ANAL YTICAL SENSITIVITY OF TSH ASSA YS BY ELISA AND ELF A [620478]

ANAL YTICAL SENSITIVITY OF TSH ASSA YS BY ELISA AND ELF A
157ABSTRACT
INTRODUCTION : The technological advances from radioimmunoassay to immunometric assays have
greatly enhanced the sensitivity of TSH assays over the last three decades and has firmly established TSH as
the single most reliable Thyroid function test in assessing the Thyroid hormone status of an individual.
AIMS AND OBJECTIVE : The objective of this current study is to compare the performance of TSH assay
based on Enzyme Linked Fluorescent Assay (VIDAS system) with Enzyme Linked Immunosorbant assay
technology (Accubind) and also to compare the analytical sensitivity of the TSH assay by the above two
technologies.
MATERIALS AND METHODS : Thyroid stimulating hormone levels have been assayed in the serum of
105 patients by both ELISA and ELFA so as to analyze the performance of the above two technologies. The
term analytical sensitivity refers to a minimal concentration of TSH that can be detected by an assay with
greater confidence.
RESUL TS : There exists statistically significant difference between assays by ELISA and ELFA in
Euthyroid (p<0.001**) and Hypothyroid samples (p<0.05). The precision CV for Hyperthyroid samples by
ELISA range from 7.7% to 39.4% whereas for ELFA the precision CV ranges from 3.4% to 14.5% thus
exhibiting the greater analytical sensitivity for ELFA.
CONCLUSION : TSH assay by ELF A has broader functional range, higher analytical sensitivity and also
higher specificity compared to TSH assay by ELISA technology .The actual purpose of this study is to fully
characterize the analytical performance of the two assays, to understand the capability and limitations of each
test so as to ensure that they are fit for routine use.
KEY WORDS : ELISA, ELFA, Thyroid stimulating hormone, analytical sensitivity
INTRODUCTION
hyroid stimulating hormone is the hetero-
dimeric glycoprotein (α and β) secreted from the
Tanterior pituitary gland. The assay of
thyrotropin is useful in detecting thyroid related
disorders. The technological advances from
radioimmunoassay to immunometric assays have
greatly enhanced the sensitivity of TSH assays over the
last three decades and has firmly established TSH as the
single most reliable Thyroid function test in assessing
1,2
the Thyroid hormone status of an individual . The
Addr ess for Corr espondence :
Dr.K.PRAMILA, M.D., Professor of Biochemistry , ICH & HC, Madras Medical College, Chennai.
Email : dr .[anonimizat] Phone no: 9444144099sensitivity of TSH assay has been further enhanced by
utilization of chemiluminescent and fluorescent signals
3,4
that has additional advantage of easier to automate .
There exists a physiological log/linear inverse
relationship between circulating TSH and FT4
5
concentrations . In fact, altered TSH level is the earliest
abnormality to be detected in a person with abnormal
6
Thyroid function . TSH assay is the excellent screening
test for the detection of Thyroid hormone status in an
individual, but not without disadvantages. In case of
National Journal of Basic Medical Sciences | Volume 6 | Issue 4 | 2016Original Article
1 2 2 3
Dr.K.Pramila , Dr.Poonguzhali Gopinath , Dr.K.Menaka Shanthi , Dr.M.Divya1 2 3
Professor , Asst.Professor , Final Year Post graduate – Department of Biochemistry
ICH & HC, Madras Medical College, Chennai.

158hypothyroidism due to hypothalamic Thyrotropin
releasing hormone deficiency the TSH assays with
current immunometric methodologies is within normal
limits. This is because TRH from Hypothalamus is
responsible for glycosylation of TSH more than release
of TSH from pituitary , so in TRH deficiency TSH is
non-glycosylated. This non-glycosylated TSH is
biologically inactive but retains immunoreactivity , so
detected by current TSH immunometric assays as
normal TSH levels even though the hormone is
7
biologically inactive .
There are various immunometric assays depending on
the signals used :
·Immunoradiometrmic assay (IRMA) –
It uses radio-isotope signals
·Immunoenzymometric assay (IEMA)-
It uses enzyme signals
·Immunoflourometric assay (IFMA)-
It uses flourophores as signals
·Immunochemiluminometric assay (ICMA)-It
uses chemiluminescent molecules as signals
·Immunobioluminometric assay (IBMA) –
It uses bioluminescent molecules as signals
The current study compares the Immunoenzymometric
assay ELISA with Immunoflourometric assay ELFA.
Enzyme linked immunosorbent assay has been proved
to be a useful test for the detection of TSH status of an
individual. Enzyme linked flourescent assay is newer ,
simple, reliable as well as sensitive method for the assay
of TSH. Both are immunometric assays, in ELISA the
enzyme hydrolyze the substrate to yield a colored
product and in ELFA the enzyme acts on the substrate to
produce a flourescent product.
MATERIALS AND METHODS
Blood samples were collected from 105 children
visiting Endocrinology OPD, Institute of child Health,
Egmore. Newborn babies and children with acute
illness were excluded from the study . Whole blood was
collected and subjected to centrifugation and the
separated serum is aliquoted in two fresh
microcentrifuge tubes– one for ELISA and other for
ELFA TSH Assays.PRINCIPLE OF ELISA :
Monoclonal biotinylated antibodies directed against
TSH are coated on the microwell plates. TSH in serum is
allowed to react simultaneously with two antibodies,
being sandwiched between solid phase and enzyme
linked antibodies(Fig 1). This sandwich complex is
immobilized to the well through high affinity
streptavidin- biotin interaction. After incubation at
room temperature for an hour, wells are washed to
remove excess antibodies. Addition of substrate results
in the formation of colored product. The enzyme and
substrate is allowed to react for 15 minutes by
incubation in room temperature. Stop solution is added
at the end of 15 minutes and the intensity of the color
developed is measured by ELISA reader set at 450nm.
FIG 1: SANDWICH ELISA
PRINCIPLE OF ELFA :
The principle of ELF A is same as that of ELISA which
utilize immunoassay sandwich methodology except
that instead of enzyme substrate reaction forming a
colored product, a final fluorescent product is formed by
reaction of the enzyme with substrate which is then
detected. The advantage of ELFA methodology is that it
can easily be automated.
REFERENCE INTER VAL FOR TSH :
The setting of reference interval for TSH is critical for
the diagnosis of mild hypo or hyperthyroidism. Current
guidelines recommend that “TSH reference intervals
should be established from the 95 percent confidence
limits of the log-transformed values of at least 120
rigorously screened normal euthyroid volunteers, who
should not have any detectable thyroid antibodies,
TPOAb or TgAb (measured by sensitive
immunoassay), do not have personal or family history of
thyroid dysfunction and have no visible or palpable
1
goiter , and are not taking any medications”. Recent
studies have suggested that TSH level increases in
National Journal of Basic Medical Sciences | Volume 6 | Issue 4 | 2016
Dr.K.PRAMILA, M.D., et.al : TSH Assays By ELISA and ELF A

159elderly and that a mild TSH elevation in elderly
8-10
individuals may even convey a survival benefit . Also
the TSH population reference range for adults will not
apply to neonates and children. Serum TSH values are
11-13
generally higher in neonates and children. . All these
studies point out the importance of establishing age
14
specific reference interval for thyroid . The reference
interval for TSH given by CALIPER study for the
children in the age group of six months to 14 years is 0.7
11
to 4.17mIU/L. Although Single nucleotide
polymorphism in genes responsible for Thyroid
hormone synthetic pathway(like PDE8B and TSH
receptor genes) may account for the euthyroid outliers
and account for skewing of established reference
15,16
intervals the utility of population based reference
range is very limited due to narrow inter individual
17 18
variability . According to Marwaha et al>95% of
Indian school children have TSH level between 5.28-
8.40µIU/mL and the mean serum TSH level is 1.83-
3.58µIU/mL. The reference range provided by the
manufacturer for ELISA kit is 0.39-6.16µIU/mL and by
ELFA it is 0.25-5.0µIU/mL. Serum TSH exhibits a
diurnal variation, that peaks between midnight to 04:00
19-21
hours . But all the blood samples are collected during
out-patient timing of 7:30 AM to 10:00 AM, hence
diurnal variation of TSH, practically has no impact on
this study . Current guidelines states that determination
of TSH levels in serum should be the first line screening
test for the detection of sub-clinical and overt hyper or
22
hypothyroidism . Hence utilization of highly sensitive
test for serum TSH determination is the need of the time
which is the purpose of this test.
RESUL TS
Out of 105 individuals, ELISA identifies 66 children to
be Euthyroid, 12 as Hyperthyroid and 27 children are
identified as being Hypothyroid (Table 1). ELFA
identifies only 63 as Euthyroid, 10 children as
Hyperthyroid and 32 children were classified as
Hypothyroid (Table 2).
The mean of Thyroid status of the individuals with
ELISA and ELFA were compared by unpaired student't'
test and p-Value was computed (Table 3). There is
statically significant difference between ELISA and **
ELFA in Euthyroid (p<0.001 ) and Hypothyroid *
(p<0.05 ) individuals. There is insignificant difference
in mean value between hyperthyroid individuals in the
study .ELISA has 27 as Hypothyroid and ELFA identifies 32 as
Hypothyroid. All the 27 patients have been compared
(Fig 2). ELISA identifies 12 as Hyperthyroid and ELFA
identifies 10 as Hyperthyroid. All the 10 patients have
been compared (Fig 3).
10 Hyperthyroid samples were selected and each
sample is run twice in ELISA and ELFA for a period of 3
days (Between run and between day variability is
determined) (Table 4). 10 Hypothyroid samples were
selected and each sample is run twice in ELISA and
ELFA for a period of 3 days (Between run and between
day variability is determined) (Table 5).
Table1: ELISA: EU/HYPO/HYPER THYROID
INDIVIDUALS
Table 2 ELFA: EU/HYPO/HYPER THYROID
INDIVIDUALS
Table 3: COMP ARISON OF THYROID STATUS BY
ELISA AND ELFA :
National Journal of Basic Medical Sciences | Volume 6 | Issue 4 | 2016
Dr.K.PRAMILA, M.D., et.al : TSH Assays By ELISA and ELF A

160FIG 2: Comparison between ELISA AND ELFA
methodology for Hypothyroid subjects:
FIG 3: Comparison between ELISA AND ELFA methodology for
Hyperthyroid subjects:
Table 5: Precision accuracy of ELISA and ELFA in Hypothyroid :Table 4: Precision accuracy of ELISA and ELFA in Hyperthyroid: DISCUSSION:
The radioimmunoassay for TSH measurement in
the initial era has limit of detection of 1 µIU/ml.
Introduction of immunometric assays have
improved the sensitivity by 10 fold, hence samples
with TSH levels as low as 0.1 µIU/ml can easily be
23
detected . The immunometric assays are inherently
more sensitive and has additional advantage that
24
they can easily be automated . However , the limit
of detection is not sufficient for TSH and even
lower levels are to be detected to differentiate
between sub-clinical and overt hyperthyroidism.
Hence it is need of time to utilize highly sensitive
assay for thyroid.
The assay of TSH by ELISA identifies 66
individuals as Euthyroid, but ELFA picked up only
63 persons to be Euthyroid, there is statistically **
significant difference(p<0.001 ) between ELISA
and ELFA for Euthyroid individuals. ELISA
identifies 27 persons as hypothyroid but ELFA
detects 32 persons as hypothyroid. The precision
CV of ELISA for hypothyroid samples ranges from
7.7% to 22.5%, whereas precision CV of ELFA for
hypothyroid samples ranges from 1.56% to 9.22%.
There is statistically significant difference as
computed by student- t test between ELISA and *
ELFA for the hypothyroid samples (p<0.05 ). There
is insignificant difference between ELISA and
ELFA for the hyperthyroid samples, possibly due to
lower sample size and the degree of significance is
not valid. The precision CV of ELISA for
hyperthyroid samples is 7.7% to 39.4% whereas for
ELFA it is 3.4% to 14.5%.
The Nomenclature Committee of the American
Thyroid Association has recommended that
Analytical detection limit of TSH assay should be
determined on the basis of Lower end inter-assay
precision characteristics. The committee reported
that precision co-ef ficient of variation at the lower
reporting limit should be 10-15%, but no worse
25
than 20% . The present study has got better lower
end precision CV for ELISA in the range of 0.2-
0.3µIU/ml whereas by ELFA technology there is
better lower end precision CV in the range of 0.05-
0.06 µIU/ml. So assay of TSH by flourescent
National Journal of Basic Medical Sciences | Volume 6 | Issue 4 | 2016
Dr.K.PRAMILA, M.D., et.al : TSH Assays By ELISA and ELF A

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26
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enzyme signals. The added advantage of
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technology provided by VIDAS system utilizes
Solid phase Receptacle (SPR) that acts both as solid
phase and pipetting device. All the required
reagents are pre-packed in the sealed reagent strips
and all the steps are carried out automatically by the
instrument. In conclusion, we found that TSH assay
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technology can detect TSH levels as low as 0.05
µIU/ml. Assay of TSH based on fluorescent signal
has higher precision and analytical sensitivity as
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of TSH by enzymatic signals.
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National Journal of Basic Medical Sciences | Volume 6 | Issue 4 | 2016Received on 04/01/2016, Modified on 06/04/2016, Accepted on 24/06/2016Dr.K.PRAMILA, M.D., et.al : TSH Assays By ELISA and ELF A