Thyroglobulin. Enzyme immunoassay for the quantitative determination of Thyroglobulin in human serum Only for in-vitro diagnostic use

Transcription

1 Thyroglobulin Enzyme immunoassay for the quantitative determination of Thyroglobulin in human serum Only for in-vitro diagnostic use Product Number: GWB-6B206E (96 Determinations)

2 1. INTRODUCTION Thyroglobulin (Tg), a glycoprotein with a molecular weight of about 660,000 Daltons, is the thyroid s main iodine protein and the most important compound of follicular colloid. Thyroglobulin is the form under which the active hormones T3 and T4 together with their immediate forerunners MIT and DIT are laid inside the thyroid gland. The clinical applications of the htg dosage seem to originate from its specificity for the thyroid and related cells. The dosage of htg can be used as support to scintigraphies or other techniques for studying pathogenesis, making a diagnosis and analyzing the course of thyroid disorders. The dosage of Tg before and after replacement treatment with L-Thyroxin cannot be established in cases of hypothyroidism due to thyroid agenesis. In cases of secondary hypothyroidism with a dysglandular goiter or ectopic thyroid, the levels of htg are normal or high. The circulating levels of htg tend to increase in several thyroid disorders such as toxic and atoxic goiter, subacute thyroiditis, Basedow s disease and carcinoma. In Basedow s disease the htg dosage is a potentially interesting index of normalization of hyperthyroidism in patients treated with anti-thyroid drugs. In the oncology field and more specifically for differentiated thyroid carcinoma, there are very promising applications linked to the ability of thyroid tumors tissues to concentrate iodine and synthesize htg as a normal thyroid. Basically the dosage of htg can be used as follows: a. Pre-operating diagnosis of thyroid tumors. This application does not allow the differentiated diagnosis of the tumor as the values of htg seen in malignant and benign nodules are superimposable. b. Post-operation monitoring In patients treated surgically or with radiotherapy, long lasting htg levels suggest the presence of a residual carcinoma and/or carcinoma with metastasis. c. Monitoring of totally thyroidectomized patients The use of circulating htg as an indicator of recurrent tumors (metastasis marker) has an established clinical value: the increase of Thyroglobulinaemia indicates the need to undergo further analysis for confirming the diagnosis. Interesting advantages can come from: a) a reduced use of scintigraphic diagnostic techniques as they imply regular suspension of replacement treatment and frequent exposure to radiation, b) and complete completion of the information obtained via scintigraphy. 2. INTENDED USE Immunoenzymatic colorimetric method (ELISA) for quantitative determination of Thyroglobulin in human serum. 3. PRINCIPLE OF THE ASSAY The essential reagents required for an immunoenzymometric assay include - an excess of high affinity and specificity antibodies (enzyme labeled and immobilized), with different and distinct epitope recognition - and native antigen (thyroglobulin). In this procedure, the microplate wells are coated with streptavidin. Mixing of monoclonal biotinylated antibodies with serum containing thyroglobulin (antigen) results in the formation of an antibody-antigen complex. The interaction is illustrated in the following equation: ka Ag(Tg) + BtAb(m) Ag(Tg)-BtAb(m) k-a BtAb(m) = biotinylated monoclonal antibody (excess quantity) Ag(Tg) = native antigen (variable quantity) Ag(Tg)-BtAb(m) = antigen-antibody complex (variable quantity) ka = rate constant of association k-a = rate constant of dissociation Simultaneously, the complex is deposited into the well through the high affinity reaction of streptavidin and biotinylated antibody. This interaction is illustrated below: Ag(Tg)-BtAb(m) + Streptavidin C.W. Immobilized complex Streptavidin C.W. = Streptavidin immobilized on well Immobilized complex = sandwich complex bound to the well 2

3 After an incubation period, the antibody-antigen bound fraction is separated from unbound antigen by a washing step. A second antibody (directed against a different epitope) labeled with an enzyme is added. This antibody binds to the already fixed complex. Excess enzyme is washed off via a wash step. A substrate is added resulting in the formation of a color that is measurable with a microplate spectrophotometer. The enzyme activity and therefore the color formation is directly proportional to the native antigen concentration. By utilizing several different serum references of known antigen concentration, a dose response curve can be generated from which the antigen concentration of an unknown can be ascertained. 4. MATERIALS 4.1. Reagents supplied Anti-Thyroglobulin Coated Wells: 12 breakapart 8-well snap-off strips coated streptavidin; in re-sealable aluminium foil. Stop Solution: 1 bottle containing 15 ml sulphuric acid, 0.15 mol/l (avoid any skin contact). Anti-Thyroglobulin - Biotin Conjugate: 1 bottle containing 13 ml of biotin labelled anti-thyroglobulin antibodies. Enzyme Reagent: 1 bottle containing 13 ml horseradish peroxidase labelled anti-thyroglobulin antibodies. TMB Substrate Solution: 1 bottle containing 12 ml 3, 3, 5, 5 -tetramethylbenzidine (H 2O 2-TMB 0.26 g/l) (avoid any skin contact). Wash Solution 50x conc.: 1 bottle containing 20 ml of a buffered solution. Thyroglobulin Standards: 6 bottles, 1 ml each of all standards. The standards have the following concentrations: Standard 0: Standard 1: Standard 2: Standard 3: Standard 4: Standard 5: 0 ng/ml 2 ng/ml 10 ng/ml 40 ng/ml 100 ng/ml 250 ng/ml 4.2. Materials supplied 1 Strip holder 1 Cover foils 1 Test protocol 1 Distribution and identification plan 4.3. Materials and Equipment needed 37 C incubator ELISA microwell plate reader, equipped for the measurement of absorbance at 405, 450 and 620 nm Manual or automatic equipment for rinsing wells Absorbent paper for blotting the microplate wells Pipettes to deliver 50 µl volumes with a precision better than 1,5% Dispenser for repetitive deliveries of 100 and 300 µl with a precision better than 1,5% Vortex tube mixer Distilled water Disposable tubes Timer 5. STABILITY AND STORAGE The reagents are stable up to the expiry date stated on the label when stored at C. Once opened the microplate and the standards are stable for another 2 months at 2 8 C. 6. REAGENT PREPARATION It is very important to bring all reagents, samples and standards to room temperature (22 28 C) for at least 30 min before starting the test run! Mix well prior to use! 6.1. 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; after first opening stable for another 2 months Anti-Thyroglobulin-Biotin Conjugate The bottle contains 13 ml of a ready to use solution with anti-thyroglobulin antibodies conjugated with biotin. 3

4 6.3. Enzyme Reagent The bottle contains 13 ml of a ready to use solution with Streptavidine conjugated with HRP Standards There is no known internationally accepted thyroglobulin standard available. The thyroglobulin used in the serum based standards is a highly purified human thyroglobulin preparation that is calibrated gravimetrically against the reference material obtained from the Community Bureau of Reference CRM 457. Once opened, the standards are stable for 2 months at 2 8 C TMB Substrate Solution The bottle contains 12 ml of a tetramethylbenzidine/hydrogen peroxide system. The reagent is ready to use and has to be stored at 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 Stop Solution The bottle contains a ready to use solution of 0.15 M sulphuric acid solution (R 36/38, S 26) Wash Solution Dilute the concentrated Wash Solution with distilled water to 1000 ml. For smaller volumes respect the 1:50 dilution ratio. Once diluted it is stable for 60 days at room temperature (22 28 C). 7. SPECIMEN COLLECTION AND PREPARATION The assay can be performed in serum samples. The usual precautions in the collection of venepuncture samples should be observed. For accurate comparison to establish normal values, a fasting morning serum sample should be obtained. The blood should be collected via a plain red-top venipuntcure tube without additives or gel barrier. Allow the blood to clot, centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2 8 C for a maximum period of 5 days. If the specimen cannot be assayed within this time, they may be stored at -20 C for up to 30 days. Avoid use of contaminated devices. Avoid repetitive freezing and thawing. When assayed in duplicate, 100 µl of the specimen is required. 8. ASSAY PROCEDURE 8.1. Test Preparation Please read the test protocol carefully before performing the assay. Result reliability depends on strict adherence to the test protocol as described. Prior to commencing the assay, the distribution and identification plan for all specimens, controls and standards 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. Please allocate at least: 1 well (e.g. A1) for blank 2 wells (e.g. B1+C1) for standard 0 2 wells (e.g. D1+E1) for standard 1 2 wells (e.g. F1+G1) for standard 2 2 wells (e.g. 1+A2) for standard 3 2 wells (e.g. B2+C2) for standard 4 2 wells (e.g. D2+E2) for standard 5 It is necessary to determine standards and patient samples in duplicate in order to improve the accuracy of the test results. 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 each patient sample. 1. Dispense 50 µl standards and samples into their respective wells. Leave well A1 for substrate blank. Add 100 µl Anti-Thyroglobulin-Biotin conjugate to each standard, control and sample. 2. Swirl the microplate gently for seconds to mix. 3. Cover wells with the foil supplied in the kit. 4. Incubate for 1.5 hour at 37 C. 5. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 350 µl diluted Wash Solution. Avoid overflows from the reaction wells. Important note: gently shake the plate for 5 seconds at each washing step to ensure proper cleaning. 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 Enzyme Reagent into all wells and cover them with the foil supplied in the kit. Do not shake the microplate after enzyme addition. 7. Incubate for 60 min at 37 C. 4

5 8. When incubation has been completed, remove the foil, aspirate the content of the wells and wash each well three times with 350 µl diluted Wash Solution. Avoid overflows from the reaction wells. Important note: gently shake the plate for 5 seconds at each washing step to ensure proper cleaning. At the end carefully remove remaining fluid by tapping strips on tissue paper prior to the next step! 9. Dispense 100 µl TMB Substrate Solution into all wells. Do not shake the microplate after enzyme addition. Cover wells with the foil supplied in the kit. 10. Incubate for exactly 15 min at room temperature (22 28 C) in the dark. 11. 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 microplate gently. Any blue colour developed during the incubation turns into yellow. 12. Measure the absorbance of the specimen at 450 nm using a reference wavelength at nm or against blank within 30 min after addition of the Stop Solution. 9. QUALITY CONTROL Each laboratory should assay controls at normal, high and low levels range of Cortisol 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. Other parameters that should be monitored include the 80, 50 and 20% intercepts of the standard curve for run-to-run reproducibility. 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. 10. RESULTS Calculation of Results A dose response curve is used to ascertain the concentration of human thyroglobulin (Tg) in unknown specimens. 1. Draw a standard curve on linear graph paper, by plotting the standard concentrations (x-axis) against the absorbance obtained for each standard (y-axis). Do not average the duplicates of the standards before plotting. 2. Draw the best fit curve through the plotted points. 3. To determine the concentration of Tg for an unknown locate the average absorbance of the duplicate for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration ( in ng/ml) from the horizontal axis of the graph (the duplicates of the unknown may be averaged as indicated). In the following example, the average absorbance (0.424) intersects the dose response curve at 25.2 ng/ml Tg concentration. Note: computer data reduction software designed for ELISA assays may also be used for the data reduction. If such software is utilized, the validation of the software should be ascertained Example of Calculation The values shown below must be considered as an example and must not be used in place of experimental data. Standard /Sample STD 0 STD 1 STD 2 STD 3 STD 4 STD 5 Sample OD 450 nm Mean OD Value Tg ng/ml

6 10.3. Quality Control Parameters In order for an assay to be considered valid, the following criteria must be met: 1. The absorbance (OD) of Standard 0 should be The absorbance (OD) of Standard 5 should be Respect the assigned ranges indicated on the certificate of analysis Reference Values Based on clinical data gathered in concordance with the published literature a normal range was established: Thyroglobulin (ng/ml) Normal (adult) ng/ml Thyroglobulin is found to be elevated in patients with thyroid follicular and papillary carcinoma, thyroid adenoma, subacute thyroiditis, Hashimoto s thyroiditis and graves disease. Low levels of Tg are an indication of thyrotoxicosis factita. Please pay attention to the fact that the determination of a range of expected values for a normal population in a given method is dependent on many factors, such as specificity and sensitivity of the method used and type of population under investigation. Therefore each laboratory should consider the range given by the manufacturer as a general indication and produce their own range of expected values based on the indigenous population where the laboratory works. 11. SPECIFIC PERFORMANCE CHARACTERISTICS 11.1 Specificity The cross-reactivity of the GenWsy Biotech, Inc. Thyroglobulin assay to selected substances was evaluated by adding the interfering substance to a serum matrix at the following concentrations. The cross-reactivity was calculated by deriving a ratio between dose of interfering substance to dose of Thyroglobulin needed to produce the same absorbance. Substance Conc. Cross-reactivity Thyroglobulin 100 ng/ml 100 % Triiodothyronine 1000 ng/dl ND Thyroxine 1000 ng/ml ND TBG 100 ng/ml ND Sensitivity The analytical sensitivity (detection limit) was calculated by determining the variability of standard 0 (0 ng/ml) and using 2 standard deviation (95% certainty) statistics to calculate the minimum dose. The assay sensitivity was found to be 0.44 ng/ml Precision The intra and inter assay precisions were determined by analyses on three different levels of pool control sera: Intra assay Serum Mean ng/ml SD % CV Replicates % % % 22 Inter assay Serum Mean ng/ml SD % CV Replicates % % % 10 6

7 11.4. Accuracy The GenWsy Biotech, Inc. Thyroglobulin ELISA assay was compared with a reference coated tube radioimmunoassay (IRMA). Biological specimens from population (symptomatic and asymptomatic) were used. The linear regression curve is: Y= *x (R 2 = 0.975) High Dose Effect Since the assay is sequential in design, high concentrations of Tg do not show the hook effect. Samples with concentration over ng/ml demonstrated extremely high levels of absorbance. 12. LIMITATIONS OF THE PROCEDURE Bacterial contamination or repeated freeze-thaw cycles of the specimen may affect the absorbance values. The results of the assay must be carefully interpreted and confirmed by clinical evaluations and further diagnostic tests. 13. PRECAUTIONS AND WARNINGS In compliance with article 1 paragraph 2b European directive 98/79/EC the use of the in vitro diagnostic medical devices is intended by the manufacturer to secure suitability, performances and safety of the product. Therefore the test procedure, the information, the precautions and warnings in the instructions for use have to be strictly followed. The use of the testkits with analyzers and similar equipment has to be validated. Any change in design, composition and test procedure as well as for any use in combination with other products not approved by the manufacturer is not authorized; the user himself is responsible for such changes. The manufacturer is not liable for false results and incidents for these reasons. The manufacturer is not liable for any results by visual analysis of the patient samples. Only for in-vitro diagnostic use. 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. Do not interchange reagents or strips of different production lots. No reagents of other manufacturers should be used along with reagents of this test kit. Do not use reagents after expiry date stated on the label. Use only clean pipette tips, dispensers, and lab ware. Do not interchange screw caps of reagent vials to avoid cross-contamination. 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. To avoid cross-contamination and falsely elevated results pipette patient samples and dispense conjugate without splashing accurately to the bottom of wells. Addition of 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 reaction. Observe the guidelines for performing quality control in medical laboratories by assaying controls and/ or pooled sera. Maximum precision is required for dispensation of the reagents. Avoid the exposure of TMB substrate to direct sunlight, metal or oxidants. Do not freeze the solution. 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. Observe the guidelines for performing quality control in medical laboratories by assaying controls and/or pooled sera. Some reagents contain small amounts of Proclin 300 as preservative. Avoid contact with skin or mucosa. Microbiologically contaminated samples should not be used in the assay. Highly lipemic or haemolysed specimens should also not be used. Plate readers measure vertically. Do not touch the bottom of the wells. It is important that the time of reaction in each well is held constant for reproducible results. Pipetting of samples should not extend beyond ten minutes to avoid assay drift. If more than 10 minutes are needed, follow the same order of dispensation. If more than one plate is used, it is recommended to repeat the dose response curve in each plate. The incomplete or inaccurate liquid removal from the wells could influence the assay precision and/or increase the background. The ELISA is only designed for qualified personnel who are familiar with good laboratory practice. 7

8 13.1. Disposal Considerations Residues of chemicals and preparations are generally considered as hazardous waste. The disposal of this kind of waste is regulated through national and regional laws and regulations. Contact your local authorities or waste management companies which will give advice on how to dispose hazardous waste. 14. LITERATURE 1. Beever K, Bradbury J, Phillips D, et al, Highly sensitive assays of autoantibodies to Thyroglobulin and Thyroid Peroxidase, Clin Chem, 35, (1989). 2. Ladenson PW, Optimal laboratory testing for diagnosis and monitoring of thyroid nodules, goiter, and thyroid cancer, Clin Chem, 42, (1996). 3. Mayo Medical Laboratories: test Catalog, Rochester, MN (1997). 4. Spencer CA, Takeucho M, Kazarosyn M, Current status and performance goals for serum thyroglobulin assays, Clin Chem, 42, (1996). 5. Tietz N. Ed: Clinical Guide to Laboratory Tests. 3rd Ed. Philadelphia. Saunders (1995). 6. Surks, MI, Chopra, IJ, Mariash, CN, American Thyroid Association guidelines for use of laboratory tests in thyroid disorders, JAMA, 263, (1990). 7. Ng, M., Rajna, A, Khalid, B, Enzyme immunoassay for simultaneous measurements of autoantibodies against thyroglobulin and thyroid microsomes in serum, Clin Chem, 33, (1987). 8. Spencer, CA, Takeucho M, Kazarosyn M, Wang CC, Guttler RB, Singer PA, et al, Serum thyroglobulin autoantibodies; prevalence, influence on serum thyroglobulin measurements, and prognostic significance in patients with differentiated thyroid carcinoma, J Clin Endocrinol Metab, 83, (1998). 9. Spencer CA, LoPresti JS, Fatemi S, Nicoloff JT, Detection of residual and recurrent differentiated thyroid carcinoma by serum thyroglobulin measurements, Thyroid, 9, (1999). 10. Schlumberger M, Baudin E, Serum thyroglobulin determinations in the follow up of patients with differentiated thyroid carcinoma, Eur J. Endocrinol, 138, (1998). 15. ORDERING INFORMATION Prod. No.: GWB-6B206E Thyroglobulin (96 Determinations) 8

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11 Symbols Key Manufactured by In Vitro Diagnostic Medical Device Lot Number Expiration Date Storage Temperature Keep away from sunlight CE Mark Catalogue Number Consult Instructions for Use Microplate Enzyme Reagent Biotin Conjugate CALl Calibrator resp. Standard Stop solution TMB Substrate solution Washing solution 50x concentrated Contains sufficient for n tests 11

12 SCHEME OF THE ASSAY Thyroglobulin Test Preparation Prepare reagents and samples as described. Establish the distribution and identification plan for all specimens and controls on the result sheet supplied in the kit. Select the required number of microtiter strips or wells and insert them into the holder. Assay Procedure blank Standard 0-5 Sample Standard µl - Sample µl Biotin-Conjugate Enzyme Reagent TMB Subst µl 100 µl Swirl the microplate gently for seconds to mix. Cover wells with foil supplied in the kit. Incubate for 1.5 hours at 37 C. Wash each well three times with 350 µl diluted Wash Solution. Important note: gently shake the plate for 5 sec. at each washing step µl 100 µl Do not shake the microplate after enzyme addition. Cover wells with foil supplied in the kit. Incubate for 60 min at 37 C. Wash each well three times with 350 µl diluted Wash Solution. Important note: gently shake the plate for 5 sec. at each washing step. 100 µl 100 µl 100 µl Cover wells with foil supplied in the kit. Incubate for exactly 15 min at room temperature (22 28 C). Stop Solution 100 µl 100 µl 100 µl Shake the microplate gently Photometric measurement at 450 nm within 30 min 12