Reverse Triiodothyronine ELISA (Reverse T3 ELISA)


Follow good laboratory practices when handling kit reagents and samples. 3. ... properly; do not use if the solution appears dark green or black in color. 19.

Reverse Triiodothyronine ELISA (Reverse T3 ELISA) For the quantitative determination of Reverse Triiodothyronine (rT3) in human serum and plasma. For Research Use Only. Not For Use In Diagnostic Procedures.

Catalog Number: Size: Version:

11-RT3HU-E01 96 wells 3.0 - ALPCO September 7, 2017

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INTENDED USE For the direct quantitative determination of Reverse Triiodothyronine (rT3) in human serum and plasma by an enzyme immunoassay. For Research Use Only. Not for use in diagnostic procedures. PRINCIPLE OF THE TEST The Reverse Triiodothyronine ELISA is a competitive enzyme immunoassay, where the antigen (rT3 present in calibrators, controls and samples) competes with a biotin-labelled antigen (rT3-Biotin conjugate) for a limited quantity of antibody which is coated on the microplate wells. After one hour incubation followed by the first washing, unbound materials are removed and a Streptavidin-HRP conjugate is added and incubated for 30 minutes. Following a second washing, the TMB substrate is added. The enzymatic reaction is terminated by addition of the stop solution, upon which the color intensity is measured with a microplate reader. The color intensity is inversely proportional to the concentration of rT3 in the sample. The set of kit calibrators that are run simultaneously with the samples is used to plot a calibration curve and determine the concentration of rT3 in samples and controls. PROCEDURAL CAUTIONS AND WARNINGS 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17.

18. 19. 20. 21.

This kit is intended for in vitro use only. Follow good laboratory practices when handling kit reagents and samples. Do not pipette by mouth. Do not smoke, drink, or eat in areas where samples or kit reagents are handled. Wear protective clothing and disposable gloves. Wash hands thoroughly after performing the test. Avoid contact with eyes; use safety glasses; in case of contact with eyes, flush eyes with water immediately and contact a doctor. Users should have a thorough understanding of this protocol for the successful use of this kit. Reliable performance will only be attained by strict and careful adherence to the instructions provided. Avoid microbial contamination of reagents. A calibrator curve must be established for every run. It is recommended to all customers to prepare their own control materials or serum pools which should be included in every run at a high and low level for assessing the reliability of results. The controls (included in the kit) must be included in every run and their results must fall within the ranges stated in the quality control certificate; a failed control result might indicate improper procedural techniques or pipetting, incomplete washing or inadequate reagent storage. When the use of water is specified for dilution or reconstitution, use deionized or distilled water. All kit reagents and samples should be brought to room temperature and mixed gently but thoroughly before use. Avoid repeated freezing and thawing of samples. Improper procedural techniques, imprecise pipetting, incomplete washing as well as improper reagent storage may be indicated when assay values for the control do not reflect established ranges. When reading the microplate, the presence of bubbles in the wells will affect the optical densities (ODs). Carefully remove any bubbles before performing the reading step. The substrate solution (TMB) is sensitive to light and should remain colorless if properly stored. Instability or contamination may be indicated by the development of a blue color, in which case it should not be used. The Biotin-rT3 conjugate solution is sensitive to light and should be of a light-yellow color if stored properly; do not use if the solution appears dark green or black in color. When dispensing the substrate and stop solutions, do not use pipettes in which these liquids will come into contact with any metal parts. To prevent contamination of reagents, use a new disposable pipette tip for dispensing each reagent, sample, calibrator and control. Do not use kit components from different kit lots within a test and do not use any component beyond the expiration date printed on the label.

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22. Kit reagents must be regarded as hazardous waste and disposed of according to local and/or national regulations. LIMITATIONS 1. All the reagents within the kit are calibrated for the direct determination of rT3 in human serum and plasma. The kit is not calibrated for the determination of rT3 in other samples of human or animal origin. 2. Do not use grossly hemolyzed, lipemic, icteric or improperly stored serum or plasma samples. 3. Samples or control sera containing azide or thimerosal are incompatible with this kit and will lead to false results. SAFETY CAUTIONS AND WARNINGS POTENTIAL BIOHAZARDOUS MATERIAL Human serum that may be used in the preparation of the calibrators and controls has been tested and found to be non-reactive for Hepatitis B surface antigen and has also been tested for the presence of antibodies to HCV and Human Immunodeficiency Virus (HIV) and found to be negative. No test method however, can offer complete assurance that HIV, HCV and Hepatitis B virus or any other infectious agents are absent. The reagents should be considered a potential biohazard and handled with the same precautions applied to blood samples. All human samples should be considered a potential biohazard and handled as if capable of transmitting infections and in accordance with good laboratory practices. CHEMICAL HAZARDS Avoid contact with reagents containing TMB, hydrogen peroxide and sulfuric acid. If contacted with any of these reagents, wash with plenty of water. TMB is a suspected carcinogen. SAMPLE COLLECTION AND STORAGE Serum: Approximately 0.2 mL of serum is required per duplicate determination. Collect 4–5 mL of blood into an appropriately labelled tube and allow it to clot. Centrifuge and carefully remove the serum layer. Store at 4°C for up to 24 hours or at -10°C or lower for longer time. Plasma: Approximately 0.2 mL of plasma is required per duplicate determination. Collect 4–5 mL of blood into EDTA plasma tubes. Store at 4°C for up to 24 hours or at -10°C or lower for longer time. Consider all human samples as possible biohazardous materials and take precautions when handling. SAMPLE PRETREATMENT This assay is a direct system; no sample pretreatment is necessary. REAGENTS AND EQUIPMENT NEEDED BUT NOT PROVIDED 1. Precision pipette to dispense 25 to 1000 μL 2. Disposable pipette tips 3. Distilled or deionized water 4. A 37°C incubator 5. Microplate reader with a filter set at 450 nm and an upper OD limit of 3.0 or greater 6. Timer

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REAGENTS PROVIDED 1. Anti-Reverse T3 Polyclonal Antibody-Coated Break Apart Well Microplate - Ready To Use Contents: One 96-well (12x8) polyclonal antibody-coated microplate in a resealable pouch with desiccant. Storage: Refrigerate at 2–8°C. Stability: 12 months or as indicated on label. 2. Reverse T3-Biotin Conjugate - Ready To Use Contents: Reverse T3-Biotin conjugate in a protein-based buffer with a non-mercury preservative. Volume: 13 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label. 3. Streptavidin-Horse Radish Peroxidase (HRP) Conjugate - Ready To Use Contents: Streptavidin-HRP conjugate in a protein-based buffer with a non-mercury preservative. Volume: 20 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months in unopened vials or as indicated on label. 4. Reverse T3 Calibrators - Ready To Use Contents: Six vials containing rT3 in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer with rT3 to the concentrations on labels. Typical calibrator concentrations*: 0, 0.02, 0.1, 0.4, 1 and 2 ng/mL. *Approximate values - please refer to vial labels for exact concentrations Volume: Calibrators A-F: 1 mL/vial Storage: Refrigerate at 2–8°C Stability: 12 months in unopened vials or as indicated on label. 5. Reverse T3 Controls - Ready To Use Contents: Two vials containing rT3 in a protein-based buffer with a non-mercury preservative. Prepared by spiking buffer with rT3 to the target concentration in QC certificate. Refer to vial labels for acceptable ranges. Volume: 1 mL/vial Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label. 6. Wash Buffer Concentrate - Requires Preparation X10 Contents: One bottle containing buffer with a non-ionic detergent and a non-mercury preservative. Volume: 50 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label. Preparation: Dilute the wash buffer concentrate 1:10 in distilled or deionized water to prepare the working wash buffer. If one whole plate is to be used dilute 50 mL of the wash buffer concentrate in 450 mL of water. 7. TMB Substrate - Ready To Use Contents: One bottle containing tetramethylbenzidine and hydrogen peroxide in buffer. Volume: 16 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label. 8. Stop Solution - Ready To Use Contents: One bottle containing 1M sulfuric acid. Volume: 6 mL/bottle Storage: Refrigerate at 2–8°C Stability: 12 months or as indicated on label. 26-G Keewaydin Drive, Salem, NH 03079│P: (800) 592-5726│F: (603) 898-6854│[email protected]│www.alpco.com Page 4 of 8

ASSAY PROCEDURE Sample Pretreatment: None. All reagents must reach room temperature before use. Calibrators, controls and samples should be assayed in duplicate. Once the procedure has been started, all steps should be completed without interruption. 1. After all kit components have reached room temperature, mix gently by inversion. Prepare the working wash buffer (see wash buffer concentrate under the section “Reagents Provided”). 2. Remove the required number of strips from the microplate and assemble into a plate frame. Reseal the bag and return any unused strips to the refrigerator. 3. Pipette 25 µL of each calibrator, control and sample (serum or plasma) into correspondingly labelled wells in duplicate. 4. Pipette 100 μL of the Reverse T3-Biotin conjugate into each well (the use of a multichannel pipette is recommended). Gently shake the microplate by hand for ten seconds to ensure complete mixing of the conjugate solution with the calibrators, controls and samples. 5. Incubate the plate at 37°C for 1 hour. Do not cover the microplate. 6. Wash the wells with 350 µL/well of working wash buffer solution 3 times. After washing, tap the plate firmly against absorbent paper to remove any residual liquid. (The use of an automatic strip washer is strongly recommended.) The accuracy of this assay depends on the correct execution of the washing procedure. 7. Pipette 150 µL of the Streptavidin-HRP conjugate into each well. (The use of a multichannel pipette is recommended.) 8. Incubate the plate at 37°C for 30 minutes. Do not cover the microplate. 9. Wash the wells 3 times using the same procedure as stated in step 6. 10. Pipette 150 µL of the TMB substrate into each well at timed intervals. (The use of a multichannel pipette is recommended.) 11. Incubate at 37°C for 15 minutes. Do not cover the microplate. 12. Pipette 50 µL of stop solution into each well at the same timed intervals as in step 10 (the use of a multichannel pipette is recommended). Gently shake the microplate by hand for ten seconds to ensure complete mixing of the stop solution in the wells. 13. Measure the absorbance at 450 nm with a microplate reader within 20 minutes after addition of the stop solution. CALCULATIONS 1. Calculate the mean optical density of each calibrator, control and sample duplicate. 2. Use a 4-parameter or 5-parameter curve with immunoassay software to generate the control and sample concentration results or draw a calibration curve on semi-log paper with the mean optical densities on the Y-axis and the calibrator concentrations on the X-axis and read the concentration of controls and samples off the calibrator curve. 3. If a sample reads greater than 2 ng/mL report the result as “> 2 ng/mL”. 4. To convert from ng/mL to ng/dL multiply the result by 100; to convert to nmol/L, multiply the ng/dL result by 0.01536 or the ng/mL result by 1.536.

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TYPICAL TABULATED DATA Sample data only. Do not use to calculate results. Calibrator

rT3 (ng/mL)

Mean OD (450 nm)

A

0

2.594

B

0.02

2.140

C

0.1

1.597

D

0.4

1.113

E

1

0.834

F

2

0.678

Unknown

0.15

1.458

TYPICAL CALIBRATOR CURVE Sample curve only. Do not use to calculate results.

rT3 (ng/mL)

PERFORMANCE CHARACTERISTICS SENSITIVITY The limit of detection (LoD) was determined from the analysis of 60 samples of the blank and a low value sample in two independent experiments and it was calculated as follows: LoD = µB + 1.645σB + 1.645σS, where σB and σS are the standard deviation of the blank and low value sample and µB is the mean value of the blank. The Limit of Detection (LoD) was determined to be 0.009 ng/mL. SPECIFICITY (CROSS-REACTIVITY) The following compounds were tested for cross-reactivity with rT3 cross-reacting at 100%: Steroid

% Cross Reactivity

rT3

100

T3

< 0.001

T4

0.005

T2

0.004

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INTERFERENT SUBSTANCES The following substances did not show significant interference with the assay: hemoglobin up to 2 g/L, free and conjugated bilirubin up to 200 mg/L, triglycerides up to 5.5 mg/mL and biotin up to 40 µg/mL. INTRA-ASSAY PRECISION Four serum samples were assayed 24 times each on the same calibrator curve. The results are tabulated below: Sample Mean (ng/mL) SD (ng/mL) CV % 1

0.089

0.0024

2.7

2

0.250

0.020

8.0

3

0.455

0.019

4.2

4

1.018

0.140

13.8

INTER-ASSAY PRECISION Four serum samples were assayed in 20 different tests in the span of ten days. The results are tabulated below: Sample

Mean (ng/mL)

SD (ng/mL)

CV %

1

0.127

0.016

12.6

2

0.304

0.038

12.5

3

0.469

0.057

12.2

4

0.847

0.083

9.8

RECOVERY Spiked samples were prepared by adding defined amounts of rT3 to three serum samples. The results are tabulated below: Obs. Result (ng/mL)

Exp. Result (ng/mL)

Recovery %

Serum Sample 1 + 0.15 ng/mL + 0.30 ng/mL + 0.45 ng/mL

0.437 0.586 0.728 0.854

0.587 0.737 0.887

99.8 98.8 96.3

Serum Sample 2 + 0.15 ng/mL + 0.30 ng/mL + 0.45 ng/mL

0.327 0.435 0.596 0.777

0.477 0.627 0.777

91.2 95.1 100.0

Serum Sample 3 + 0.15 ng/mL + 0.30 ng/mL + 0.45 ng/mL

0.149 0.287 0.460 0.614

0.299 0.449 0.599

96.0 102.4 102.5

Sample

COMPARATIVE STUDIES The Reverse T3 ELISA kit (y) was compared with the leading competing technology: Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) (x). The comparison of 40 serum samples yielded the following linear regression results: y = 0.79x + 0.01, r = 0.96. 26-G Keewaydin Drive, Salem, NH 03079│P: (800) 592-5726│F: (603) 898-6854│[email protected]│www.alpco.com Page 7 of 8

REFERENCES 1. van den Beld AW, et al. Thyroid Hormone Concentrations, Disease, Physical Function and Mortality in Elderly Men. J Clin Endocrinol Metab. 2005; 90(1):6403–9. 2. Holtorf K. Thyroid Hormone Transport into Cellular Tissue. Journal of Restorative Medicine. 2014; 3(1):53–68. 3. Holtorf K Peripheral Thyroid Hormone Conversion and Its Impact on TSH and Metabolic Activity. Journal of Restorative Medicine. 2014; 3(1):30–52. 4. Senese R et al. Thyroid: Biological Actions of “Non-classical” Thyroid Hormones. J Endocrinol. 2014; 221(2):R1–12. 5. Warner MH, Beckett GJ. Mechanisms Behind the Nonthyroidal Illness Syndrome: An Update. J Endocrinol. 2010; 205(1):1–13. 6. Peeters RP, et al. Tissue Thyroid Hormone Levels in Critical Illness. J Clin Endocrinol Metab. 2005; 90(12):6498–507. 7. Friberg L, et al. Association Between Increased Levels of Reverse Triiodothyronine and Mortality after Acute Myocardial Infarction. Am J Med. 2001; 111(9):699–703. 8. Pimentel, CR et al. Reverse T3 as a Parameter of Myocardial Function Impairment in Heart Failure. Int J Cardiol. 2010; 145(1):52–3. 9. Economidou F, et al. Thyroid Function During Critical Illness. Hormones (Athens). 2011; 10(2):117–24. 10. Thyroid Hormone Transport. National Academy of Hypothyroidism. http://www.nahypothyroidism.org/thyroidhormone-transport/#reverseT3

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