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TIIIITIINllll. . .111 nil
Enzyme llnud lmmuno,ortient Assay (ELISA) for Quantitative Determination of
Total Triiodothyronlnt (T3) In Human Serum
FOR IN VITRO DIAGNOSTIC USE ONLY
SIM! al 2'C to 8' C
IIIT!NDEOU~
Quallsa• n Competitive B.JSA test is intended for lhe qvanlitative clelenrinatlon oflolal Trilldolhylo
ltine (T3) In hinan
serum. For In Vilto Dlagr,osllc Use only.

INTIIOOUCTIOII
The IDmones llyroxile (T4) and lriodolhyloltirte (T3) drcwle in lhe bloodslream . mosly boond to lhe plasma
tiyroxile tmding gtoblirl (TBG). The concenlrallon of T3 is much less lhan Iha! of T4, but its melabofic llfOlea\,
polency is much
greater. T3 determinationis an impoltlnt factor in the diagnosis of thyroid disease. tis measurement has uncovered
avariant
of hypertllyroidlsm In thyrotoxic patients wilh elevated T3 vakJes and normal T4 values. T3 determination
Is also useful In
monitoring both patients under lleatmectlor hype,11\yroidsm and patients who ha...e disoonllued ar.t>-thytoid
drug \herapy. In
addition tohype!1hyrokism. T3 IM!s are elevaled In women Vllto are preQnan~ and in women receiwlg oral
contraceptives or
estrogen treatment.

PRINCIPLE OF THE ASSAY

Qullisa'" T3 Quantitative Tesl Kil is a Cornpeilive enzym&-inked imroonosorbenl assay. In \tis lesl.
acertain all'Ol'lt of r i
T3 antibody is coated on JTicrotiter wells. Ameasured amo\111 of patiert serum, and a conllanl am0llll ol T3
conjugated wilh
horseradish peroxidase are added lo the microliter wells. During lno.ibalion, T3 antibody In the samples and
conjugated T3
compete for the limited bindilig sites on 111e antl-T3 antibody of the wells. Alter lncuballon lhe wells a111 washed
and bound
enzyme is detected by additg S11bstrate. The reaction is stopped aner specified time w1lll stop solution and
absorbance Is
detemined for eadl wet using an ruSA reader. The intansily ol lhe color lormed is popcnomll to the ammt
of enzyme
presenl and is inversely related lo lhe amount of unlabeled T3 In\he sample. By reference lo
aseries of T3 slaftdards assayed
in lhe sameway, lheCllllcenlration of T3in the unknown sample isquanUfied .

IIATERJALS AHO COMPONENTS

llatnJs plO'lldtd Wilfl Ille lest ldts:
1. Coated Mia'owells: Microwells coated wilh Anti- T3 antibody.
2. T3 HRPO Conjugate Diluent
3. T3 HRPO Enzyme Conjugate concen1ra1e (20X~
4. TMB Subsnte. Ready to use.
5. Stop Solution. Ready to use.
6. T3Standard setof6 Slandardslabeled as Ato Fin liquid form.Ready to use. For Standard Coocentrations
referviallabet
7. Wash BufferConcen1rat1 (20X~

~ · t9q11nd but nol provldtd

1. Predslon p~es: 10-100µ1, 50-200µ1, 100-1000µ1
2. Disposable pipette tips
3. Distilled water
4. Disposable Gbves
5. ELISA reader
6. ELISA washer

:, r0UGE ANO STABlUTY

n
1. Qudsa• kl Is stable at 2-8'C uploexpiry date prinled on lhe label.
2. Coaled mlcrowells should be used within one month upon opening lhepouch provided lhat once opened, the
pouch must
be resealed loprolectfrom moisture. lllhecolourofthe dessicant has changed lrom blue to white at 111etime
of opening
the pouch, anoll1ercoaled miaoweb poocn should be used.
l DilnedWash Bcllerisstable uptooneweek VlhenQ>l8dat 2-a•c .

SAMPLE COLLECTION
1. CoUect Blood spedmen by venipuncll.Jre according lo lhe standard procedure.
2. Onlyserumshouklbe used.
3. A'lllid grossly hemolytic. llpemicor llmd samples.
4. Preferably use fresh samples. However Specimens can be stored up to 48 hours at 2-8'C,for short duration.

~ - -- - - - -
~ 4~-- - -

. ;::-_-_ · _ ._-- -~--=--~ _- -- - - - - ~ ··_-

· _ - ~--=- ~ - -- -
5. f orlonger s~g e, specimens can be frozen at
6. Do not heat11actlvate before use. -20•C.Thawed samples ITIIJstbe mixed prior to
leslinn
1• S~ en contalning precipitate or particulate •·
matl
8. Spec,menshll\lld befreelrompartlculatemattera ershll\llcl be clarified bycen~gallon prior 10~se.
ndmicrobialoontamlrlatlon.
PRECAUTIONS
1. Bring an reagents and spedmon lo room
2. Do not p(pel\e any material by mou t&mperoture Defore use.
lh.
3. Do not ea~ drink or smoke in lhe area
where testing ls done.
4. Use protective clothil1g and wear glov
es when haruling samples.
5. Use absolbent sheet 10 CC1Vef the work
illg area .
6. lmmedialely dun up 1111y spl1s with sod
un l\ypoctllorile.
7. All speci'nens and standards should
8. Neolrallze acid oontainlng waste before
be oons idered pdenUally infectill\ls and discarded a
adding hypcchlorite. p~y
9. Do not use kit after the elll)iry date. ·
10. Do not nu compooenlS ol one kit with anoth
er.
11. #wa ys use new lip for each specimen
and reagent.
12. Do not allow liquid from or,e wel lo ITix with
oeher well\.
13. Do nol lel the strips dry in between the steps
.
~EM.ENT PREPAI IATION
t. All reagents should be brought to room tempetaw
re (U-25•C) and mixed by genly inverting or
notInduce foaming. swlr1ing prior to use. Do
2. DIiute Wash Buffer 20 limes (for exan
ple add 5ml conc:ennted butler to 95 ml dis\ll
before use. ed or deionlled water). Mix wel
3. DIiute Enzyme ~ug ate with Corjugal
e Diluent acco,dilg lo the requirement as shown
for each assay. below. Prepare atrash dilution
No. of Strips 0.5 1 2 3 4 5 6 7
Enzyme Conjugate (µI) 12.5 25
8 9 10 11 12
50 75 100 125 150 175 200 225 :I.SO
Conjugate DIiuent (µI) 250 500 1000 2.75 300
1500 200) 2500 3000 3500 4000 4500
5000 5500 6000
TEST PROCEDURE
1. Secure the desied number ol coaled wets
In the holder. Dispense 25 µI of Standards and
wells. Serums into the appropriate
2. Oispe(lse 50 µI of diuled Coo1Jgale into each
well. lnOlbale at room temperature (1 &-2S•c1.
3. Remove 1he Incubation mixture by empt
ying the plate contentinto awaste conlain«.Rins
•s
for minules.
5 limes -.it! Wash &lie r ( IX). Strike tile micm e and e~ llle mic:rotiter plate
liler plate shatply onto Ille absorbent paper or
residualwaterdlllplets. paper towels to remove an
4. Dispense 100 µI ol lMB Substrate into
eachwal. lnQlbate al room temperature(! 9-25
5. Slop the reacUon by adding 100 µI of Slop •ci in lhe datk. for 20 minutes.
Solution to each well Gently mix for 10 seconds
changes to yelow. untjl the blue colorcomplelely
6. Read Ille optical density at450/630 nm
willl amlaotiterplalereaderwllhin 15 minutes.

Add 25 µI Standards/sampes into 11:e respe

clive Miclowel

Add 50 µI dllle d Conju!jale in each well

~rJ. y p1a1e sealer .-.id incl.bale lo( 45 minll

tes at 1a.2 s•c

Wash MiClll'MllS S tl!IIIS wt, 350 µI of diluted

wash bu!ll!f

lncubate for 20 Minutes al 11·25'C in dark

Add 100 µI slop solution in each well

Read results
CALCULATION OF RESUlTS
Construd a slandard curve by plolti119 the absorbance obtain
ng/ni on lhe graph paper, with absorbance values on 1he venic:r~~ ~tlerence s_ landard against 11s concentration In
the absorbance values for each specimen 10 de axJS an conce,,1rat10ns on the horizontal or Xaxis. use
curve.Anydllutedspedmensmustbecorrectedby~l:~P:P:~~iiu'"t!'fuC:,cen1tati011 olT3 In ng/ml rrorn the standard

Example of Standard curve

Results of a typical slandard run with optical d sity 1
concenlrationsshown intheXaxis. en read ng 31 45onm (ref SOO-l00nm) shown in the Y axis against T3
Suggest Uae4-Parameter Slandardcurveto calculate sample values.
T3 Values (ng/ml) Absorbance
A 2.127
B 1.587
C 1.360
D 0.804
E 0.492
F 0,205

I\
"'-..
~

0.0
.........
-- --
This Slandatd curve Is for the purpose or illuSlratiln only and !Sli011ld not be used to calculate samples,Each user shoo Id oblaln
his or her own standard curve and dala.

Expec:wd Ranges ofvalues

Normal Range: 0.&-2.0 ng/ml
The rrinimal deteclableconcenllation orT3 by lhls assay is estimated 1o be 025 ng/ml.

PERFORMANCE CHARACTERISTICS
A) Internal Evaluation:
1. Accuracy: In an intemal study Quallsa'" T3 was evaluated against commercially available licensed kit with 90 random
cinlcal samples, & Quallaa'" T3 has demoostrated 100% cllnJcal correlatioo with the convnercially availablelicensed kil

2. Precision: Quallo• n was evaluated with licensed external Quality controls ror Precision Swdles & following Is the
data:
Controls No. of testing's Mean Control values with Quallaa"' T3 Coefficient or Variable (CV)
Level 1 10 0.942 3.40
Level 2 10 2,252 ,.o,
Level 3 10 3.204 423

BJ External Evaluation:
Quallsa'" Tl ELISA has been evaluated by a NABL accredited Jab agalnst lhelr reference method. In this evaluation
Qualisa'" T3 ELISA has demonsllaled 100%correlatlonwi1h 1he reference method.
'Data file: ZephyrBiomedicals (A Division of Tutip Diagnostics PvL Lid.).
 IMPORTANT NOTE
1. The T3 assay Is alemperalure sensi6ve assay. The besllemperalllre condition lor1his assay is from 1B"C11122•c.
2. The wash procedtre is aillcal. lnsuftrJent washlngwill lBSllllln poor precision and lal&elyeleva\ed ab$01banco readll9s.
3. II ls recomm.ended lo use the multi cllannel pipettes ID avoid time effecl.Alul plate of 96 wells may be used WaulOmated
pdpettillg Is avalable.
4. Oupdicallon ofslandards& samples rs not mandatory but may provide informa1Jon on reprodudbflijy &application error.;.

UMITATI0NS0FTHEASSAY
1. As with all lfiagnostic tests,a definlte clinlcaf diagnosis should not be based on the results or a single test but slloukl only
be made by the physlcian after al dinlcal and laboratory li1.dlngs have been evaluated.
2. The activity of the enzyme used is temperarure-dependenl and the OD values may vary. The higher1he room temperature
(+1B"C to +25' C) during substrate incubaUon, the greateir will be the OD values. Corresponding variations apply also to
the incubation ~mes. HoweYer, the standards are subject to the same inftuences, with the result Iha\ such variations wil
be largely compensated In the calculation of the result.
3. Adaptation of this assay for use wi1h automated sample procesSOIS and other liquid handllng devices, in whole orln part.
may yield drtterences in lest results from those obtained uslng the manual procedure. It Is the respons;blllly of each
laboratory to validate lhat their automated procedure yields lest results within acceptable limits.
4. ln~\lifJQent washing (e.g., less than 5wash cycles, too small wash buffer volumes, or shortened reaction limes) can lead
lo incorrect OD values.

BIBLIOGRAPHY'
1. Walker W.H.C. lntroduction:AnApproadl 1D Immunoassay. Clin. Chem. 19TT; 23: 384
2. KirkegaardC.,FriisT.andSier.;back•NlelsenK. AdaEndocrinot 1974;TT:71 .

...
Manufacl~red by:
Zephyr Biomedicals
-ffi ADivision ol Tulip Di~noslb {P) Ud.
M46-47. Phase 1118, Vema Industrial Estate, Vema. Goa · .W3 722. INDIA.
"-Od, Office: Git.anjali, Tulip Blodl, Or.Antonio Do~• Bagh. Allo SanlaauZ.
~
Ba1m0llmComplex P.O., Goa · 4D3 2ll2. INPIA.
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