Safety Notes

T3 Do not swallow the reagents. Avoid contact with eyes, skin and
mucous membranes, All patient specimens and ICAll should be
ELISA Test for the Quantitative handled as potentially infectious. ICAL! have been checked on
Determination of Total Triiodothyronine (T3) donor level for HCV and HIV-1/2 antibodies and HBsAg and found
negative. Wear protective clothing and disposable gloves according
in Human Serum or Plasma to Good Laboratory Practices,
All materials contaminated with patient specimens or ICAll should
Package Size be inactivated by validated procedures (autoclaving or chemical
IREFI 54010 treatment) in accordance with applicable regulations.
IivDI ISTopl irritates eyes, skin and mucous membranes, Upon contact,
rinse thoroughly with copious amounts of water and consult a
Intended Use doctor.
Triiodothyronine (T3) is a hormone synthesised and stored in the
thyroid gland. More than 99% of T3 in the blood is bound reversibly Stability
to plasma proteins. The concentration of T3 is much lower than that The reagents are stable up to the stated expiry dates on the
of T4, but its metabolic potency is much greater. T3 determination individual labels when stored at 2 ..,8C,
is an important tool in thyroid disease diagnosis. Its measurement After opening reagents have to be stored at 2 ...8C and used within
has uncovered a variant of hyperthyroidism where thyrotoxic 60 days (see also "Note").
patients present elevated T3 values with normal T4 values (T3-
hyperthyroidism). An increase in T3 without an increase in T4 is
IMlcI
sealed in an aluminium bag with a desiccant.
frequently a forerunner of recurrent thyrotoxicosis in previously
before opening, the strips must be at room temperature.
treated patients. The clinical significance of T3 is also evident in
unused: return to the zip-lock bag with the desiccant. Strips
patients in whom euthyroidism is attributable only to normal T3,
stored in this way at 2 ...8C can be used until the expiration date
though their T4 values are subnormal. T3 determination is also
(see also "Note"),
useful in monitoring both patients under treatment for hyper-
- Do not touch the upper rim or the bottom of the wells with fingers,
thyroidism and patients who have discontinued antithyroid drug
therapy. It is especially valuable in distinguishing between euthyroid
Reagent Preparation
and hyperthyroid subjects. In addition to hyperthyroidism, T3 levels
Bring all reagents to room temperature (15 ...25C) before use.
increase during pregnancy, oral contraception or estrogen
Reagents not in use should always be stored at 2 ...8C.
treatment, paralleling TBG (Thyroxine Binding Globulin) increases
in a manner analogous to T4. Likewise, a decrease in TBG concen-
Working conjugate solution IWCONI
tration decreases T3 concentration. These changes in the T3 level,
Dilute ICONI1 + 10 with IC-Dlll: e.g. dilute 160 ~llcONI with 1.6 ml
however, are not a true reflection of thyroid status. Best diagnostic
IC-DILIfor 16 wells.
information about the thyrostasis in such situations can be obtained
by the TRH test. Stability: 24 h at 2, ..8C.
Working Wash Solution IWASHI
Principle - Competitive EIA - faint turbidity, which may appear in the concentrate ~' will
The T3 ELISA is based on the principle of competitive binding
completely dissolve on dilution.
between T3 in a test specimen and T3-peroxidase conjugate for a
dilute ~ to 1000-IDLw.ilh fresh, deionised water in a suitable
limited number of binding sites on the anti-T3 (sheep) coated well.
container. Rinse vial several times.
Thus the amount of T3-peroxidase conjugate bound to the well is
Stability: up to 60 days at 15 ...25C.
inversely proportional to the concentration of T3 in the specimen.
After incubation of specimen and T3-peroxidase conjugate un- Substrate Working Solution ISUBI
bound enzyme conjugate is removed in the equilibrium state by wa- for longer periods of usage: prepare needed amount by mixing
shing. TMB/Substrate solution is added (step 2), and a blue colour equal portions of ~ and ~. Use only a clean plastic vial pre-
develops. The intensity of this colour, which changes to yellow after viously rinsed with deionised water.
stopping the reaction, is inversely proportional to the amount of T3 for use within 30 days: pour contents of the vial ~ in vial ~ mix
in the specimen. and store at 2 ...8C,
The absorbance of calibrators and specimen is determined by handle ~ carefully and avoid contamination! Do not use, if it
using ELISA microplate readers or automated ELISA systems (like looks blue!
HUMAN's HUMAREADER or ELiSYS line). Specimen's concentra- Store protected from bright light.
tion is extrapolated from a dose response curve generated by Stability: 30 days at 2,..8C
utilising serum calibrators of known antigen concentrations.
Specimen
Reagents and Contents Serum or plasma (EDTA, Heparin)
~ 12 Microtiter Strips (in 1 strip holder) Do not use highly lipemic or hemolysed specimens.
8-well snap-off strips, coated with anti-T3 (sheep) Specimens may be stored for 48 hours at 2 ...8C, up to 30 days at
~ A -F Calibrators (white cap) -20C. Freeze and thaw once only. Thawed specimen must be
6x2.0ml Ready to use, in human serum homogenised. Eliminate particulate matter by centrifugation or
T3 level: 0 (A), 0.50 (8), 1.00 (C), 2.50 (D), 5.00 (E) filtration.
and 7.50 (F) ng/ml
Procedure
IcoNI 1.5 ml Enzyme - antigen conjugate (white cap)
Follow the procedure exactly as described.
T3-HRP-conjugate, coloured yellow pH 7.45 0.1
in a protein stabilising matrix 1% Procedural Notes
IC.DILI 13 ml Conjugate buffer (white cap) P1: Do not mix or use components with different lot numbers. Do
not mix caps of vials (risk of contamination). Do not use reagents
Phosphate buffer, coloured red
after their expiration date.
~ 20 ml Wash Solution (black cap)
P2: Do not use reagents that could be contaminated or look or
Concentrate for ca. 1000 ml pH 8.8 0.4
smell different than usual.
MOPS buffered saline 5 mmol/I P3: Record ICAl!. specimens and controls carefully on the spread
~ 7.0 ml Substrate Reagent A (yellow cap) pH 3.5 0.1 sheet supplied with the kit.
3,3', 5,5'-tetramethylbenzidine (TMB) 4 mmol/I P4: [9 - select the required number and place firmly in the holder.
Sodium acetate buffer 0.05 molll
P5: Run duplicates for ICAl!. controls and specimens. Pipette
~ 7.0 ml Substrate Reagent 8 (blue cap) pH 4.5 0.1
them on the bottom in the microwells.
Urea hydrogen peroxide 10 mmol/I
P6: Always add reagents in the same order and timing to
Sodium acetate buffer 0.05 mol/I
minimise reaction time differences between wells. This is
ISTOpl 7.5 ml Stop solution (red cap) important for reproducible results. Pi petting of specimens should
Sulphuric acid not exceed 10 minutes. Otherwise pipette the calibration curve in
Adhesive strip the indicated positions at half way time of the series. If more than 1
Preservatives: Total concentration < 0.04%. plate is used, repeat the dose response curve for each plate.
P7: Avoid/remove air bubbles prior to incubations and reading Expected Values
absorbance. Results from a study with euthyroid subjects:
P8: ISUBI initiates and ISTOpl terminates a kinetic reaction. Avoid
bright light during colour development. Mean (X) 1.36 ng/ml
P9: [9 - rock gently for 20-30 sec. after each pipetting step Standard Deviation (S.D.) 0.33 ng/ml
without spilling the solutions to ensure thorough mixing. If available Expected Ranges ( 2 S.D.) 0.69 - 2.02 ng/ml
mix on a plate shaker.
Each laboratory should establish its own Expected Values utilising
Wash Procedure instrumentation, blood collection methods and testing techniques
The wash procedure is critical. Insufficient washing will result in commonly used in that laboratory as the T3 levels are much
poor precision or falsely high absorbance. influenced by geographical and dietary factors.
W1: Remove adhesive strips, aspirate off the contents, add IWASH!.
aspirate off after 30 sec. soak time and repeat washing twice. Performance Characteristics
W2: In case of automatic washers fill and prime with IWASHI. The T3 ELISA test has a analytic sensitivity of about 0.05 ng/ml T3.
Subsequently wash strips 3 times. Ensure the washer fills all wells Specimens with T3 concentrations above 7.5 ng/ml may be diluted
completely and aspirates off efficiently after 30 sec. (remaining with ICAll A and reassayed. To obtain the sample's concentration
liquid: < 15 Ill). multiply by the dilution factor.
W3 After washing, remove remaining liquid by tapping the plate
upside down on tissue paper. Typical performance data can be found in the Verification Report,
Pipetting Scheme accessible via
www.human.de/data/gb/vr/el-t3.pdf or
Reagents and specimens should be at room temperature before www.human-de.com/data/gb/vr/el-t3.pdf
use.
Step 1 Well [Ill] Note
E2 ... The components of the kit are stable until the expiry date even after
A1 ... D2
opening. However, a potential contamination is directly related to
Calibrators Specimen
the number of samplings. The 60 days limit after first use is set for
ICAll A-F; in duplicate 50 -- safety reasons.
Specimens, Controls; in duplicate -- 50 The handling should always be in compliance with common GLP
IWCONI 100 100 requirements (*)! The validation criteria must be met!
(*This includes: Proper caps being replaced on the vials and firmly tightened / Remove
Mix and cover IMlcl with Adhesive Strip
only reagents required for a run from stock solutions if they could come into contact with
Incubate 60 min. at 20 ...25C other contaminating solutions like patient specimens etc. / Stock solutions always
returned to 2...8C when not in use.)
Wash 3 times as described (see W1 - W3)
IWASHI 300 300
Step 2
ISUBI 100 100
Incubate 15 min. at 20 ...25C (see P8)
\sTopl I 50 I 50
Mix carefully
Measure the absorbance at 450 nm as soon as possible or
within 10 min. after terminating of reaction, using a reference
wavelength of 630-690 nm (if available).

Validation of the Test

The test results are valid provided the following criteria are met:
Maximum absorbance (calibrator 2A) 0.0. ~ 1.5
T3 concentration at 80% maximum absorbance = 0.60 0.20 ng/ml References
1. Barker, S.B., Determination of Protein Bound Iodine, Journal
T3 concentration at 50% maximum absorbance = 1.85 0.35 ng/ml
Biological Chemistry 173, 175 (1948)
T3 concentration at 20% maximum absorbance = 5.50 1.25 ng/ml
2. Chopra, I.J. ef a/., A Radioimmunoassay of Triiodothyronine, J.
Calculation Clinical Endocrino!. 33, 865 (1971)
A dose response curve is used to interpolate the concentration of 3. Young, D.S. ef a/., Effects of Drugs on Clinical Laboratory
triiodothyronine in unknown specimens. Tests, Clinical Chemistry 21, 3660 (1975)
1. ICAll - Plot the absorbance for each duplicate versus the 4. Sterling, L., Diagnosis and Treatment of Thyroid Disease,
corresponding T3 concentration in ng/ml on linear graph paper Cleveland CRC Press, p. 19 - 51 (1975)
(do not average the duplicates of the calibrators before
plotting).
2. Draw the best-fit curve through the plotted points.
3. To determine the concentration of T3 for an unknown sample
(S), locate the average absorbance of the duplicates on the
vertical axis of the graph, find the intersecting point on the
curve, and read the concentration (in ng/dl) from the horizontal
axis of the graph.

Interpretation of Results
Total serum triiodothyronine concentration is dependent upon a
multiplicity of factors: thyroid gland function and its regulation, TBG
concentration, and the binding of triiodothyronine to TBG3,4
Thus, total triiodothyronine concentration alone is not sufficient to
assess the clinical status.
Total serum triiodothyronine values may be elevated under condi-
tions such as pregnancy or administration of oral contraceptives. A

.......
&.7
..&_
decrease in T3 values is found with protein-wasting diseases, cer-
tain liver diseases and administration of hormones and drugs3.
EL-T3
INF 5401001 GB
05-2006-16
CE
Human Gesellschaft fUr Biochemica und Diagnostica mbH
Max-PlanckRing 21 - 065205 Wiesbaden - Germany
Telefon: +49 6122 9988 0 - Telefax: +49 6122 9988100 - eMail: human@human.de