the analogues.D. Keep the kit at 37oC for 3 days. The loss rate was determined by accelerated thermal degradation test. Note: To minimize extra influence on the performance. Note: Limited by current skills and knowledge. which was calculated by the Arrhenius equation. operation procedures and lab conditions. The results were demonstrated by the percentage of calculated concentration to the expected. (referring from China Biological Products Standard. and compare O. For ELISA kit.

as well as incorrect parameter setting for the plate reader may lead to incorrect results. sensitivity and color developing time. .uscnk. Each kit has been strictly passed Q. Incubate 1 hour at 37oC. However. Wrong operations during the reagents preparation and loading. In order to get better reproducible results. Do not remove microtiter plate from the storage bag until needed. Add 50µL Stop Solution. Protect all reagents from strong light during storage and incubation. 2. 2. www. Even the same operator might get different results in two separate experiments. and clothing protection when using this material. Do not mix or substitute reagents from one kit lot to another. 6.D. 8. 4. 9. hand. 7. So there might be some qualitative and technical risks to use the kit. There may be some foggy substance in the wells when the plate is opened at the first time.uscnk. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism.htm). Please make sure that sufficient samples are available. Incubate 30 minutes at 37oC. Wear eye. The final experimental results will be closely related to validity of the products. Intra-assay variance among kits from different batches might arise from above factors.C Please read the instruction carefully and adjust the instrument prior to the experiment. operation skills of the end users and the experimental environments. Limited by the current condition and scientific technology.uscnk. a preliminary experiment before assay for each batch is recommended. It will not have any effect on the final assay results. the operation of every step in the assay should be controlled. Incubate 2 hours at 37oC. Incubate 15-25 minutes at 37oC. samples and standards. 3. [ PRECAUTION ] The Stop Solution suggested for use with this kit is an acid solution. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O. 8. Aspirate and wash 5 times. 5. www. Use only the reagents supplied by manufacturer. 3. please refer to the operation video (http://www. since we haven’t compared our products with other manufacturers. Aspirate and wash 3 times. face. results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Add 100µL standard or sample to each well. is only for information. Furthermore. 5. 11.uscnk. Kits from different batches may be a little different in detection range. 7. Add 100µL prepared Detection Reagent B. Read at 450nm immediately. The instruction manual also suits for the kit of 48T. Add 100µL prepared Detection Reagent A. [ IMPORTANT NOTE ] 1. 10. Kits from different manufacturers with the same item might produce different results. Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website (www. 4. but all reagents of 48T kit are reduced by half. Valid period: six months. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. For more information.[ ASSAY PROCEDURE SUMMARY ] 1. 12. Add 90µL Substrate Solution. Prepare all reagents. we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers.