ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit Instructions for Use For the quantitative measurement of Human Thyroid Stimulating Hormone (TSH) in plasma and serum. This product is for research use only and is not intended for in vitro diagnostic use. www.abcam.com
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Table of Contents 1. Introduction 3 2. Assay Summary 4 3. Kit Contents 5 4. Storage and Handling 6 5. Additional Materials Required 6 6. Preparation of Reagents 6 7. Preparation and Collection of Specimens 8 8. Assay Method 9 9. Data Analysis 12 10. Limitations 15 11. Specificity 16 12. Troubleshooting 17 2
1. Introduction ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit is intended for the quantitative determination of Thyroid Stimulating Hormone (TSH) in human plasma (citrate) or serum. Thyroid-stimulating hormone (TSH or thyrotropin) is a glycoprotein hormone synthesized and secreted by the anterior lobe of the pituitary gland, which regulates the endocrine function of the thyroid. Structurally, the glycoprotein hormones are related heterodimers comprised of a common alpha subunit and a hormone-specific beta subunit, which are non-covalently bound to one another. The alpha subunit of TSH is identical to that of human chorionic gonadotropin (HCG), luteinising hormone (LH) and follicle-stimulating hormone (FSH). The beta subunit is TSH-specific, and therefore determines its function. 3
2. Assay Summary ab108682 is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes two monoclonal antibodies specific for distinct antigenic determinants on the TSH beta subunit. The first antibody is immobilized on the surface of the microtiter wells. The second antibody is conjugated to horseradish peroxidase. The test sample is allowed to react simultaneously with the two antibodies, resulting in the TSH molecules being sandwiched between the solid phase and the enzyme-linked antibodies. After an incubation step, the wells are washed with Washing Solution to remove all unbound material. The immune complex is visualized by adding Tetramethylbenzidine (TMB) substrate which gives a blue reaction product. The intensity of this product is proportional to the concentration of TSH in the specimen. Sulphuric acid is added to stop the reaction. This produces a yellow endpoint colour. Absorbance at 450 nm is read using an ELISA microwell plate reader. The color intensity is directly proportional to the concentration of TSH present in the test sample. 4
3. Kit Contents Antibody-Coated Wells: 12 break-apart 8-well snap-off strips coated with anti-tsh mouse monoclonal antibodies; in resealable aluminium foil. Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.2 mol/l; ready to use; red cap. Washing Solution (20x conc.): 1 bottle containing 50 ml of a 20- fold concentrated buffer for washing the wells; ph 7.2 ± 0.2; white cap. contains 0.1 % Bronidox L after dilution. Anti-TSH Conjugate: 1 bottle containing 15 ml of peroxidase labelled monoclonal antibodies to TSH; coloured red; ready to use; black cap. Contains 0.2 % Bronidox L. TMB Substrate Solution: 1 bottle containing 15 ml 3,3,5,5 - tetramethylbenzidine (TMB); ready to use; yellow cap. TSH Standard Set: colored yellow, ready to use; yellow cap (1 set, 1 ml/vial); contains 0, 0.2, 0.5, 2.5, 5.0, 10.0 and 20 µlu/ml. Contains 0.1 % Kathon. 1 Strip holder. 1 Cover foil. 5
4. Storage and Handling The reagents are stable up to the expiry date stated on the label when stored at 2-8 Keep microtiter plate in a sealed bag with desiccant to minimize exposure to damp air. 5. Additional Materials Required ELISA microwell plate reader, equipped for the measurement of absorbance at 450/620 nm Incubator 37 C Manual or automatic equipment for rinsing wells Pipettes to deliver 50 and 100 µl Vortex tube mixer Deionised or (freshly) distilled water Timer 6. Preparation of Reagents 1. All reagents should be allowed to reach room temperature (20-25 C) before use. 2. Coated Snap-off Strips: The ready to use break-apart snap-off strips are coated with monoclonal antibodies to TSH. Store at 2 6
- 8 C. Immediately after removal of strips, the remaining strips should be resealed in the aluminum foil along with the desiccant supplied and stored at 2-8 C; stability until the expiry date.. 3. Anti-TSH Conjugate: The bottle contains 15 ml of a solution with anti-tsh horseradish peroxidase, buffer, stabilizers, preservatives and an inert red dye. The solution is ready to use. Store at 2-8. After first opening stability until the expiry date when stored at 2-8 C. 4. Standards: The vials labelled with Standard A, B, C, D, E, F and G contain a ready to use standard solution. The standards, calibrated in accordance with the WHO 3 rd IS for htsh (81/565), have the following concentrations: Standard A: Standard B: Standard C: Standard D: Standard E: Standard F: Standard G: 0.0 µiu/ml 0.2 µiu/ml 0.5 µiu/ml 2.5 µiu/ml 5.0 µiu/ml 10.0 µiu/ml 20.0 µiu/ml The solutions have to be stored at 2-8 C and contain 0.1 % Kathon. After first opening stability until the expiry date when stored at 2-8 C. 7
5. Washing Solution (20x conc.): The bottle contains 50 ml of a concentrated buffer, detergents and preservatives. Dilute Washing Solution 1 + 19; e. g. 10 ml Washing Solution + 190 ml fresh and germ free redistilled water. The diluted buffer will keep for 5 days if stored at room temperature. Crystals in the solution disappear by warming up to 37 C in a water bath. After first opening stability until the expiry date when stored at 2 to 8 C. 6. TMB Substrate Solution: The bottle contains 15 ml of a tetramethylbenzidine/hydrogen peroxide system. The reagent is ready to use and has to be stored at 2-8 C, away from the light. The solution should be colorless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away. After first opening stability until the expiry date when stored at 2-8 C. 7. Stop Solution: The bottle contains 15 ml 0.2 M sulphuric acid solution. This ready to use solution has to be stored at 2-8 C. After first opening stability until the expiry date. 7. Preparation and Collection of Specimens 1. Use human serum or plasma (citrate) samples with this assay. 2. If the assay is performed within 5 days after sample collection, the specimens should be kept at 2-8 C; otherwise they should 8
be aliquoted and stored deep-frozen (-20 - -70 C). If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. 3. Heat inactivation of samples is not recommended. 8. Assay Method Test Preparation: Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described. If performing the test on ELISA automatic systems we recommend increasing the volume of Washing Solution from 300 µl to 350 µl to avoid washing effects. Prior to commencing the assay, the distribution and identification plan for all specimens and standards should be carefully established. Select the required number of microtiter strips or wells and insert them into the holder. Please allocate at least: 1 well (e. g. A1) for the substrate blank, 7 wells (e. g. B1, C1, etc.) for Standard A, B, C, D, E, F and G It is recommended to determine standards and samples in duplicate. Perform all assay steps in the order given and without any appreciable delays between the steps. 9
A clean, disposable tip should be used for dispensing each standard and sample. Adjust the incubator to 37 ± 1 C. Assay Procedure: 1. Dispense 50 µl of each Standard (A, B, C, D, E, F and G) and samples into their respective wells. Leave well A1 for the substrate blank. 2. Dispense 100 µl of Conjugate into each well except for the blank (e. g. A1). 3. Cover wells with the foil supplied in the kit. 4. Incubate for 1 hour ± 5 min at 37 ± 1 C. 5. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well five times with 300 µl of Washing Solution. Avoid overflows from the reaction wells. The soak time between each wash cycle should be > 5 sec. At the 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. 6. Dispense 100 µl of TMB Substrate Solution into each well. 7. Incubate for exactly 15 min at room temperature (20-25 C) in the dark. 10
8. Dispense 100 µl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. Any blue color developed during the incubation turns into yellow. 9. Measure the absorbance of the specimen at 450/620 nm within 30 min after addition of the Stop Solution. Measurement: Adjust the ELISA Microwell Plate Reader to zero using the standard 0. If - due to technical reasons - the ELISA reader cannot be adjusted to zero using the substrate blank in well A1, subtract the absorbance value of well A1 from all other absorbance values measured in order to obtain reliable results! Measure the absorbance of all wells at 450 nm and record the absorbance values for each standard and sample in the distribution and identification plan. Dual wavelength reading using 620 nm as reference wavelength is recommended. Where applicable calculate the mean absorbance values of all duplicates. 11
9. Data Analysis A. Assay Validation CriteriaI In order for an assay to be considered valid, the following criteria must be met: Standard Absorbance Substrate Blank <0.100 A <0.100 B > Standard A C > Standard B D > Standard C E > Standard D F > Standard E G >1.000 B. Calculation of Results In order to obtain quantitative results in µiu/ml plot the (mean) absorbance values of the Standards A G (y-axis) on graph paper in 12
a system of coordinates against their corresponding concentrations (x-axis) and draw a standard calibration curve. Read results from this standard curve employing the (mean) absorbance values of each patient specimen. All suitable computer programs available can be used for automated result reading and calculation. C. Typical Calibration Curve D. Interpretation of results Normal value ranges for this ELISA assay should be established by each laboratory. The following values should be considered as a guideline: Normal TSH range 0.3 to 4.5 µiu/ml. 13
E. Sensitivity The analytical sensitivity - defined as the apparent concentration of the analyte that can be distinguished from the zero calibrator - is < 0.1 µiu/ml. F. Precision Intra-assay n Mean (E) CV (%) Serum 1 24 0.519 6.7 Serum 2 24 0.207 5.4 Serum 3 24 0.064 10.5 Inter-assay n Mean (µiu/ml) CV (%) Serum 1 12 5.42 5.2 Serum 2 12 2.45 4.0 Serum 3 12 1.40 4.0 G. Correlation ab108682 Thyroid Stimulating Hormone (TSH) Human ELISA kit was compared with a reference electrochemiluminescence 14
immunoassay. 47 biological specimens were used (the values ranged from approx. 0 - approx. 11 µiu/ml). The linear regression curve was calculated. Y= 1.06 x -0.11 r 2 = 0.97 H. Interferences Interferences with hemolytic, lipemic or icteric sera are not observed up to a concentration of 10 mg/ml hemoglobin, 5 mg/ml triglycerides and 0.5 mg/ml bilirubin. I. Hook Effect No hook effect was observed applying up to 5,000 µiu/ml Thyroid Stimulating Hormone. 10. Limitations Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values. 15
11. Specificity The following hormones were tested for cross-reactivity at concentrations up to the levels indicated below. No cross-reactivity was observed for any of the components. Hormone tested LH (WHO 2 nd IS 80/552) hcg (WHO 5 th IS 07/364) FSH (WHO IS 83/575) Concentration [miu/ml] 70 175 350 1,000 5,000 25,000 10 20 50 Produced intensity equivalent to TSH [µiu/ml] - - <0.1 - - <0.1 - <0.5 <0.5 16
12. Troubleshooting Problem Cause Solution Poor standard curve Improper standard dilution Standard improperly reconstituted (if applicable) Standard degraded Curve doesn't fit scale Confirm dilutions made correctly Briefly spin vial before opening; thoroughly resuspend powder (if applicable) Store sample as recommended Try plotting using different scale Low signal Incubation time too short Try overnight incubation at 4 C Target present below detection limits of assay 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 Decrease dilution factor; concentrate samples Increase dilution factor of sample Detection may be reduced or absent in untested sample types Ensure proper sample preparation/dilution Large CV Bubbles in wells Ensure no bubbles present prior to reading plate 17
High background Low sensitivity All wells not washed equally/thoroughly Incomplete reagent mixing Inconsistent pipetting Inconsistent sample preparation or storage Wells are insufficiently washed Contaminated wash buffer 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) Check that all ports of plate washer are unobstructed/wash wells as recommended Ensure all reagents/master mixes are mixed thoroughly Use calibrated pipettes and ensure accurate pipetting Ensure consistent sample preparation and optimal sample storage conditions (eg. minimize freeze/thaws cycles) Wash wells as per protocol recommendations Make fresh wash buffer 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 For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 18
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