El i aad go
Enzyme Immunoassay for quantitative
‘cantration in human eerum and plasma, (for
Jinvltrodiagnostic use only)
‘SUMMARY:
Thyroxine hormone is secreted in the thyroic
important comaonent of
important
hyperthyrol
isease,
in those with myxedema,
the Mleveliedecreased
_ AEST PRINCIPLE:
Competitive E1A (Quanta
)
In a competive EIA, enzyme
competes
ized antibody coated on the micro wel
Unbound antigen fraction is then washed away.
The enzyme actvity in the antivody bound
ion which is inversely proportional to the
weasured by
ing calibrators
fof known antigen values, dose response cune
‘may be generated. from which the antigen
‘coneentrationafan unknown can be obtained
KIT CONTENTS:
41, TéReaction Coated Microplate (96 well)
ne 96-weil microplate coated with sheep ant
{thyroxine serum and packaged in an aluminum
Daguithacessicant Storeat23°C,
2. Té-Enzyme Conjugate (1.Smilvial)-[2)
‘One vial of thyroxine-horseradsh peroxidase
(HRP) conjugate ina bovine burst
‘alr A preservative has been added. Store at
26
Btoreat 28°C.
of serum reference fr tyro
tions of 0 (4A) 2.0 (4B). 5:0
*5.0(dE]and25.0[@F gi. Store
28°C, Apreservativehas baen added.
5. Substrate A (7mivial)-[5]
‘One botle containing tetramethylbenzidine
(TMia)inbuter. Store at23°C.
6. Substrate 8 (7mivial)-[6)
‘One bats containing hydrogen peroxide (+.0,)
Inbufler. Storost2 °C.
7. Wash Solution Concentrate (2%
‘One val containing a surfactant in buflored
saline. preservative has been added, Store at
230°C.
8. Stop Solution (@mitvial) [8]
(One botie containing a strong acid (1N HCI.
Storeat 230°C.
9, Productinser.
PRECAUTIONS:
4. Al Reagents are for in vo diagnostic uso
only
2. Please handle all reagants and controls
provided in the kit 25 potenlaly infectous
although they are non reactive for HIV 1 and
2, HBsAg and HCV ay FDA appraveditests,
3. The stop solution providedinthe ktisastrong
acid (1N HC). Please wear gloves and face
‘mask avoid contact with the skin,
4. Please use a disposable container or acid
vwashed tubes for preparing the substrate
vith INHOlor INH
2, Micro plate washer or a squeeze bottle
(optiona,
‘Quality control material
Timer.
Micro plate cover for incubation steps.
‘Storage container for storage of wash
bur.
7. Vacuum aspirator (optional) for wash steps.
8. Deionized water.
tes capable of delivering 25
1004
10. Dispensers) for repetitive dl
‘and 0.3 mi. volumes witha pri
than 1.5% (optional.
11, Containers for mixing reagents.
‘SAMPLE COLLECTION AND PREPARATION :
‘Serum or plasma may be used forthe test, Serum
jasma should be prepared trom a whole blood
specimen obtained by approved aseptic
technique. If testing cannot be done wi
hour after sample collection, refigerate
‘maximumof 48 hours) the specimen mec
PREPARATION OF REAGENTS AND
STORAGE:
1. Working Conjugate Solution
lugate solution ty
‘The prepared reagents should be slored at2.
8°C and must be used
preparation
2. Wash Solution
Preservedat21-25°Cfor60 days
When stored at 2.8°C the wash solution
concentrate may get crystalized, use that
cnly after thawing properly by keeping at
37°C forsome time.
3. Working Substrate Solution
Determine the amount ofreagentneededand
Brepare by mixing equal portions of Substrate
and Substrate Bin a sutable container. For
example, add Iml ofA and tml of B per two
{2)eight well strips (A sight excess of solution
Ismade, Discarc the unused portion).
Note: Do not use the working substrate if
Itlooks blue.
ASSAY PROTOCOL:
Before starting the assay, allow al the reagents,
serum references & controls 10 reach room
temperature,
1. Format the micro plate wells for each
callorator and patient specimen to be
assayed. Replace the unused
2. Add 25 ul of the calibrator and the patient
specimen to the assigned wes
enzyme conjugate
reagentto the bottom
3. Shake the microplate gently for 20-30
‘seconds & cover
6. Incubate for 60 min. at controlled room
temperature (21-25°C),
7. Aspirate the contents ofthe wells & f
complotely (approximately 300 pl) with
diluted- washing solution. Repeat the
aspiration and washing procedure two mere
times. After the ast wash, blotthe micro plat
on absorbent issue fo remove excess quid
is. Aways add reagent inthe same
mize reaction time aiference
9. Incubate at controled room temperature (21
25°C} forfteen minutes,
10, Add 80 lof Stop Solution to each well and
Wor 15-20 seaonds,
11, Read the absorbance in each well at 450 mm
{using a reference nave length of 620-630
‘Amo minimize well imperfections) in a micro
plate reader.
(QUALITY PARAMETERS:
Every control with known concent
hypothyroid, euthyroid and hyperthyroid range
‘ust be included in every run. Each laboratory
must establish lls own acceplable assay
performance limits. Run to. run reproducibly
Must be observed in a batch. If there is any
‘deviation from tne established data, t could be
‘due to degradation in the kit components. or
change inthe experimental cond
The absorbance ofthe calibrator 4A (0.0)
should be> 1.3 or an assay tobe valid
CALCULATION OF RESULTS:
A
& samples
reference
B. Plot @ point to
absorbance of
against concentration of each calibrator on
thexaxle
3 absorbance value for each sample
responding concentration
obiaines,
GUIDELINES:
A. Performanceoftheassay
1. Same sequence of reagent addition should
be maintained throughout
assay deitcan be avoided
2. Donottouchthebottom ofthe wells
3. Improperwashing may leactofaultyresuts.
4. Lipemic, hemolyzed_ and contaminated
specimen should not be used,
8B. Interpretation of results
1. The concentration obtained ofthe calibrators
valuesinygid
2 Total serum thyroxine concentration
obiainedis reguated by many factorsike theTBG. Thus total thyroxine concentration
ne Ie nat sufiient to assess clinical
Slandard deviation,
Intra Assay Reproducibility:
(Valve in ygial)
3 sets pooled samples (24 in each) were studied
‘and gave a coeficient of
3 sets of pooled samples were studied for inter
assay reproducblly and gave a coeficient of
‘variation of 8.3% for low value samples and <5%
for normal andhigh samples,
Accuracy:
‘Stacan"™ Té results wore compared with 2 coated
‘The correlation coefficient and least square
regression equation were completed for Sasa”
Téin comparison wit the reference method
‘A very high degree of correlation was found
between Btaeen'T4 and ine reference method
‘SENSITIVITY AND SPECIFICITY:
Sensitivity
80 pg. Equivalent to a sample containig a
concentration of.4 us,
Specificity
The extent of cross reactivity of Etseea™ 4 with
&
interfering substances was!
{L-Thyroxine 1.00
- Thyroxine -0.98
4- Trlodathyronine- 1.5% increase in 0.0.
1 Triodothyronine - 3% increase in O.D. at 100
Indio beas follows,
roxine” Clinical
Endocrinol, 33,865
2. Storing, L., Diagnosis and Treatment of
Thyroid Disease, Cleveland CRC Press, P.
3, Charkes ND, "The many causes of subclinical
hyperthyroidism. "Thyroid 1996;6:301-306,
Manutaetred by
urbe