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Thyretain TSI Reporter BioAssay

2-Replicate Samples IFU


The synthesis and secretion of thyroid hormone by the thyroid gland is controlled by thyroid stimulating hormone (TSH), also called thyrotropin. TSH, secreted by the anterior pituitary, binds to the thyroid stimulating hormone receptors (TSHR), or thyrotropin receptors (TR), on the cells of the thyroid gland stimulating the synthesis and secretion of thyroid hormones.
Hyperthyroidism is characterized by an excessive synthesis and secretion of thyroid hormones. In the majority of cases this overproduction of hormones is caused by a class of autoantibodies to TSHR which mimic the action of TSH. These stimulating autoantibodies are referred to as thyroid stimulating immunoglobulins (TSI) and, when present, are indicative of GD.
In a normal functioning system, homeostasis is maintained by the hypothalamic-pituitary-thyroid axis. The hypothalamus senses low circulating levels of thyroid hormone and responds by releasing thyrotropin releasing hormone (TRH). The TRH stimulates the pituitary to produce and secrete TSH. The TSH, in turn, stimulates the thyroid to produce and release thyroid hormone until levels in the blood return to normal. Thyroid hormone exerts negative feed back control over the hypothalamus as well as the anterior pituitary thus controlling the release of both TRH from hypothalamus and TSH from anterior pituitary gland.

The two major hormones produced by the thyroid are thyroxine (T4) and triiodothyronine (T3). T3 is formed through deiodination of T4 and is the most active of the thyroid hormones, regulating most bodily processes. The TSI present in patients with GD mimic TSH causing an over-production of both hormones, leading to hyperthyroidism.

GD, one of the most common forms of hyperthyroidism, has an incidence of approximately 5 in 10,000 people per year, affecting 13 million, and targets women seven times as often as men.1 Although there is currently no cure for GD, it is treatable by anti-thyroid drug therapies, radioactive iodine ablation or surgical removal of the thyroid gland, as cited by American Association of Clinical Endocrinologist guidelines.2 Though the presence of TSI in serum of patients known to have GD is significant to the disease, the direct screening for this autoantibody has not been used as a primary tool in its diagnosis. The diagnosis of GD is typically derived from a panel of diagnostic tests, which includes the measurement of serum levels of TSH, T3, T4, and thyroid receptor antibodies (TRAb). There are two types of TRAb, however, TSI and Thyroid Blocking Immunoglobulins (TBI). The TBI binds to the TSHR and prevents or inhibits the stimulation and secretion of thyroid hormones by TSH, leading to hypofunctioning of the thyroid or hypothyroidism. The measurement of serum TRAb is flawed by its inability to distinguish TSI from TBI.
Thyretain™ TSI Reporter BioAssay (TSI Reporter) is a cell-based assay (or “bioassay”) which utilizes a genetically engineered cell line capable of specifically detecting serum TSI.


The Thyretain™ TSI Reporter BioAssay (TSI Reporter) utilizes a patented bioassay technology to detect TSI in human serum. Genetically engineered Chinese hamster ovary (CHO) cells, expressing a chimeric form3 of the human TSHR and a cyclic adenosine monophosphate (cAMP) induced luciferase reporter gene, are cryogenically preserved and provided in measured aliquots. The cells are seeded and grown for 15 to 18 hours to a confluent monolayer in a 96-well plate.
Patient sera, reference control, positive and normal controls are diluted with a proprietary Reaction Buffer, added to the cell monolayers and allowed to react with the cells for 3 hours. During this induction period, TSI, if present in the patient serum, bind to the chimeric human TSHR on the cell surface. This binding event induces a signaling cascade resulting in increased production of intra-cellular cAMP. This increased production of cAMP is evidenced by increased production of luciferase. At the conclusion of the 3 hour induction period the cells are lysed. Luciferase levels are then measured using a luminometer. A significant increase in luminescence over the Reference Control indicates the presence of TSI antibodies in the sample.


  1. Thyretain™ TSI Reporter BioAssay Kit Components

  1. CHO Mc4 FreshFrozenCells®: Cryovials containing CHO Mc4 cells cryogenically preserved in cryoprotective medium containing DMSO. Reagent is stored at -70oC or lower.

  2. Cell Attachment Solution, 200-mL: A proprietary reagent that promotes rapid cell attachment is used to treat the wells of a 96-well plate prior to planting the cells.. Reagent is stored at 2 to 30C.

  3. Growth Medium, 200-mL: Hamm’s F-12 cell culture medium containing 10% FBS. Reagent is stored at 2o to 8oC.

  4. Reaction Buffer, 500-mL: A proprietary buffer that augments the reaction of TSI with the TSHR. Reagent is stored at 2o to 8oC.

  5. Control Set:

    1. Positive Control, 0.5-mL: TSI-containing human serum which yields a value that is ≥140% of the Reference Control. Reagent is stored at -70°C or lower.

    2. Reference Control, 0.5-mL: A bTSH-containing solution against which controls and test specimens are compared. Reagent is stored at -70°C or lower.

    3. Normal Control, 0.5-mL: Human serum that is negative for the presence of TSI which yields a value that is <140% of the Reference Control. Reagent is stored at -70°C or lower.

  6. Luciferase Assay Reagent Set:

    1. Luciferase Substrate, 1 vial: A lyophylized beetle luciferin substrate which is converted by luciferase to oxyluciferin and light. Reagent is stored at -20°C or lower.

    2. Luciferase Assay Buffer Solution, 1 vial, 10-mL: A cell culture lysis buffer.

B. Reagent Storage Instructions

Table 1: Reagent Storage Conditions

    1. CHO Mc4 FreshFrozenCells®

Store at -70°C or lower

    1. Control Set (Positive, Reference, and Normal)

    1. Cell Attachment Solution

NOTE: The solution may show precipitation if stored at refrigerated temperatures. The material may be dissolved when the solution is warmed in a 35° to 37°C water bath. It is recommended that this solution be stored at room temperature to avoid precipitation.

Store at 2° to 30°C.

    1. Growth Medium

Store at 2° to 8°C.

    1. Reaction Buffer

    1. Luciferase Assay Reagent Set

Store at -20°C or lower.

C. Stability

Reagents and components will retain their full potency through the expiration date shown on the label of each bottle when stored at recommended temperatures.


For in vitro diagnostic use

  1. This Kit contains materials of human (e.g., human serum) and bovine (e.g., bTSH) origin. All bovine materials have been certified to be of United States origin. All human serum controls have been tested for HBsAg, HIV-1, -2 and HCV antibodies and found to be negative. Despite this screening, all human serum controls and patient samples should be considered potentially hazardous and handled with extreme care.

  2. No known test method can offer complete assurance that infectious agents are absent; therefore, all human blood derivatives, reagents and human specimens should be handled as if capable of transmitting infectious disease. It is recommended that reagents and human specimens should be handled in accordance with the OSHA Standard on Bloodborne Pathogens.4

    1. Cell cultures may have some potential to be hazardous. Personnel working with these cultures must be properly trained in safe handling techniques5 and have experience with tissue culture before attempting this procedure.

    2. All procedures must be conducted in accordance with the CDC 5th edition Biosafety in Microbiological and Biomedical Laboratories, 2007, and CLSI Approved Guideline M29-A, Protection of Laboratory Workers from Instrument Biohazards and Infectious Disease Transmitted by Blood, Body Fluids, and Tissue.

  3. All specimens and materials used to process them should be considered potentially infectious and handled in a manner which prevents infection of laboratory personnel.

    1. Biosafety Level 2 or other appropriate biosafety practices should be used when handling these materials.

    2. Decontamination of specimens and cultures is most effectively accomplished using a 1:10 dilution of household bleach.

    3. Although Control reagents have been shown to be non-infectious (Positive and Normal Controls) and of United States origin (Reference Control), the same precautions taken in handling and disposing of other infectious materials should be employed in their use.

  4. Never pipette reagents or clinical samples by mouth; avoid all contact of clinical samples with broken skin.

  5. Avoid splashing and the generation of aerosols with clinical samples.

  6. Use aseptic technique and sterile equipment and materials for all cell culture procedures.

  7. The CHO Mc4 FreshFrozenCells® must be properly stored (-70o C or below) at all times to maintain optimum performance. The swift transfer of freezer vials to and from the freezer or liquid nitrogen storage is mandatory. Repeated exposure to temperature fluctuations may affect cell viability and/or assay performance.

  8. The CHO Mc4 FreshFrozenCells® are single-use only and cannot be re-frozen once thawed.

  9. Extreme care should be taken to ensure that the level of CO2 in the incubator is accurately calibrated to 5%. Prolonged exposure to excessively high (>5.5%) or low (<4.5%) CO2 conditions could affect assay performance.

  10. The handling and preparation of the CHO Mc4 FreshFrozenCells® and cell culture reagents must be performed using aseptic technique, unless otherwise noted.

  11. All reagents should be pre-warmed to ambient temperature before use. This includes Growth Medium, Reaction Buffer, and Luciferase Assay Reagent Set.

  12. The TSI Reporter Controls are supplied at working strength. Any dilution of these reagents will decrease sensitivity.

  13. Reagents should be used prior to their expiration date.

  14. Each multi-well plate should be used only once. Do not re-use previously assayed plate.

  15. Microbial contamination of the CHO Mc4 FreshFrozenCells® and cell culture reagents may cause a decrease in sensitivity.

  16. Use of other reagents than those specified with the components of this Kit, especially those that contain sodium azide, may lead to erroneous results.

  17. The Growth Medium contains the pH indicator phenol red. Repeated exposure of the medium to the air may cause an increase in pH, evidenced by an increasingly deep red color. Limit the exposure of the medium to air as pH levels above 7.9 may affect the assay performance.


Proper collection and handling of the patient specimen are the most important factors in successful TSI detection. Specimen collection and processing should be attempted only by personnel trained in such procedures. Care should be taken during all specimen collection and handling to avoid generation of aerosols.
Serum is required for the TSI Reporter. For additional specimen collection and processing recommendations please refer to CLSI Document H3-A66.

A. Specimen Transport and Storage

Serum specimens should be transported to the laboratory at 2° to 8°C using cold packs, wet ice, foam refrigerant, or other coolants. The specimen should be processed and tested as soon as possible or stored for up to 72 hours at 2° to 8°C before testing. If testing does not occur before 72 hours the specimen may be aliquoted and frozen at -20o C for up to 2 months. Extended storage, beyond 2 months, should occur at temperatures that are -70°C or lower or in a liquid nitrogen Dewar.

  • Repeated freezing and thawing of serum samples should be avoided since this may affect specimen biological activity, leading to erroneous results.

  • Samples may go through a maximum of three freeze/thaw cycles.

  • Samples collected for retrospective analysis should be aliquoted upon receipt and immediately frozen.

All potentially infectious agents should be transported according to International Air Transport Association (IATA), International Civil Aviation Organization, (ICAO), Titles 42 and 49 of the US Code of Federal Regulations, or other regulatory requirements, as may be applicable.


A. Materials Provided

  1. CHO Mc4 FreshFrozenCells®, 1.0-mL each.

  2. Cell Attachment Solution (CAS), 200-mL each.

  3. Growth Medium (GM), 200-mL each.

  4. Reaction Buffer (RB), 500-mL each.

  5. Control Set:

  1. Positive Control, 0.5-mL each.

  2. Reference Control, 0.5-mL each.

  3. Normal Control, 0.5-mL each.

  1. Luciferase Assay Reagent Set

    1. Luciferase Substrate, 1 vial.

    2. Luciferase Assay Buffer Solution, 1 vial, 10-mL.

B. Materials Required but Not Provided

  1. -70°C or lower freezer or liquid nitrogen Dewar

NOTE: A chest freezer is preferred over an upright freezer. Assay failure is a potential issue due to repeated exposure of the cells to temperature changes over time when an upright freezer is used.

  1. Humidified, 5% CO2, 35°C to 37°C Incubator

  2. Bio-safety Cabinet Class II

  3. Luminometer capable of reading a 96 multi-well plate (MWP)

  4. Luminometer calibrator plate

  5. Microscope, inverted

  6. Calibrated Pipettes

    1. Multi-Channel 20 to 200-L

    2. Single 20 to 200-L

    3. Single 100 to 1000-L

    4. Sterile Pipette Tips

    5. 5, 10, 25-mL Sterile Serological Pipettes

  1. Sterile Transfer Pipette

  2. Pipetman® variable volume pipetters

  3. 96 MWP, Black, Clear Bottom (Costar #3603)

  4. Sterile Reagent Reservoirs

  5. Sterile Absorbent Pad

  6. Water bath, 35C to 37°C

  7. Sterile screw-cap tubes (15- or 50-mL)

  8. 13-mm Test Tubes

  9. Vortex Mixer

  10. Timer

  11. Household Bleach

C. Preliminary Comments and Precautions

  1. Adhere to the recommended volumes and times in the following procedure to ensure that accurate results are obtained.

  2. When medium is decanted from the cells, it is important that all medium is removed. Decant medium with enough force to completely remove it from each well. Visually examine the wells for medium removal before proceeding.

NOTE: It is imperative that all medium be removed from the wells before moving to the next step in the procedure.


  1. To prevent aspiration of water from the water bath into the vial, do not allow the water bath level to reach the junction of the vial/cap.

  2. Aseptic technique must be used throughout the first 15 to18 hours of the procedure. Microbial contamination increases the risk of assay failure.

  3. Do not allow the monolayers to dry between steps; this can be avoided by handling one plate at a time.

  4. Uniform heating of the cells is a requirement. The plates are to be placed side-by-side in the incubator rather than stacked. Stacking the plates will cause poor assay performance and greatly increase the risk for both inter- and intra-plate variation as well as assay failure.

  5. The plates must be carefully handled in order to avoid uneven distribution of the cells. Use of non-vibrating surfaces is a necessity to ensure uniform distribution of the cells in the wells.

  6. A confluent monolayer is one where cells are in contact with each other forming a continuous sheet of adherent cells on the bottom of the plate well. The confluency of the monolayer is assessed prior to use with a microscope at 100X magnification.


  1. It is good practice to examine the results of the Positive and Normal Controls before examining the test results of the specimens. However different lots might have a different positive control range. Please check the positive control reference range label for the test range before the test. If one or both of the controls fail to perform as expected, review the steps and conditions under which the test was performed to determine the cause(s). Do not report results until controls perform as expected.

D. TSI Reporter Procedure

Day 1: Carry out aseptically

  1. Calculate the number of plates needed to perform the assay – each plate can support 21 specimens tested along with the Positive, Reference and Normal Controls. All testing is performed in duplicate.

  2. Aliquot 5-mL per plate of Growth Medium (GM) into a suitable, sterile container (i.e., 50-mL centrifuge tube).

  3. Place aliquoted GM into a 35° to 37°C water bath 5 minutes prior to use.

  4. Add 100-L of Cell Attachment Solution (CAS) using a multi-channel pipette to each of the 48 inner wells of each plate, and treat for 1 to 10 minutes at 20° to 25°C.

  5. Decant the CAS from the wells onto a sterile absorbent pad inside the biological hood.

  6. Thaw one vial of CHO Mc4 FreshFrozenCells® per plate in a 35° to 37°C water bath for 2 to 4 minutes.

  7. Transfer the entire volume of thawed cells to the pre-warmed GM using a sterile transfer pipette. Rinse transfer pipette into the GM by aspirating and expelling 3 times.

  8. Close the tube and mix the cell suspension by inverting several times.

  9. Pour the suspension to a sterile reagent reservoir.

  10. Add 100-L of the cell suspension to each of the inner 48 wells of each plate using a 100-L multi-channel pipette. It is necessary to mix the suspension in the reagent reservoir by pipetting up and down frequently to ensure the cells stay in suspension and are uniformly distributed among the wells.

  11. Incubate all seeded plates for 15 to 18 hours in a humidified, 5% CO2, 35 to 37C incubator. In order to avoid microbial contamination, handle the plates so that the lid is not opened outside of the Bio-safety Cabinet.

Day 2: Steps 1-16 can be carried out on the benchtop

  1. Place Reaction Buffer (RB) in a 35° to 37°C water bath 5 minutes prior to use.

  2. Remove the plate(s) from the incubator.

  3. Examine the monolayers in each well microscopically using a magnification of 100X to 200X.

    1. Plates exhibiting signs of microbial contamination should be discarded.

    2. Monolayers must be confluent to be used in the assay. Mark any wells for disqualification based on sub-confluence.

NOTE: Plates can remain in the incubator for up to 18-hours or until confluency is reached (a minimum of 15-hours). Plates that fail to reach confluency should be disqualified from use.

    1. Individual wells containing piled or layered cells should be disqualified from use based on over-confluence.

  1. Return plate(s) to the incubator following examination.

  2. Calculate the amounts of serum specimen, control and Reaction Buffer (RB) needed to perform the assay as follows:

    1. Prepare a 1:11 dilution of each specimen as follows:

  1. Label a 13-mm test tube with patient identifier.

  2. Add 400-L of RB to each tube.

  3. Thaw frozen specimens in a 35° to 37°C water bath for 7 to 10 minutes.

  4. Vortex vigorously for 15 seconds.

  5. Add 40-L of each serum to the RB in the appropriately labeled tube.

  6. Vortex vigorously for 15 seconds.

    1. Prepare a 1:11 dilution of each control and reference as follows (multiply the volumes listed in Steps 5.b.iii and 5.b.v (below) by the number of plates tested to prepare final volume needed for the run):

  1. Thaw the appropriate volume of Controls and Reference in a 35° to 37°C water bath for 7 to 10 minutes.

  2. Label a 13-mm test tube with control identifier.

  3. Add 400-L of RB to each tube.

  4. Vortex controls and reference vigorously for 15 seconds.

  5. Add 40-L of each serum to the RB in the appropriately labeled tube.

  6. Vortex vigorously for 15 seconds.

The following Steps 6 through 16 are to be performed one plate at a time. They should also be performed sequentially in order to avoid drying of the cell monolayer.

  1. Remove the plate(s) from the incubator.

  2. Decant the GM from the plate into an appropriate waste container.

  3. Rinse the cells by adding 100-L of pre-warmed RB to each well using a multi-channel pipette. The medium should be gently dispensed down the side of the well in order to avoid disruption of the monolayer.

  4. Decant the RB into an appropriate waste container.

  5. Add 100-L of pre-warmed RB to each well using a multi-channel pipette. The medium should be gently dispensed down the side of the well in order to avoid disruption of the monolayer.

  6. Add 100-L of the diluted controls, reference and specimens (prepared above) to the appropriate RB containing wells (in duplicate) using a 20 to 200-L pipette.

  7. Incubate each plate for 3 hours in a humidified, 5% CO2, 35 to 37C incubator.

  8. Remove an appropriate number of Luciferase Assay Reagent Set kits from the freezer approximately 30 minutes prior to use. Two kits provide enough substrate for 5 plates.

    1. Thaw the Luciferase Assay Buffer Solution (buffer) in a 35° to 37°C water bath for 15 minutes. Store at 20° to 25°C until used.

    2. Store the Luciferase Substrate at 20° to 25°C until used.

  9. Pour the buffer (white bottle) into the lyophilized Luciferase Substrate (amber bottle). Replace the cap, and invert gently 6 times to mix.

  10. Transfer the substrate solution to a reagent reservoir and cover to keep the substrate solution in the dark. Minimize exposure of the substrate solution to light as this may cause a decrease in activity.

  11. Process each plate separately to completion as follows:

NOTE: The lysis process for the next plate should not be carried out until the previous plate is completed and enough time has elapsed to allow for the previous plate to be analyzed in the luminometer.

    1. Decant the contents of the plate into an appropriate waste container.

    2. Remove the remaining volume of medium from the wells by blotting the plate upside down on an absorbent pad.

    3. Add 75-L of Luciferase Substrate to each well using a multi-channel pipette.

    4. Cover the MWP and allow to stand at 20° to 25°C for 10 minutes.

NOTE: It is critical to maintain the cell lysis temperature above 20°C. The test result will be affected if the lysis temperature falls to 19°C or less.

    1. Read the plate in a luminometer that is programmed to read the inner 48 wells of the plate at an integration time of 1 second per well.

E. Quality Control

  1. Positive and Normal Controls should be run and calculated with each plate of specimens to confirm the assay performance.

  2. A reference range is provided with each Control Set which establishes the maximum and minimum acceptable values for the Positive Controls when it is compared to the Reference Control. The positive control range may change with each lot of the positive control. Please check the value for the positive control on the positive control reference range label prior to evaluating control validity.

  3. If assay controls fail to perform correctly (i.e., above or below established range), results for that plate are considered invalid. Contact Diagnostic Hybrids, Inc. Technical Support if an assay run is invalid.


A. Calculation of Results

  1. The average Relative Light Unit (RLU) of each specimen is calculated using the values from the duplicate wells.

Average RLU = (Well 1 RLU + Well 2 RLU) / 2


































































































(19228+21009)/2 = 20119

(8478+8696)/2 = 8587

  1. Calculate the coefficient of variation (CV) % for each test specimen and control using the following equation:

CV% = ([StDev Test Specimen RLU] / [Average Test Specimen RLU])*100%

Example: (1259/20119)*100% = 6.3%

  1. Calculate the Sample to Reference Ratio (SRR) % using the following equation:

SRR% = ([Average Test RLU] / [Average Reference Control RLU])*100%

Example: (20119/8587)*100% = 234%

B. Interpretation of Results (See Decision Matrix below)

  1. A specimen must have a CV of less than 15% between the duplicate RLU values to be considered valid. All specimens with an invalid (Null) result must be repeated.

  2. A positive result is one in which the Specimen – Reference Ratio (SRR%) is greater than or equal to (≥) 140% of the Reference Control.

  3. A negative result is one in which the SRR% that is less than (<) 140% of the Reference Control.


  1. This assay requires serum samples only. Use of plasma or whole blood may result in assay failure.

  2. Serum must be free of particulate matter before analysis can commence. The presence of particulate matter may affect the sensitivity of the assay.

  3. The testing of serum that is visibly icteric, hemolytic or lipemic may lead to decreased sensitivity in the detection of TSI.

  4. Incubation times or temperatures other than those cited in the test instructions may give erroneous results.

  5. Detection of TSI can vary depending upon the specimen quality and subsequent handling. A negative result does not exclude the possibility of the presence of TSI. Results of the test should be interpreted in conjunction with information available from other clinical information, such as physical symptoms and thyroid hormone testing, as recommended by the American Thyroid Association (ATA).

  6. The TSI Reporter assay is intended for the qualitative detection of TSI. It is not intended for use in monitoring a patient’s treatment. The effects of various drug therapies on the performance of this Kit have not been established.

  7. This is a functional bioassay for the detection of serum TSI. Sample dilutions are not advisable as there is a non-linear relationship between antibody concentration and signal (Relative Light Unit, RLU).

  8. Performance of the Kit can only be assured when components used in the assay are those supplied by Diagnostic Hybrids, Inc.

  9. False-positive results may occur when serum TSH levels are greater than (>) 350 mU/L. Specimens with TSH levels above this range should be disqualified for use.


1AARDA, American Autoimmune Related Diseases Association; NWHIC, National Women’s’ Health Information Center.

2AACE Medical Guidelines for Clinical Practice for the Evaluation and Treatment of Hyperthyroidism and Hypothyroidism. Endocrine Practice, Vol. 8, No. 6. Nov./Dec. 2002.

3Tahara K., Ban T., Minegishi T., Kohn L.D. Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients. Biochem Biophys Res Commun. 1991 Aug 30; 179(1):70-7.

4US Department of Labor, Occupational Safety and Health Administration, 29 CFR Part 1910.1030, Occupational safety and health standards, bloodborne pathogens.

5Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th edition, 2007, CDC-NIH manual. []

6CLSI. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard, 5th Edition. CLSI document H3-A6 (ISBN 1-56238-650-6). CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2003.

From PI-317-1en_Thyretain_TSI_Reporter_BioAssay_2-Replicates_40-xx000-v2_v2010JUN02

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