For the quantitative measurement of Human Thyroid Stimulating Hormone (TSH) in plasma and serum

Transcription

1 ab 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 diagnostic use.

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3 Table of Contents 1. Introduction 3 2. Assay Summary 6 3. Kit Contents 7 4. Storage and Handling 8 5. Additional Materials Required 9 6. Preparation of Reagents Preparation and Collection of Specimens Assay Method Data Analysis Specificity Limitations Troubleshooting 21 2

4 1. Introduction ab 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 glycopolipeptide hormone synthesized and secreted by the anterior pituitary gland which regulates the endocrine function of the thyroid gland. TSH consists of two subunits, the alpha and the beta subunit. The α subunit is identical to that of human chorionic gonadotropin (HCG), luteinising hormone (LH), follicle-stimulating hormone (FSH). The β (beta) subunit is unique to TSH, and therefore determines its function. TSH production is controlled by a Thyrotropin Releasing Hormone, (TRH), which is manufactured in the hypothalamus and transported to the pituitary gland, where it increases TSH production and release. Somatostatin is also produced by the hypothalamus, and has an opposite effect on the pituitary production of TSH, decreasing or inhibiting its release. TSH stimulates the thyroid gland to secrete the hormones thyroxine (T4) and triiodothyronine (T3). 3

5 Release of TSH is regulated by the circulating free fraction of thyroid hormones in the blood. TSH levels are depressed when peripheral concentrations of the free fraction of thyroid hormones are high. Conversely, TSH levels are high when peripheral concentrations of thyroid hormones are low. This effect creates a regulatory negative feedback loop. TSH levels are tested in the blood of patients suspected of suffering from excess (hyperthyroidism), or deficiency (hypothyroidism) of thyroid hormone. Generally, a normal range for TSH is between 0.3 and 3.0 miu/ml, but the interpretation depends also on what the blood levels of thyroid hormones (T3 and T4) are. Higher than normal levels of TSH combined with high levels of thyroid hormone (T3 and T4) may indicate dysfunction of the hypothalamus and pituitary gland. In these case, a high TSH is often produced by a benign tumour of the pituitary (adenoma). Conversely, low levels of TSH, while blood levels of T3 and T4 are also low, indicates abnormally low function of the pituitary, known as hypopituitarism. On the other hand, due to the negative feedback described above, abnormally high levels of Thyroid hormone, due to overproduction in the thyroid, results in low TSH levels. This occurs in diseases such as hyperthyroidism or Grave s disease. 4

6 Conversely, an underproduction of T3 and T4 caused by diseases such as congenital hypothyroidism (cretinism), hypothyroidism or thyroid hormone resistance, gives rise to an increase in the measured TSH. Clearly both TSH and T3 and T4 should be measured to ascertain where a specific thyroid disfunction is caused by primary pituitary or by a primary thyroid disease 5

7 2. Assay Summary TSH calibrators, patient specimens and/or controls containing the native antigen are first added to streptavidin coated wells. Biotinylated monoclonal and horseradish peroxidase (HRP) labelled antibodies are added and the reactants are mixed. The different types of antibodies used have high affinity and specificity and are directed against distinct and different epitopes of TSH. Reaction between the various TSH antibodies and native TSH occurs in the microwells without competition or sterics hindrance forming a soluble sandwich complex. The interaction is illustrated by the following equation: HRP-Ab(p) + AgTSH + BtnAb(m) Ka K-a HRP-Ab(p)-AgTSH-BtnAb(m) BtnAb(m) Biotinylated Monoclonal Antibody (Excess Quantity) AgTSH Native Antigen (Variable Quantity) HRP-Ab(p) HRP labeled Antibody (Excess Quantity) HRP-Ab(p)-AgTSH-BtnAb(m) Antigen-Antibodies Sandwich Complex Ka Rate Constant of Association K-a Rate Constant of Dissociation Simultaneously, the complex is fixed to the well through the high affinity reaction of streptavidin and biotinylated antibody. This interaction is illustrated below: HRP-Ab(p)-AgTSH-BtnAb(m) + Streptavidin CW CW Immobilized Complex Streptavidin CW Immobilized Complex Streptavidin immobolized on well. Antibodies-Antigen sandwich bound. 6

8 After equilibrium is attained, the antibody-bound fraction is separated from unbound antigen by aspiration. The native antigen concentration is directly proportional to the HRP activity in the antibody-bound fraction. The activity of the conjugated HRP is quantitated by reaction with TMB substrate to produce blue colour. The reaction is terminated by adding stop solution which turns the blue colour into yellow. The absorbance is measured on a plate reader. 3. Kit Contents Streptavidin Coated Wells: 12 breakapart 8-well snap-off strips coated with streptavidin, in resealable aluminium foil. Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.15 mol/l (avoid any skin contact). Conjugate: 1 bottle containing 13 ml of goat anti-tsh-horseradish peroxidase labelled antibodies and mouse anti-tsh biotinylated antibodies. TMB Substrate Solution: 1 bottle containing 15 ml 3, 3, 5, 5 -tetramethylbenzidine (H 2 O 2 -TMB 0.26 g/l) (avoid any skin contact). Wash Solution (50x): 1 bottle containing 20 ml (NaCl 45 g/l, Tween g/l). 7

9 TSH control: 1 bottle containing 1 ml of a lot-specific control solution. The concentration is indicated on the label of the bottle. TSH Standards: 7 bottles 1ml each. The standards are calibrated against the (WHO 2 nd IRP 80/558) and have approx. the following concentrations: Standard 0: 0.0 miu/l Standard 1: 0.2 miu/l Standard 2: 0.5 miu/l Standard 3: 2.5 miu/l Standard 4: 5.0 miu/l Standard 5: 10.0 miu/l Standard miu/l 1 Strip holder 1 Cover foils 4. Storage and Handling The reagents are stable up to the expiry date stated on the label when stored at 2-8 C Keep microtiter plate in a sealed bag with desiccant to minimize exposure to damp air. 8

10 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 9

11 6. Preparation of Reagents All reagents should be allowed to reach room temperature (20-28 C) before use. Mix well. 1. Coated snap-off Strips: The ready to use break apart snap-off strips are coated with streptavidin. Store at 2-8 C. Immediately after removal of strips, the remaining strips should be resealed in the aluminium foil along with the desiccant supplied and stored at 2-8 C; stability until expiry date. 2. Conjugate: The bottle contains 13 ml of a solution with goat anti-tsh horseradish peroxidase labelled antibodies and mouse anti-tsh biotinylated antibodies. 3. Standards: The 7 vials contain standard solutions of the concentration mentioned in Section 3. The standards are ready to use. After first opening stable for another 6 months at 2-8 C. 4. TSH control: The bottle contains 1 ml of a lot-specific control solution. The concentration is indicated on the label. 5. 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 in the dark. The solution should be colourless or could have a slight blue tinge. If the substrate turns into blue, it may have become contaminated and should be thrown away. 10

12 6. Stop Solution: The bottle contains 15 ml 0.15 M sulphuric acid solution. This ready to use solution has to be stored at 2-8 C. After first use stable until expiry date. 7. Wash Solution (50x): Before use dilute 20 ml concentrated Wash Solution with 980 ml distilled water. For smaller volumes respect the 1:50 ratio. The diluted wash solution is stable for 30 days at 2-8 C. 7. Preparation and Collection of Specimens 1. Use Human serum or plasma with this assay. 2. If the assay is performed within 48 hours after sample collection, the specimens should be kept at 2-8 C; otherwise they should be aliquoted and stored deep-frozen. If samples are stored frozen, mix thawed samples well before testing. Avoid repeated freezing and thawing. 3. Samples with TSH concentrations greater than 20 miu/l should be diluted with standard 0. Correct the result using an appropriate dilution factor. 11

13 8. Assay Method General Considerations Caution: The reagents contain Proclin 300 R as a preservative. Do not use heavily haemolysed samples Maximum precision is required for reconstitution and dispensation of the reagents. Avoid the exposure of TMB substrate to direct sunlight, metal or oxidants. The TMB Substrate contains an irritant, which may be harmful if inhaled, ingested or absorbed through the skin. To prevent injury, avoid inhalation, ingestion or contact with skin and eyes. The Stop Solution consists of a diluted sulphuric acid solution. Sulphuric acid is poisonous and corrosive and can be toxic if ingested. To prevent chemical burns, avoid contact with skin and eyes. Addition of the TMB Substrate solution initiates a kinetic reaction, which is terminated by the addition of the Stop Solution. Therefore, the TMB Substrate and the Stop Solution should be added in the same sequence to eliminate any time deviation during the reaction. 12

14 Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera. Microbiologically contaminated samples should not be used in the assay. Highly lipemeic or haemolysed specimens should also not be used. Plate readers measure vertically. Do not touch the bottom of the wells. This method allows the determination of TSH from 0.2 miu/l to 20.0 miu/l. Test Preparation Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described. It is recommended to use internal controls in every assay. Control results should be within established ranges. Prior to commencing the assay, the distribution and identification plan for all specimens, standards and controls should be carefully established on the result sheet supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than one plate is used, it is recommended to repeat the dose response curve. 13

15 Please allocate at least: 2 wells (e.g. A1+B1) for standard 0 2 wells (e.g. C1+D1) for standard 1 2 wells (eg. E1+F1) for standard 2 2 wells (eg. G1+H1) for standard 3 2 wells (eg. A2+B2) for standard 4 2 wells (eg. C2+D2) for standard 5 2 wells (eg. E2+F2) for standard 6 2 wells (e.g. G2+H2) for control 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. A clean, disposable tip should be used for dispensing each standard and sample. 14

16 Assay Procedure: 1. Pipette 50 μl standards, controls and samples into their respective wells. 2. Dispense 100 μl conjugate into the wells. 3. Cover wells with the foil supplied in the kit. 4. Incubate for 1 hour at room temperature (22-28 C) (to increase sensibility read the absorbance after 120 min of incubation). 5. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well six times with 300μl diluted Wash Solution. Avoid overflows from the reaction wells. The soak time between each wash cycle should be >5sec. 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 TMB Substrate Solution into all wells. 7. Cover wells with the foil supplied in the kit. 8. Incubate for exactly 20 min at room temperature (22-28 C) in the dark. 15

17 9. Dispense 100 μl Stop Solution into all wells in the same order and at the same rate as for the TMB Substrate Solution. Shake the plate gently to mix the solutions. Any blue colour developed during the incubation turns into yellow. 10. Measure the absorbance of the specimen at 450 nm within 30 min after addition of the Stop Solution. 9. Data Analysis A. Calculation of Results Calculate the mean 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.Cubic Spline, Four Parameter Logistic). Interpolate the values of the samples on the standard curve to obtain the corresponding values of the concentrations expressed in miu/l. 16

18 B. Reference Values Serum samples of apparently healthy women and men were assayed using the TSH ELISA, with the following results: Mean (miu/l) Range (miu/l) TSH C. Quality Control Each laboratory should assay controls at normal, high and low levels range of TSH for monitoring assay performance. These controls should be treated as unknowns and values determined in every test procedure performed. Quality control charts should be maintained to follow the performance of the supplied reagents. Pertinent statistical methods should be employed to ascertain trends. The individual laboratory should set acceptable assay performance limits. In addition, maximum absorbance should be consistent with past experience. Significant deviation from established performance can indicate unnoticed change in experimental conditions or degradation of kit reagents. Fresh reagents should be used to determine the reason for the variations. If computer controlled data reduction is used to calculate the results of the test, it is imperative that the predicted values for the calibrators fall within 10% of the assigned concentrations. 17

19 D. Precision Intra Assay Variation: Within run variation was determined by replicate determination (16x) of two different control sera in one assay. The within assay variability is 4.6 %. Inter Assay Variation: Between run variation was determined by replicate measurements (12x) of three different control sera in different lots. The between assay variability is 10.8 %. E. Sensitivity On the basis of results of 16 replicate determinations of standard 0, the minimum TSH concentration detectable by the present method is 0.01 miu/l with a confidence limit of 95%. 18

20 F. Correlation ab was compared with two other commercially available TSH assays. The total number of such specimens was 54. The two linear regression curves were calculated: Y 1 = 0.96x r 2 = Y 2 = 0.87x r 2 = G. Hook Effect ab shows no Hook Effect up to 500 miu/l. H. Recovery The recovery of miu/l of TSH added to samples gave an average value (±SD) of 85.85% ± 2.62% with reference to the original concentrations. 19

21 10. Specificity The cross-reactivity of the thyrotropin ELISA method to selected substances was evaluated by adding the interfering substance to a serum matrix at various concentrations. The cross-reactivity was calculated: Chemical % Reactivity Thyrotropin (htsh) 100 % Thyrotropin-alpa (htsh-α) 9.3 % Thyrotropin-beta (htsh-β) 152 % Follicle-stimulating hormone (hfsh) 1.0 % Luteinising Hormone (hlh) 1.0 % Chorionic Gonadotropin (hcg) <0.1 % 11. Limitations Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values. 20

22 12. Troubleshooting Problem Cause Solution Poor standard curve Low signal High background Improper standard dilution Standard improperly reconstituted (if applicable) Standard degraded Curve doesn't fit scale Incubation time too short 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 Wells are insufficiently washed Contaminated wash buffer Waiting too long to read plate after adding STOP solution Confirm dilutions made correctly Briefly spin vial before opening; thoroughly resuspend powder (if applicable) Store sample as recommended Try plotting using different scale Try overnight incubation at 4 C 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 Wash wells as per protocol recommendations Make fresh wash buffer Read plate immediately after adding STOP solution 21

23 Large CV Low sensitivity Bubbles in wells All wells not washed equally/thoroughly Incomplete reagent mixing Inconsistent pipetting Inconsistent sample preparation or storage Improper storage of ELISA kit Using incompatible sample type (e.g. Serum vs. cell extract) 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 and ensure accurate pipetting Ensure consistent sample preparation and optimal sample storage conditions (e.g. minimize freeze/thaws cycles) 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 or phone (select contact us on for the phone number for your region). 22

24 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中 國 聯 通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.