rev: June 17, 2005
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FREE Triidothyronine (fT3) Microplate EIA
Intended Use: The Quantitative Determination of Free Triiodothyronine Concentration in Human Serum by a Microplate Enzyme Immunoassay. Levels of fT3 are thought to reflect the amount of T3 available to the cells and may therefore determine the clinical metabolic status of T3.
same as RDI-FT3075 $281.00/kit
SUMMARY AND EXPLANATION OF THE TEST
Triiodothyronine, a thyroid hormone, circulates in blood
almost completely bound (>99.5%) to carrier proteins (1,2). The main transport
protein is thyroxine-binding globulin (TBG). However, only the free (unbound)
portion of triiodothyronine is believed to be responsible for the biological
action. Further, the concentrations of the carrier proteins are altered in
many clinical conditions, such as pregnancy. In normal thyroid function as
the concentrations of the carrier proteins alters, the total triiodothyronine
level changes so that the free triiodothyronine concentration remains constant.
Thus, measurements of free triiodothyronine concentrations correlate more
reliably with clinical status than total triiodothyronine
For example, the increase in total triiodothyronine levels associated with pregnancy, oral contraceptives and estrogen therapy result in higher total T3 levels while the free T3 concentration remains basically unchanged.
This microplate enzyme immunoassay methodology provides the technician with optimum sensitivity while requiring few technical manipulations in a direct determination of free T3. In this method, serum reference, patient specimen, or control is first added to a microplate well. Enzyme-T3 conjugate (analog method) is added, then the reactants are mixed. A competition reaction results between the enzyme conjugate and the free triiodothyronine for a limited number of antibody combining sites immobilized on the well.
After the completion of the required incubation period, the antibody bound enzyme-triiodothyronine conjugate is separated from the unbound enzyme-triiodothyronine conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce color.
The employment of several serum references of known free triiodothyronine concentration permits construction of a graph of activity and concentration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with free triiodothyronine concentration.
Competitive Enzyme Immunoassay � Analog Method for Free T3
essential reagents required for a solid phase enzyme immunoassay include
immobilized T3 antibody, enzyme-T3 conjugate and native free T3 antigen.
The enzyme-T3 conjugate should have no measurable binding to serum proteins
especially TBG and albumin. The method achieves this goal.
Upon mixing immobilized antibody, enzyme-T3 conjugate and a serum containing the native free T3 antigen, a competition reaction results between the native free T3 and the enzyme-T3 conjugate for a limited number of insolubulized binding sites. The interaction is illustrated by the followed equation:
EnzAg + Ag + AbC.W. AgAb C.W. + EnzAgAbC.W AgAbC.W..
AbC.W. = Monospecific Immobilized Antibody (Constant Quantity)
Ag = Native Free Antigen (Variable Quantity)
EnzAg = Enzyme-T3 antigen Conjugate (Constant Quantity)
AgAbC.W. = AntigenAntibody Complex
EnzAg AbC.W. = Enzyme-antigen Conjugate Antibody Complex
k a = Rate Constant of Association
k a = Rate Constant of Disassociation
K = k a / k-a = Equilibrium Constant
After equilibrium is attained, the antibodybound fraction is separated from unbound antigen by decantation or aspiration. The enzyme activity in the antibodybound fraction is inversely proportional to the native free 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.
REAGENTS FOR 96-well MICROPLATE
1. Human Serum References -- 1.0 ml/vial - Icons A-F
Six (6) vials of serum reference for free triiodothyronine at approximate* concentrations of 0 (A), 0.4 (B), 1.2 (C), 4.5 (D), 8.0 (E) and 18.0 (F) pg/ml. Store at 28�C. A preservative has been added. * Exact levels are given on the labels on a lot specific basis.
For SI units: 1pg/ml x 1.536 = pmol/L
2. Enzyme-antigen Conjugate � 1.0ml/vial - Icon
One (1) vial of triiodothyronine-horseradish peroxidase (HRP) conjugate in a bovine albumin-stabilizing matrix. A preservative has been added. Store at 28�C.
3. Enzyme-antigen Conjugate Buffer-- 11 ml - Icon
One (1) bottle reagent containing buffer, blue dye, and preservative. Store at 28�C.
4. Antibody Coated Microplate -- 96 wells - Icon
One 96-well microplate coated with sheep anti-triiodothyronine serum and packaged in an aluminum bag with a drying agent. Store at 28�C.
5. Wash Solution Concentrate -- 20ml - Icon
One (1) vial containing a surfactant in phosphate buffered saline. A preservative has been added. Store at 2-30�C.
6. Substrate A --7.0ml/vial - Icon SA
One (1) bottle containing tetramethylbenzidine (TMB) in buffer. Store at 28�C.
7. Substrate B -- 7.0ml/vial - Icon SB
One (1) bottle containing hydrogen peroxide (H2O2) in buffer. Store at 28�C.
8. Stop Solution -- 6.0ml/vial - Icon
One (1) bottle containing a strong acid (1N HCl). Store at 230�C.
Note 1: Do not use reagents beyond the kit expiration date.
Note 2: Opened reagents are stable for sixty (60) days when stored at 28�C.
For In Vitro Research Use Only
Not for Internal or External Use in Humans or Animals
All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, "Biosafety in Microbiological and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.
SPECIMEN COLLECTION AND PREPARATION
Collect sample(s) by venipuncture in ten (10) ml silicone evacuated tube(s). The usual precautions in the collection of venipuncture samples should be observed. Separate the red blood cells by centrifugation use serum for the free T3 procedure. Specimen(s) may be refrigerated at 28(C for a maximum period of 48 hours. If the specimen(s) can not be assayed within 48 hours, the sample(s) may be stored at temperatures of �20�C for up to 30 days. When assayed in duplicate, 0.10ml of the specimen is required.
The crossreactivity of the triiodothyronine antibody to selected substances was evaluated by adding the interfering substance to a serum matrix at various concentrations. The cross-reactivity was calculated by deriving a ratio between dose of interfering substance to dose of Triiodothyronine needed to displace the same amount of tracer.
Substance Cross Reactivity Concentration
I-Triiodothyronine 1.0000 -
I-Thyroxine < 0.0002 10�g/ml
lodothyrosine < 0.0001 10�g/ml
Diiodothyrosine < 0.0001 10�g/ml
Diiodothyronine < 0.0001 10�g/ml
Phenylbutazone < 0.0001 10�g/ml
Sodium Salicylate < 0.0001 10�g/ml
1. Six (6) vials of free triiodothyronine human serum references.
2. One (1) vial of T3-enzyme conjugate concentrate.
3. One (1) vial of Free T3/T4 conjugate buffer.
4. One 96-well Antibody coated microplate.
5. One (1) bottle of Wash buffer concentrate.
6. One (1) bottle of Substrate A.
7. One (1) bottle of Substrate B.
8. One (1) bottle of Stop solution.
Required But Not Provided:
1. Pipette capable of delivering 50�l volumes with a precision of better than 1.5%.
2. Dispenser(s) for repetitive deliveries of 0.100ml and
0.300ml volumes with a
precision of better than 1.5%.
3. Adjustable volume (20-200�l) and (200-1000�l) dispenser(s) for conjugate and substrate dilutions.
4. Microplate washer or a squeeze bottle (optional).
5. Microplate Reader with 450nm wavelength absorbance capability.
6. Test tubes for dilution of enzyme conjugate and substrate A and B.
7. Absorbent Paper for blotting the microplate wells.
8. Plastic wrap or microplate cover for incubation steps.
9. Vacuum aspirator (optional) for wash steps.
11. Quality control materials.
1. Working Reagent 1 - Triiodothyronine-enzyme Conjugate Solution
Dilute the Triiodothyronine-enzyme conjugate 1:11 with Triiodothyronine-enzyme conjugate buffer in a suitable container. For example, dilute 160�l of conjugate with 1.6ml of buffer for 16 wells (A slight excess of solution is made). This reagent should be used within twenty-four hours for maximum performance of the assay. Store at 2-8(C.
Amount of Buffer required = Number of wells * 0.1
Quantity of Enzyme conjugate necessary = # of wells * 0.01
i.e. = 16 x 0.1 = 1.6ml for T3 Conjugate Buffer
16 x 0.01 = 0.16ml (160�l) for T3 enzyme conjugate
2. Wash Buffer
Dilute contents of Wash Concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature until expiration date printed on concentrate label.
3. Working Substrate Solution - Prepare immediately before use
Determine the amount of reagent needed and prepare by mixing equal portions of Substrate A and Substrate B in a suitable container. For example, add 1ml of A and 1ml of B per two (2) eight well strips (A slight excess of solution is made). Use within one (1) day of preparation for maximum performance of the assay.
Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 - 30�C).
1. Format the microplates� wells for each serum reference, control and patient specimen to be assayed in duplicate.
2.Pipette 0.050 ml (50�l) of the appropriate serum reference, control or specimen into the assigned well.
3.Add 0.100 ml (100�l) of Working Reagent 1, Triiodothyronine-enzyme conjugate solution, to all wells (see Reagent Preparation Section).
4.Swirl the microplate gently for 20-30 seconds to mix and cover.
5.Incubate 60 minutes atroom temperature.
6.Discard the contents of the microplate by decantation or aspiration. If decanting, blot the plate dry with absorbent paper.
7. Add 300�l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two (2) additional times for a total of three (3) washes. An automatic or manual plate washer can be used. Follow the manufacturer�s instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat two (2) additional times.
8. Add 0.100 ml (100�l) of working substrate solution to all wells (see Reagent Preparation Section). Always add reagents in the same order to minimize reaction time differences between wells.
9. Incubate at room temperature for fifteen (15) minutes.
10. Add 0.050ml (50�l) of stop solution to each well and gently mix for 15-20 seconds. Always add reagents in the same order to minimize reaction time differences between wells.
11. Read the absorbance in each well at 450nm in a microplate reader. The results should be read within thirty (30) minutes of adding the stop solution.
Each laboratory should assay controls at levels in the hypothyroid, euthyroid and hyperthyroid range 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.
A dose response curve is used to ascertain the concentration of free triiodothyronine in unknown specimens.
1. Record the absorbance obtained from the printout of the microplate reader as outlined in Example 1.
2. Plot the absorbance for each duplicate serum reference versus the corresponding fT3 concentration in pg/ml on linear graph paper (do
not average the duplicates of the serum references before plotting).
3. Draw the best-fit curve through the plotted points.
4. To determine the concentration of fT3 for an unknown, locate the average absorbance of the duplicates for each unknown on the vertical axis of the graph, find the intersecting point on the curve, and read the concentration (in pg/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 1.799 (intersects the standard curve at (1.2 pg/ml) fT3 concentration (See Figure 1).
WELL SERUM REFERENCES ABSORBANCE
1 0.0 pg/ml 2.639
2 0.0 pg/ml 2.615
3 0.4 pg/ml 2.330
4 0.4 pg/ml 2.259
5 1.2 pg/ml 1.835
6 1.2 pg/ml 1.753
7 4.5 pg/ml 0.939
8 4.5 pg/ml 0.975
9 8.0 pg/ml 0.537
10 8.0 pg/ml 0.569
11 18.0 pg/ml 0.301
12 18.0 pg/ml 0.287
Well I. D. O.D. O.D. Value
13 Unknown #1 1.876
14 Unknown #1 1.721 1.799 1.2 pg/ml
The data presented in Example 1 and Figure 1 are for illustration only and should not be used in lieu of a standard curve prepared with each assay.
Maximum Absorbance (O calibrator) = 1.5 - 2.7
LIMITATIONS OF PROCEDURE
A. Assay Performance
Sample(s), which are contaminated microbiologically, should not be used in the assay. Highly lipemeic or hemolysed specimen(s) should similarly not be used.
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 (10) minutes to avoid assay drift. If more than one (1) plate is used, it is recommended to repeat the dose response curve.
Addition of the substrate solution initiates a kinetic reaction, which is terminated by the addition of the stop solution. Therefore, the addition of the substrate and the stopping solution should be added in the same sequence to eliminate any time deviation during reaction.
Plate readers measure vertically. Do not touch the bottom of the wells.
Failure to remove adhering solution adequately in the aspiration or decantation wash step(s) may result in poor replication and spurious results.
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.
Several drugs are known to effect the binding of Triiodothyronine to the thyroid hormone carrier proteins or its metabolism to T3 and complicate the interpretation of free T3 results (3).
Circulating autoantibodies to T3 and hormone-binding inhibitors may interfere (4).
Heparin has been reported to have in vivo and in vitro effects on free T3 concentration (5). Therefore, do not obtain samples in which this anti-coagulant has been used.
In severe nonthyroidal illness (NTI), the assessment of thyroid status becomes very difficult. TSH measurements are recommended to identify thyroid dysfunction (6).
Familial dysalbuminemic conditions may yield erroneous results on direct free T3 assays (7).
"NOT INTENDED FOR NEWBORN SCREENING"
EXPECTED RANGES OF VALUES
A study of euthyroid adult population was undertaken to determine expected values for the fT3 EIA Test System. The mean (R) values, standard deviations (S.D.) and expected ranges (�2 S.D.) are presented in Table 1.
Expected Values for the Free T3 EIA Test System
(110 specimens) (75 specimens)
Mean (X) 2.8 3.0
Deviation (S.D.) 0.7 0.6
(�2 S. D.) 1.4 � 4.2 1.8 � 4.2
It is important to keep in mind that establishment of a range of values which can be expected to be found by a given method for a population of "normal"persons is dependent upon a multiplicity of factors: the specificity of the method, the population tested and the precision of the method in the hands of the analyst. For these reasons each laboratory should depend upon the range of expected values established by the Manufacturer only until an inhouse range can be determined by the analysts using the method with a population indigenous to the area in which the laboratory is located.
The within and between assay precision of the fT3 Microplate EIA Test System were determined by analyses on three different levels of pool control sera. The number, mean values, standard deviation and coefficient of variation for each of these control sera are presented in Table 2 and Table 3.
Within Assay Precision (Values in pg/ml)
Sample N X S.D. C.V.
Low 20 1.37 0.16 11.9%
Normal 20 4.21 0.17 4.1%
High 20 7.1 0.17 2.4%
Between Assay Precision (Values in pg/ml)
Sample N X S.D. C.V.
Low 10 1.4 0.15 10.7%
Normal 10 4.4 0.23 5.2%
High 10 7.0 0.30 4.2%
*As measured in ten experiments in duplicate over a ten day period.
The fT3 Microplate EIA Test System was compared with a coated tube radioimmunoassay analog method. Biological specimens from hypothyroid, euthyroid and hyperthyroid populations were used (The values ranged from 0.1pg/ml � 14pg/ml). The total number of such specimens was 85. The least square regression equation and the correlation coefficient were computed for this fT3 EIA in comparison with the reference method. The data obtained is displayed in Table 4.
Method Mean (x) Analysis Coefficient
This Method 3.4 y = 0.15+0.925(x) 0.955
Only slight amounts of bias between this method and the reference method are indicated by the closeness of the mean values. The least square regression equation and correlation coefficient indicates excellent method agreement.
The Triiodothyronine procedure has a sensitivity of 0.05 pg/ml. The sensitivity was ascertained by determining the variability of the 0 pg/ml serum calibrator and using the 2( (95% certainty) statistic to calculate the minimum dose.
1. Pederson, K.O, Scand. J. Clin. Lab Invest, 34, 247 (1974)
2.Wild, D., Immunoassay Handbook, Stockton Press, 339 (1994).
3. Wenzel, K.W., Metabolism, 30, 717 (1981)
4. Bhagat,C.,et.al, Clin Chem, 29, 1324 (1983-).
5. Lundberg, P.R., et.al, Clin Chem, 28, 1241 (1982).
6. Melmed, S. et.al, J Clin Endocrinol Metab, 54, 300 (1982).
7. Lalloz M.R., et al, Clin Endocrinol, 18, 11 (1983).
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