TSH Receptor Autoantibody Coated Tube Kit from RSR ...

1 RiaRSR TRAb CT SYMBOLS. TSH Receptor Autoantibody Coated Symbol Meaning Tube Kit Instructions for use EC Declaration of Conformity RSR Limited Avenue Park Pentwyn Cardiff IVD In Vitro Diagnostic Device CF23 8HE United Kingdom Tel.: +44 29 2073 2076 Fax: +44 29 2073 2704 REF Catalogue Number Email: Website: LOT Lot Number INTENDED USE. The RSR TSH Receptor Autoantibody (TRAb) Coated Consult Instructions Tube (CT) kit is intended for use by professional persons only for the quantitative determination of Manufactured by thyrotropin Receptor autoantibodies in human serum. Hyperthyroidism in Graves' disease is due to the Sufficient for presence of autoantibodies to the TSH Receptor and measurement of these autoantibodies can be useful in Expiry Date disease diagnosis and management. REFERENCES Store J. Sanders et al The Interaction of TSH Receptor Autoantibodies Negative Control with 125I-Labelled TSH Receptor J Clin Endocrinol Metab 1999 84: 3797-3802 Positive Control PATENTS MATERIALS REQUIRED AND NOT SUPPLIED.

2 European patent 1021721 B1, US patent 6,844,162 tubes for total counts B1 and Japanese patent 4331403 apply. Suitable rack for assay tubes ASSAY PRINCIPLE Pipettes capable of dispensing 50 L, 100 L and In RSR's TRAb CT kit TSH Receptor autoantibodies in 1 mL. patient sera, calibrators and controls are allowed to Means of diluting wash buffer interact with TSH Receptor Coated onto plastic tubes . Pure water After a 2 hour incubation, the samples are discarded Orbital shaker Gamma counter leaving TRAb bound to the immobilised TSH Receptor . Porcine TSH labelled with 125I is added in a 2nd MATERIALS SUPPLIED IN 60 and 100 TUBE. incubation step, where it interacts with TSH receptors KITS. which have not been blocked by bound TRAb. Any MATERIAL 60 Tube 100 Tube unbound 125I-labelled TSH is then removed from the 1 x mL 1 x 11 mL. 125. tubes by a wash step prior to counting on a gamma I-labelled TSH 100kBq/bottle 180kBq/bottle (at manufacture) (at manufacture).

3 Counter. A lower level of radioactivity bound indicates the presence of TRAb in the test sample. The TSH Receptor Coated 3 x 20 tubes 5 x 20 tubes measuring range is 1 40 u/L (NIBSC 08/204). tubes Start Buffer 1 x 10 mL 1 x 10 mL. STORAGE AND PREPARATION OF TEST SERUM Calibrators 4 x mL 4 x mL. SAMPLES Negative Control 1 x mL 1 x mL. Sera to be analysed should be assayed soon after Positive Control 1 x mL 1 x mL. separation or stored, preferably in aliquots, at or Concentrated Wash 1 x 50 mL 1 x 50 mL. below 20oC. mL is sufficient for one assay Solution (duplicate 100 L determinations are recommended). PREPARATION OF REAGENTS SUPPLIED. Repeated freeze thawing or increases in storage Store kits and all kit components (A-G) at 2 8oC. temperature must be avoided. Incorrect storage of TSH Receptor Coated tubes serum samples can lead to loss of TRAb activity. Do 20 tubes sealed in each foil bag. (A small not use lipaemic or haemolysed serum samples.)

4 Do amount of white residue may be present in not use plasma in the assay. When required, thaw test the tubes but this does not affect assay sera at room temperature (20 25 C) and mix gently performance.). to ensure homogeneity. Centrifuge the serum prior to A. After opening return any unused tubes to assay (preferably for 5 minutes at 10 15,000g in a the original foil packet and seal. Then microfuge) to remove any particulate matter. place foil bag in the self-seal plastic bag, with desiccant provided, and store at 2- 8oC for up to 2 weeks. 18th April 2016 Page 1 of 3 RSR/09c Rev 14. Start Buffer against the % binding on the y-axis (linear scale). 10 mL Alternatively, % Inhibition (I) can be calculated using B. Coloured yellow the formula;. Ready for use B . %I 100 x 1 - . Negative Control Bo . C mL Other data reduction methods can be used. Samples Ready for use with high TRAb concentrations can be diluted in kit Calibrators negative control (C).

5 For example 20 L of sample 1, 2, 8, and 40 units/L plus 180 L of negative control to give a 10x dilution. D1-4 (units are NIBSC 08/204) Other dilutions ( 100x) can be prepared from a 4 x mL 10x dilution or otherwise as appropriate. Some sera Ready for use will not dilute in a linear way and we suggest that the Positive Control (See label for range) dilution giving a value closest to 50% inhibition is E mL used for calculation of TRAb concentration. The Ready for use negative control has a concentration of 0 units/L, but 125. I-Labelled TSH can be assigned a value of units/L to facilitate F Coloured red computer processing of data. Ready for use Concentrated Wash Solution TYPICAL RESULTS (example only, not for calculation 50 mL of actual results). G %B %I units/L. Dilute to 500 mL with pure water before use. Store at 2 8oC up to kit expiry. Total Counts 84,530. Control C 0. ASSAY PROCEDURE D1 14 1. Allow all reagents to stand at room temperature (20 D2 24 2.)

6 25oC) for at least 30 minutes before use. A repeating D3 64 8. type Eppendorf pipette is recommended for steps 1, D4 90 40. 4, 5, 6 and 8. Control E 43 1. Pipette 50 L of start buffer (B) into each tube (A) to be used. 2. Pipette 100 L of negative control (C), 100. calibrators (D1-4), positive control (E) and patients' sera into each tube. 75. 3. Cover the tubes and incubate for 2 hours at room temperature on an orbital shaker % Inhibition (200 shakes per min.). 50. 4. Pipette 1 mL of diluted wash solution (G). into each tube and aspirate or decant each 25. tube to waste. If decanting, allow the tubes to drain onto a clean, dry, absorbent 0. surface for 2 minutes. 1 10 100. 5. Repeat wash step 4. Concentration (u/L). 6. Pipette 100 L of 125I-labelled TSH (F) into each tube and into two empty tubes for ASSAY CUT OFF. total counts. units/L. 7. Cover the tubes and incubate for 1 hour at Negative 1. room temperature on an orbital shaker Borderline Positive > (200 shakes per min.

7 Positive > 8. Repeat wash step 4 twice (except tubes This cut off has been validated at RSR. However each for total counts). If decanting, allow the laboratory should establish its own normal and tubes to drain as above for 5 minutes. 9. Count each tube including total count pathological reference ranges for TRAb levels. Also it tubes for 125I for 1 minute. is recommended that each laboratory include its own panel of control samples in the assay. RESULT ANALYSIS. Express the counts bound to each tube (B) as a CLINICAL EVALUATION. percentage of the counts bound in the presence of the Clinical Specificity negative control (Bo) giving the % binding. A 242 samples from healthy blood donors were assayed calibration curve can be established by plotting in the TRAb CT kit. 242 (100%) were identified as being negative for TSH Receptor autoantibodies. calibrator concentration on the x-axis (log scale). 18th April 2016 Page 2 of 3 RSR/09c Rev 14.

8 Clinical Sensitivity SAFETY CONSIDERATIONS. 50 samples from patients diagnosed with Graves' Follow the instructions carefully. Observe expiry dates disease were assayed using the TRAb CT kit. 46 stated on the labels and the specified shelf life for (92%) were identified as being positive for TSH diluted reagents. Refer to Safety Data Sheet for more Receptor autoantibodies. detailed safety information. The kit contains Lower Detection Limit radioactive material. Users should make themselves The kit negative control was assayed 20 times and aware of and observe any national and local legislation the mean and standard deviation calculated. The and codes of practice governing the use, storage, lower detection limit at 2 standard deviations was transportation and disposal of radioactive materials. units/L. Avoid all actions likely to lead to ingestion. Avoid Inter Assay Precision (n=20) contact with skin and clothing. Wear protective Sample units/L CV (%) %I CV (%) clothing and where appropriate personal dosimeters.

9 1 17 12 13 Radioactive materials should only be used by 2 13 27 authorised personnel and in designated areas. Wash 3 57 hands thoroughly after handling. Monitor hands and 4 13 15 74 clothing before leaving the designated area. Materials 5 24 16 84 of human origin used in the preparation of the kit have Intra Assay Precision (n=25) been tested and found non reactive for HIV1 and 2, Sample units/L CV (%) %I CV (%) HCV antibodies and HbsAg but should none the less 6 21 be handled as potentially infectious. Wash hands 7 45 thoroughly if contamination has occurred and before Clinical Accuracy leaving the laboratory. Sterilise all potentially Analysis of sera from patients with autoimmune contaminated waste, including test specimens before diseases other than Graves' disease indicated no disposal. Material of animal origin used in the interference from autoantibodies to thyroglobulin; preparation of the kit has been obtained from animals thyroid peroxidase; glutamic acid decarboxylase; 21- certified as healthy.

10 These materials should be hydroxylase; acetylcholine Receptor ; dsDNA or from handled as potentially infectious. Some components rheumatoid factor. contain small quantities of sodium azide as preservative. With all kit components, avoid ingestion, Interference inhalation, injection and contact with skin, eyes and No interference was observed when samples were clothing. Avoid formation of heavy metal azides in the spiked with the following materials; haemoglobin up drainage system by flushing any kit component away to mg/mL; bilirubin up to mg/mL; Intralipid with copious amounts of water. up to 1000 mg/dL; human LH up to 10 u/mL; hCG up to 160 u/mL; human FSH up to 15 u/mL and human TSH up to u/L. ASSAY PLAN. Allow all reagents and samples to reach room temperature (20 25 oC) before use Pipette: 50 L start buffer Pipette: 100 L negative control, calibrators, positive control, patients' sera Incubate 2 hours at room temperature on an orbital shaker at 200 shakes/min Pipette: 1 mL wash solution Aspirate/Decant: tubes Pipette: 1 mL wash solution Aspirate/Decant: tubes (Drain for 2 minutes).