ELISA Kit for Anti-Sperm Antibody (AsAb)
Contents
1. 3 Incubation To ensure accurate results proper adhesion of plate sealers during incubation steps is necessary Do not allow wells to sit uncovered for extended periods between incubation steps Once reagents are added to the well strips DO NOT let the strips DRY at any time during the assay Incubation time and temperature must be controlled 4 Washing The wash procedure is critical Complete removal of liquid at each step is essential for good performance After the last wash remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate Insufficient washing will result in poor precision and false elevated absorbance reading 5 Controlling of reaction time Observe the change of color after adding TMB Substrate e g observation once every 10 minutes if the color is too deep add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading 6 TMB Substrate is easily contaminated Please protect it from light 7 The environment humidity which is less than 60 might have some effects on the final performance therefore a humidifier is recommended to be used at that condition TEST PRINCIPLE The microtiter plate provided in this kit has been pre coated with an antigen Standards or samples are then added to the appropriate microtiter plate wells with a Horseradish Peroxidase HRP conjugated secondary antibod2. The linearity of the kit was assayed by testing samples spiked with appropriate concentration of AsAb and their serial dilutions The results were demonstrated by the percentage of calculated concentration to the expected PRECISION Intra assay Precision Precision within an assay 3 samples with low middle and high level AsAb were tested 20 times on one plate respectively Inter assay Precision Precision between assays 3 samples with low middle and high level AsAb were tested on 3 different plates 8 replicates in each plate CV SD meanX100 Intra Assay CV lt 10 Inter Assay CV lt 12 STABILITY The stability of ELISA kit is determined by the loss rate of activity The loss rate of this kit is less than 5 within the expiration date under appropriate storage condition To minimize extra influence on the performance operation procedures and lab conditions especially room temperature air humidity incubator temperature should be strictly controlled It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards 2 Add 100uL standard or sample to each well Incubate 2 hours at 37 C 3 Aspirate and add 100uL prepared Detection Reagent A Incubate 1 hour at 37 C 4 Aspirate and wash 5 times 5 Add 90uL Substrate Solution Incubate 15 25 minutes at 37 C 6 Add 50uL Stop S
3. desiccant pack and reseal along entire edge of zip seal Note It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit For the expiration date of the kit please refer to the label on the kit box All components are stable until this expiration date SAMPLE COLLECTION AND STORAGE Serum Use a serum separator tube and allow samples to clot for two hours at room temperature or overnight at 4 C before centrifugation for 20 minutes at approximately 1000xg Assay freshly prepared serum immediately or store samples in aliquot at 20 C or 80 C for later use Avoid repeated freeze thaw cycles Seminal plasma Collect semen samples using a sterile container then allow it to liquefy at room temperature or 37 C After liquefaction centrifuge at 4000 rom for 10 min Remove the supernate and assay immediately or store samples at 20 C Note 1 Samples to be used within 5 days may be stored at 4 C otherwise samples must be stored at 20 C lt 1 month or 80 C lt 2 months to avoid loss of bioactivity and contamination 2 Sample hemolysis will influence the result so hemolytic specimen should not be detected 3 When performing the assay bring samples to room temperature REAGENT PREPARATION 1 Bring all kit components and samples to room temperature 18 25 C before use 2 Standard Reconstitute the Standard with 0 4mL of Standard Diluent kept for 1
4. with a bandwidth of 10nm or less and an optical density range of 0 3 O D or greater at 450 10nm wavelength is acceptable for use in absorbance measurement Please read the instruction carefully and adjust the instrument prior to the experiment For more information please refer to the operation video http www uscnk com homepage operate elisa htm Even the same operator might get different results in two separate experiments In order to get better reproducible results the operation of every step in the assay should be controlled Furthermore a preliminary experiment before assay for each batch is recommended Each kit has been strictly passed Q C test However results from end users might be inconsistent with our in house data due to some unexpected transportation conditions or different lab equipments Intra assay variance among kits from different batches might arise from above factors too 10 Kits from different manufacturers with the same item might produce different results since we haven t compared our products with other manufacturers 11 The instruction manual also suits for the kit of 48T but all reagents of 48T kit are reduced by half PRECAUTION The Stop Solution suggested for use with this kit is an acid solution Wear eye hand face and clothing protection when using this material TROUBLE SHOOTING Curve Inadequate reagent volumes added to wells Calibrate pipettes and Add adequate reagents Incorre
5. 0 minutes at room temperature shake gently not to foam The concentration of the standard in the stock solution is 100ng mL Please prepare 7 tubes containing 0 25mL Standard Diluent and produce a double dilution series according to the picture shown below Mix each tube thoroughly before the next transfer Set up 7 points of diluted standard such as 100ng mL 50ng mL 25ng mL 12 5ng mL 6 25ng mL 3 12ng mL 1 56ng mL and the last EP tubes with Standard Diluent is the blank as Ong mL 250uL 250uL 250uL 250uL 250uL 250uL A LALM A Stock Standard gt 1 _ Srandil i f w V V Tube 1 2 3 4 5 6 T 8 ng mL 100 50 25 12 0 6 25 3 12 1 56 0 3 Detection Reagent A Briefly spin or centrifuge the stock Detection A before use Dilute to the working concentration with working Assay Diluent A respectively 1 100 4 Wash Solution Dilute 20mL of Wash Solution concentrate 30x with 580mL of deionized or distilled water to prepare 600mL of Wash Solution 1x 5 TMB substrate Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again Note 1 The standard for this kit is liquid Due to its small volume maybe invisible to the eye 2 Making serial dilution in the wells directly is not permitted 3 Prepare standard within 15 minutes before assay Please do not dissolve the reagents at 37 C directly 4 Please carefully reconstitute Standards or working Detec
6. AEA465Hu 96 Tests Enzyme linked Immunosorbent Assay Kit For Anti Sperm Antibody AsAb Organism Species Homo sapiens Human Instruction manual FOR IN VITRO AND RESEARCH USE ONLY NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES 11th Edition Revised in July 2013 INTENDED USE The kit is an enzyme immunoassay for in vitro quantitative measurement of ASAb in human serum and seminal plasma REAGENTS AND MATERIALS PROVIDED Pre coated ready to use 96 well strip plate Plate sealer for 96 wells Standard Standard Diluent 1x20mL Detection Reagent A 1x120uL Assay Diluent A 1x12mL TMB Substrate Stop Solution 1x6mL Wash Buffer 30 x concentrate 1x20mL Instruction manual MATERIALS REQUIRED BUT NOT SUPPLIED 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 STORAGE OF THE KITS 1 For unopened kit All the reagents should be kept according to the labels on vials The Standard Detection Reagent A and the 96 well strip plate should be stored at 20 C upon receipt while the others should be at 4 C 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
7. Substrate Solution to each well Cover with a new Plate sealer Incubate for 15 25 minutes at 37 C Don t exceed 30 minutes Protect from light The liquid will turn blue by the addition of Substrate Solution 6 Add 50uL of Stop Solution to each well The liquid will turn yellow by the addition of Stop solution Mix the liquid by tapping the side of the plate If color change does not appear uniform gently tap the plate to ensure thorough mixing 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 Then run the microplate reader and conduct measurement at 450nm immediately Note 1 Assay preparation Keep appropriate numbers of wells for each experiment and remove extra wells from microplate Rest wells should be resealed and stored at 20 C 2 Samples or reagents addition Please use the freshly prepared Standard Please carefully add samples to wells and mix gently to avoid foaming Do not touch the well wall For each step in the procedure total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes This will ensure equal elapsed time for each pipetting step without interruption Duplication of all standards and specimens although not required is recommended To avoid cross contamination change pipette tips between additions of standards samples and reagents Also use separated reservoirs for each reagent
8. ct incubation times Ensure sufficient incubation times Low OD Incorrect incubation temperature Reagents balanced to room temperature Guise Conjugate or substrate reagent failure Mix conjugate amp substrate color should develop immediately
9. og of the data to establish standard curve for each test is recommended Typical standard curve below is provided for reference only 120 100 80 60 40 20 Concentration ng mL 0 0 5 4 1 5 2 2 9 3 3 5 Optical Density Typical Standard Curve for AsAb Human ELISA DETECTION RANGE 1 56 100ng mL The standard curve concentrations used for the ELISA s were 100ng mL 50ng mL 25ng mL 12 5ng mL 6 25ng mL 3 12ng mL 1 56ng mL SENSITIVITY The minimum detectable dose of AsAb is typically less than 0 59ng mL The sensitivity of this assay or Lower Limit of Detection LLD was defined as the lowest protein concentration that could be differentiated from zero It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration SPECIFICITY This assay has high sensitivity and excellent specificity for detection of ASAb No significant cross reactivity or interference between AsAb and analogues was observed Note Limited by current skills and knowledge it is impossible for us to complete the cross reactivity detection between AsAb and all the analogues therefore cross reaction may still exist RECOVERY Matrices listed below were spiked with certain level of recombinant AsAb and the recovery rates were calculated by comparing the measured value to the expected amount of AsAb in samples 1 LINEARITY
10. olution Read at 450nm immediately IMPORTANT NOTE 1 Limited by the current condition and scientific technology we can t completely conduct the comprehensive identification and analysis on the raw material provided by suppliers So there might be some qualitative and technical risks to use the kit The final experimental results will be closely related to validity of the products operation skills of the end users and the experimental environments Please make sure that sufficient samples are available Kits from different batches may be a little different in detection range sensitivity and color developing time Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for information Do not mix or substitute reagents from one kit lot to another Use only the reagents supplied by manufacturer Protect all reagents from strong light during storage and incubation All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism There may be some foggy substance in the wells when the plate is opened at the first time It will not have any effect on the final assay results Do not remove microtiter plate from the storage bag until needed Wrong operations during the reagents preparation and loading as well as incorrect parameter setting for the plate reader may lead to incorrect results A microplate plate reader
11. r kits e g antibody targets conformational epitope rather than linear epitope some native or recombinant proteins from other manufacturers may not be recognized by our products 6 Influenced by the factors including cell viability cell number or sampling time samples from cell culture supernatant may not be detected by the kit 7 Fresh samples without long time storage is recommended for the test Otherwise protein degradation and denaturalization may occur in those samples and finally lead to wrong results ASSAY PROCEDURE 1 Determine wells for diluted standard blank and sample Prepare 7 wells for standard 1 well for blank Add 100uL each of dilutions of standard read Reagent Preparation blank and samples into the appropriate wells Cover with the Plate sealer Incubate for 2 hours at 37 C 2 Remove the liquid of each well don t wash 3 Add 100uL of Detection Reagent A working solution to each well Incubate for 1 hour at 37 C after covering it with the Plate sealer 4 Aspirate the solution and wash with 350uL of 1x Wash Solution to each well using a squirt bottle multi channel pipette manifold dispenser or autowasher and let it sit for 1 2 minutes Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper Totally wash 5 times After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against absorbent paper 5 Add 90uL of
12. tion Reagent A according to the instruction and 9 6 avoid foaming and mix gently until the crystals are completely dissolved To minimize imprecision caused by pipetting use small volumes and ensure that pipettors are calibrated It is recommended to suck more than 10uL for once pipetting The reconstituted Standards and Detection Reagent A can be used only once If crystals have formed in the Wash Solution concentrate 30x warm to room temperature and mix gently until the crystals are completely dissolved Contaminated water or container for reagent preparation will influence the detection result SAMPLE PREPARATION 1 Cloud Clone Corp is only responsible for the kit itself but not for the samples consumed during the assay The user should calculate the possible amount of the samples used in the whole test Please reserve sufficient samples in advance 2 Please predict the concentration before assaying If values for these are not within the range of the standard curve users must determine the optimal sample dilutions for their particular experiments 3 If the samples are not indicated in the manual a preliminary experiment to determine the validity of the kit is necessary 4 Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals 5 Due to the possibility of mismatching between antigen from other origin and antibody used in ou
13. y After TMB substrate solution is added those wells that contain AsAb will exhibit a change in color The enzyme substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm 10nm The concentration of AsAb in the samples is then determined by comparing the O D of the samples to the standard curve CALCULATION OF RESULTS Average the duplicate readings for each standard control and samples and subtract the average zero standard optical density Construct a standard curve by plotting the mean O D and concentration for each standard and draw a best fit curve through the points on the graph or create a standard curve on log log graph paper with AsAb concentration on the y axis and absorbance on the x axis Using some plot software for instance curve expert 1 30 is also recommended If samples have been diluted the concentration read from the standard curve must be multiplied by the dilution factor TYPICAL DATA In order to make the calculation easier we plot the O D value of the standard X axis against the known concentration of the standard Y axis although concentration is the independent variable and O D value is the dependent variable However the O D values of the standard curve may vary according to the conditions of assay performance e g operator pipetting technique washing technique or temperature effects plotting l
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