ab178661 TSH Receptor Autoantibody ELISA Kit Instructions for Use For the quantitative measurement of TSH Receptor Autoantibodies in Human serum. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 4 December 2013
Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 3 GENERAL INFORMATION 3. PRECAUTIONS 4 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 6 7. LIMITATIONS 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION 8 10. SAMPLE COLLECTION AND STORAGE 9 11. PLATE PREPARATION 9 ASSAY PROCEDURE 12. ASSAY PROCEDURE 10 DATA ANALYSIS 13. CALCULATIONS 12 14. TYPICAL DATA 13 15. TYPICAL SAMPLE VALUES 14 16. ASSAY ANALYTICAL SPECS 14 RESOURCES 17. INTERFERENCES 15 18. TROUBLESHOOTING 15 19. NOTES 17 Discover more at www.abcam.com 1
INTRODUCTION 1. BACKGROUND Abcam s anti-tsh Receptor Autoantibody in vitro ELISA (Enzyme- Linked Immunosorbent Assay) kit is designed for the accurate quantitative measurement of TSH Receptor autoantibodies in Human serum. A 96-well plate has been precoated with TSH Receptor to bind cognate antibodies. Controls, standards or test samples are added to the wells and incubated. Following washing, biotin labelled TSH is added to the wells, which binds to immobilized TSH Receptors which have not been blocked by the bound TSH Receptor autoantibodies from controls, standards or test samples. Streptavidin Peroxidase is then added which binds specifically to biotin. Excess unbound streptavidin is then washed away and the addition of tetramethylbenzidine (TMB) substrate results in formation of a blue colour that changes to yellow after adding an acidic stop solution. The density of yellow coloration is inversely proportional to the amount of TSH Receptor autoantibody sample captured in plate. Hyperthyroidism is a disease that produces an increase in blood circulating levels of thyroid hormones. In some cases, as in Graves' disease is caused by autoantibodies against the TSH receptor (TSHr). Autoantibodies mimic the effect of TSH on the thyroid gland causing a rise in blood levels of T3 and T4. The determination of these autoantibodies (TRAb) may be useful for the diagnosis of the disease and its treatment. The main treatment for Graves disease is represented by use of antithyroid drugs (propylthiouracil or methimazole), I131 and surgery. The dosage of TRAb is therefore useful in the course or at the end of therapy. Discover more at www.abcam.com 2
INTRODUCTION 2. ASSAY SUMMARY Prepare all reagents, samples, controls and standards as instructed. Add samples, standards, controls and start solution to appropriate wells. Incubate at room temperature Wash wells and then add prepared Biotinylated Antibody to each well. Incubate at room temperature. Wash wells and then add the prepared Streptavidin- Peroxidase conjugate to each well. Incubate at room temperature. After washing, add TMB substrate solution to each well. Incubate at room temperature. Add Stop Solution to each well. Read immediately. Discover more at www.abcam.com 3
GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at 2-8 C immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in section 9. Reagent Preparation. Discover more at www.abcam.com 4
GENERAL INFORMATION 5. MATERIALS SUPPLIED Item Amount Storage Condition (Before Preparation) TSH Receptor Coated Microplate (12 x 8 wells) 96 Wells 2-8 C Stop Solution 10 ml 2-8 C 20X Streptavidin Peroxidase Concentrate 750 µl 2-8 C Streptavidin Peroxidase Diluent 15 ml 2-8 C Biotin (lyophilized) 3 bottles 2-8 C Biotin Diluent 15 ml 2-8 C TMB Substrate Solution 15 ml 2-8 C 10X Washing Solution 100 ml 2-8 C Start Buffer 10 ml 2-8 C TSH Receptor Standard 1-1 U/L 1 ml 2-8 C TSH Receptor Standard 2-2 U/L 1 ml 2-8 C TSH Receptor Standard 3-8 U/L 1 ml 2-8 C TSH Receptor Standard 4-40 U/L 1 ml 2-8 C TSH Receptor Positive Control 1 ml 2-8 C TSH Receptor Negative Control 1 ml 2-8 C Strip Holder 1 unit 2-8 C Cover Foil 1 unit 2-8 C Discover more at www.abcam.com 5
GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Microplate reader capable of measuring absorbance at 450 nm or 620 nm Multi and single channel pipettes to deliver volumes between 10 and 1,000 µl Optional: Automatic plate washer for rinsing wells Vortex tube mixer Deionised or (freshly) distilled water Disposable tubes Timer 7. LIMITATIONS ELISA kit intended for research use only. Not for use in diagnostic procedures All components of Human origin used for the production of these reagents have been tested for anti-hiv antibodies, anti-hcv antibodies and HBsAg and have been found to be non-reactive. Nevertheless, all materials should still be regarded and handled as potentially infectious Use only clean pipette tips, dispensers, and lab ware. Do not interchange screw caps of reagent vials to avoid crosscontamination Close reagent vials tightly immediately after use to avoid evaporation and microbial contamination After first opening and subsequent storage check conjugate and control vials for microbial contamination prior to further use Discover more at www.abcam.com 6
GENERAL INFORMATION To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells 8. TECHNICAL HINTS Avoid foaming or bubbles when mixing or reconstituting components Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps Complete removal of all solutions and buffers during wash steps is necessary for accurate measurement readings This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions Discover more at www.abcam.com 7
ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents, samples and controls to room temperature (18-25 C) prior to use. 9.1 1X Washing Solution Prepare 1X Washing Solution by diluting 20X Washing Solution with deionized water. To make 200 ml 1X Washing Solution combine 10 ml 20X Washing Solution with 190 ml deionized water. Mix thoroughly and gently. 9.2 1X Biotin Solution Reconstitute each vial of the lyophilized Biotin with 4.5 ml Biotin Diluent. When more than one vial is to be used, pool the vials and mix gently before use. Store at 2-8 C for up to 6 months after reconstitution. 9.3 1X Streptavidin Peroxidase Solution Prepare 1X Streptavidin Peroxidase Solution by diluting the 20X Streptavidin Peroxidase Concentrate Dilute with Streptavidin Peroxidase Diluent. To make 10 ml 1X Streptavidin Peroxidase Solution, dilute 500 µl of 20X Streptavidin Peroxidase Concentrate with 9.5 ml of Streptavidin Peroxidase Diluent. Store at 2-8 C for up to expiry date of the kit. All other solutions are supplied ready to use Discover more at www.abcam.com 8
ASSAY PREPARATION 10. SAMPLE COLLECTION AND STORAGE Use Human serum samples with this assay. Sera to be analysed should be assayed soon after separation or stored, preferably in aliquots, at 20 C or below. Incorrect storage of serum samples can lead to loss of TSH Receptor Autoantibody activity. Do not use highly lipaemic or haemolysed serum samples. Do not use plasma in the assay. If samples are stored frozen, mix thawed samples well before testing to ensure homogeneity. Avoid repeated freezing and thawing. 11. PLATE PREPARATION The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents Unused well strips should be returned to the plate packet and stored at 4 C For each assay performed, a minimum of 1 well must be used as a blank, omitting sample and conjugate from well addition For statistical reasons, we recommend each standard and sample should be assayed with a minimum of two replicates (duplicates) Discover more at www.abcam.com 9
ASSAY PROCEDURE 12. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. Please read the test protocol carefully before performing the assay. Reliability of results depends on strict adherence to the test protocol as described. If performing the test on ELISA automatic systems we recommend increasing the washing steps from three to five and the volume of washing solution from 300 µl to 350 µl to avoid washing effects. Assay all standards, controls and samples in duplicate. All controls (TSH Receptor Positive and TSH Receptor Negative) must be included with each assay performed to determine test results. 12.1. Prepare all reagents, working standards, and samples as directed in the previous sections. 12.2. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, reseal and return to 4 C storage. 13.1. Add 75 µl of each standard, control or sample into their respective wells. Add 75 µl of Start buffer. Leave a blank well for substrate blank. 12.3. Cover wells with the foil supplied in the kit. 12.4. Incubate for 120 minutes at room temperature (22-28 C) while shaking at >500 rpm. Alternatively incubate without shaking for at least 180 minutes at room temperature (22-28 C). 12.5. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well with 300 µl of 1X Washing Solution, drain the wash completely. Avoid overflows from the reaction wells. The soak time between each wash cycle should be > 5 seconds. At the Discover more at www.abcam.com 10
ASSAY PROCEDURE end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step. Note: Washing is critical. Insufficient washing results in poor precision and falsely elevated absorbance values. 12.6. Add 100 µl 1X Biotin Solution into all wells except for the blank well. Cover with foil. 12.7. Incubate for 25 minutes at room temperature (22-28 C). Do not expose to direct sunlight. 12.8. Repeat step 12.5. 12.9. Add 100 µl 1X Streptavidin Peroxidase Solution into all wells except for the blank well. 12.10. Incubate for 20 minutes at room temperature (22-28 C). 12.11. Repeat step 12.5 three times. 12.12. Add 100 µl TMB Substrate Solution into all wells. 12.13. Incubate for exactly 30 minutes at room temperature in the dark. 12.14. Add 50 µl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. Shake the microplate gently. Any blue color developed during the incubation turns into yellow. 12.15. Measure the absorbance of the specimen at 450 nm against Blank within 20 minutes of addition of the Stop Solution. Discover more at www.abcam.com 11
DATA ANALYSIS 13. CALCULATIONS Calculation of Results Calculate the mean background subtracted absorbance for each point of the standard curve and each sample. Plot the mean value of absorbance of the standards against concentration. Draw the best-fit curve through the plotted points. (e. g.: Four Parameter Logistic). Interpolate the values of the samples on the standard curve to obtain the corresponding values of the concentrations expressed in U/mL. Results can also be expressed as inhibition (%I) of TSH binding calculated using the formula; 100 x [ 1 Sample absorbance value Negative Control absorbance value] Samples with high TSH Receptor Autoantibody concentrations can be diluted in TSH Receptor Negative control. For example, 20 µl of sample plus 180 µl of TSH Receptor Negative control to give a 10x dilution. Other dilutions (e.g. 100x) can be prepared from a 10x dilution or otherwise as appropriate. Some sera will not dilute in a linear way and we suggest that the dilution giving a value closest to 50% inhibition is used for calculation of TSH Receptor Autoantibody concentration. Interpretation of Results Normal value ranges for this ELISA should be established by each researcher. The following values should be considered as a guideline only: Negative Inconclusive (Grey zone): Reactive: 1 U/mL 1.1 1.5 U/mL > 1.5 U/mL Discover more at www.abcam.com 12
DATA ANALYSIS 14. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. Standard 1 Standard 2 Standard 3 Standard 4 Test # 1 U/l 2 U/l 8 U/l 40 U/l O.D. %B/B0 O.D. %B/B0 O.D. %B/B0 O.D. %B/B0 1 2,039 100 1,688 82 0.901 44.2 0.094 4.6 2 1,720 100 1,292 7.1 0.617 35.9 0.115 6.7 3 1,849 100 1,495 80.9 0.843 45,6 0.122 6.6 4 1,945 100,0 1,655 85.1 0.927 47.7 0.126 6.5 5 1,929 100 1,650 85.5 0.793 41.1 0.092 4.8 6 1,648 100 1,214 73.7 0.794 48.2 0.119 7.2 n 6 6 6 6 6 6 6 6 Media 1.86 100 1.5 80.5 0.81 43.8 0.11 6.1 d.s. 0.15 0 0.2 0.05 0.11 0.05 0.01 0.01 %C.V. 7.9 0 13.6 6.3 13.6 10.6 13.2 18 Figure 1. Acceptable ranges are calculated as the mean of each Calibrator point response ± 3 S.D. Discover more at www.abcam.com 13
DATA ANALYSIS 15. TYPICAL SAMPLE VALUES PRECISION Intra-Assay Inter-Assay n= 16 16 %CV 7.6 6.5 16. ASSAY ANALYTICAL SPECS SPECIFICITY - 154 samples from healthy blood donors were assayed in the TSH Receptor Autoantibody ELISA Kit. 152 (99%) were identified as being negative for TSH Receptor autoantibodies. SENSITIVITY - 50 samples from patients diagnosed with Graves disease were assayed using the TSH Receptor Autoantibody ELISA Kit. 49 (98%) were identified as being positive for TSH Receptor autoantibodies. 1 sample (2%) was identified as being within the equivocal range. The kit negative control was assayed 32 times and the mean and standard deviation calculated. The lower detection limit at 2 standard deviations was 0.21 U/ml. Discover more at www.abcam.com 14
RESOURCES 17. INTERFERENCES No interference was observed when samples were spiked with the following materials: haemoglobin (5 mg/ml), bilirubin (20 mg/dl), intralipid (10 mg/ml). 18. TROUBLESHOOTING Problem Cause Solution Low signal Large CV Incubation time to short Precipitate can form in wells upon substrate addition when concentration of target is too high Using incompatible sample type (e.g. serum vs. cell extract) Sample prepared incorrectly Bubbles in wells All wells not washed equally/thoroughly Incomplete reagent mixing Inconsistent pipetting Inconsistent sample preparation or storage Try overnight incubation at 4 C Increase dilution factor of sample Detection may be reduced or absent in untested sample types Ensure proper sample preparation/dilution Ensure no bubbles present prior to reading plate Check that all ports of plate washer are unobstructed/wash wells as recommended Ensure all reagents/master mixes are mixed thoroughly Use calibrated pipettes & ensure accurate pipetting Ensure consistent sample preparation and optimal sample storage conditions (e.g. minimize freeze/thaws cycles) Discover more at www.abcam.com 15
RESOURCES Problem Cause Solution Wells are insufficiently washed Wash wells as per protocol recommendations High background Contaminated wash buffer Make fresh wash buffer Low sensitivity Waiting too long to read plate after adding stop solution Improper storage of ELISA kit Using incompatible sample type (e.g. Serum vs. cell extract) Read plate immediately after adding stop solution Store all reagents as recommended. Please note all reagents may not have identical storage requirements. Detection may be reduced or absent in untested sample types Discover more at www.abcam.com 16
RESOURCES 19. NOTES Discover more at www.abcam.com 17
RESOURCES Discover more at www.abcam.com 18
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