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American Journal of  Medical 
Science and Innovation (AJMSI) 

Comparison of  ELISA & ICT Methods Determining Hepatitis B Surface in Suspected 
Patient Attending at Bangladesh Institute of  Health Science (BIHS)

General Hospital, Dhaka
Shohanur Rahaman1, Sadia Islam2, KM. Rashidul Islam Sakib3, Khaleda Ferdous4, Md. Nazmul Islam5

Md. Al-Amin Hossen6, Md. Ashiqur Rahman7*

Volume 2 Issue 2, Year 2023
ISSN: 2836-8509 (Online)

DOI: https://doi.org/10.54536/ajmsi.v2i2.1743
https://journals.e-palli.com/home/index.php/ajmsi

Article Information ABSTRACT

Received: May 20, 2023

Accepted: June 12, 2023

Published: July 18, 2023

Hepatitis B virus (HBV) is a common blood borne infectious agents causing high morbidity 
and mortality that constitute the major global health problems. Infection with HBV results 
in a wide spectrum disease from subclinical to fulminant hepatitis leading to death. The most 
important laboratory screening test for the diagnosis of  early HBV is the immunoassay for 
HBsAg. Different analytical methods are being used now a days for the diagnosis of  hepatitis 
namely, ICT, ELISA, CMIA and PCR. The aim of  the study to determine viral hepatitis 
(HBV) seromarkers in suspected patients using different methods ICT and ELISA. This 
cross sectional study was conducted at the Immunology laboratory, BIHS General Hospital, 
Dhaka. A total 240 of  HBsAg test results of  the patients were collected from Laboratory 
register book. All tests were carried out by ELISA & ICT methods and compare between 
them. Majority (76.3%) of  the donors was within the age group of  19-30 years. Among the 
donors, male donors were predominant (93.75%) than female (6.25%). A total number of  
240 HBsAg tests were done in ICT method and found 6 Positive (2.5%) of  them. Then we 
done correspondence test to confirm these by ELISA method and found 100% negative 
results. This study shows ICT method were able to determine HBsAg negative samples 
reasonably well that was detected negative by ELISA. ELISA method is more specific and 
sensitive than that of  ICT.

Keywords
HBsAg, ELISA, ICT, HBV

1 Diabetic Association of  Bangladesh (BADAS) COVID-19 Diagnostic Laboratory, Bangladesh
2 Bangladesh Specialized Hospital Limited (BSHL), Bangladesh
3 ZH Sikder Womens Medical College and Hospital, Bangladesh
4 Bangladesh University of  Health Sciences (BUHS), Bangladesh
5 Opsonin Pharma Limited, Bangladesh
6 United Hospital Limited, Bangladesh
7 Novus Clinical Research Services Limited (NCRSL), Bangladesh
* Corresponding author’s e-mail: ararashiqur@gmail.com

INTRODUCTION
Hepatitis B virus (HBV) is a common blood borne 
infectious agents causing high morbidity and mortality 
that constitute the major global health problems. 
Approximately one fifth of  the world populations 
are being chronically infected with HBV. Death of  1.5 
million people every year attributed to HBV related 
chronic liver diseases (Hayder et al. 2012). Infection with 
HBV results in a wide spectrum disease from subclinical 
to fulminant hepatitis leading to death. Hepatitis due to B 
virus often progress to chronic active hepatitis, cirrhosis 
of  liver with development of  hepatocellular carcinoma. 
Since these virus are mostly transmitted by transfusion 
of  contaminated blood and blood product, however, 
other subjected modes of  transmission like intravenous 
drug abuse, close personal contact, use of  shared needle, 
razor etc cannot be ignored (Ahmed et al. 2009). It 
commonly causes asymptomatic infection but chronic 
infection causes scarring of  the liver which is generally 
apparent after many years. Approximately, 75% acutely 
infected patients develop chronic hepatitis B infection 
that commonly progresses to liver cirrhosis and hepatic 
malignancy (Ahmed et al. 2009).
The HBV is found to be rapidly spreading in the 

developing countries including Bangladesh. It found to 
be 50 to 100 times more infectious than HIV and an 
important occupational hazard for health workers. About 
2 billion people worldwide have been infected with 
the virus and more than 350 million live with chronic 
lifelong infection (WHO, 2000). HBsAg appears during 
the incubation and becomes detectable in most cases 
during the prodrome and acute stage of  the disease. It 
usually disappears from the blood within 5-6 months in 
young adults. However, prolonged persistence of  the 
HBsAg for more than six months indicates the carrier 
state. These viruses are still causing the most clinically 
significant transfusion transmissible infections with a per 
unit risk of  1: 82,000 (Riskin et al. 2009). Looking into 
the data on the prevalence of  the transfusion transmitted 
infections (TTIs), specifically HBV among blood donors 
permits an assessment of  the occurrence of  infections in 
the blood donor population and consequently the safety 
of  the collected donations. It also gives an idea of  the 
epidemiology of  these diseases in the community. Most 
of  the studies conducted in Bangladesh are limited among 
the professional blood donors, drug addicts, commercial 
sex workers or hospitalized patients (Islam et al. 1984). 
The rapid spread of  HBV infection and the changed 

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scenario in blood donation practice has inspired us to 
depict the trends of  the prevalence of   HBsAg infection 
among the healthy blood donors, and to describe their 
socio-demographic background at Hospitals in Dhaka 
city in Bangladesh. Such information could be a guide 
for planning and implementing programs for preventive 
measures in Bangladesh.The most important laboratory 
screening test for the diagnosis of  early HBV is the 
immunoassay for HBsAg. Different analytical methods 
are being used now a day for the diagnosis of  hepatitis 
namely, ICT, ELISA, CMIA and PCR. The ELISA, 
CMIA and PCR methods are found to be expensive and 
are used in advanced laboratories and major tertiary care 
hospitals. ICT, using by the most of  the laboratories as a 
rapid diagnostic analytical method are now considered a 
good choice because of  its low price, less involvement of  
skilled technician and infrastructure (Rahman et al. 2008). 
The rapid ICT kits are known to have less sensitivity 
and specificity than ELISA. The aims of  this study is to 
determine HBsAg in patients’ blood by using ICT and 
ELISA. ELISA is a wet lab type analytical assay that uses 
a solid phase enzyme immunoassay to detect the presence 
of  an antigen or antibody in a liquid phase. Along with 
the enzyme-labeling of  antigens or antibodies, the 
technique involves an immune reaction, enzymatic 
chemical reaction, signal detection and quantification in 
combination which make it one of  the most specific and 
sensitive than other immunoassays to detect the biological 
molecule. Although confirmation of  hepatitis B infection 
is based on advanced immunological, molecular and 
histological techniques (Mustafa et al. 1989) laboratory 
use immune-chromatographic test strips to screen 
hepatitis. Their mode of  action is based on common 
principle of  antibody present in the test serum or plasma 
reacting with the protein coated particle and migrating 
upward on a membrane chromatographically by capillary 
action to react with recombinant antigen present on the 
membrane thereby generating a color line in the test 
region. These test strips are one step rapid test. The test 
strips manufactures also claim that these test strips have 
relatively high sensitivity, specificity and accuracy but 
controversy often arises regarding these claims (Ahmad 
et al. 1991). Reports from various studies suggest that this 
rapid test method not always confirms the accuracy of  the 
test results, increasing the risk of  blood borne infection 
during blood transfusion. Very early stages of  infection 
and patients in the recovering stages usually have low viral 
titers, reflected by low optical density (Low SOD) in the 
test results. These low positive cases may not be detected 
by rapid screening test like ICT, these can be detected 
by ELISA. However it should be kept in mind that 
method standardization is always crucial before diagnosis 
of  an infection. A major concern in utilizing screening 
tests is that these tests should have a high performance 
in detecting infections irrespective of  stages of  disease. 
Therefore the intent of  this study is to compare rapid 
test strip screening method with advanced immunological 
techniques and to recommend for a reliable, cost-effective 

and less time consuming laboratory method for detection 
of  HBV seromarkers in donors’ blood.
The aim of  this study is to determine hepatitis B surface 
antigen (HBsAg), by ELISA and ICT method among 
patients and compare the test results of  HBsAg seromarker 
obtained by the above methods for evaluation in respect 
of  sensitivity, specificity and accuracy of  those methods. 
This study will help to evaluate the methods, comparatively 
better for the detection of  viral hepatitis seromarkers.

MATERIALS AND METHODS
This is a cross-sectional study. The study was conducted 
by obtaining the blood samples from donors attending at 
the  BIHS General Hospital Ltd, Dhaka and Bangladesh 
University of  Health Sciences (BUHS). The study was 
conducted during the period of  From February to 
October 2018. Patients qualifying criteria for the donation 
were included in the study. The qualifying criteria for the 
participants were: age between 18 to 60 years with clinical 
feature for hepatitis infection. Participants having low 
body weight, low blood pressure and anemia, professional 
blood donors, drug abusers, pregnant women, etc. were 
excluded from the study. Persons having positive history of  
HBV, HCV, HIV and venereal disease were also excluded 
from the study. A purposive sampling was followed. The 
entire samples collected from the laboratory record book 
those are fulfill the inclusion criteria of  study.

Collection of  Blood Sample, Processing and 
Preservation
Five milliliters of  blood were collected by standard aseptic 
technique in the coded vacutainer. The blood was kept 
stand still to allow clotting and the serum was separated 
by centrifugation at 4000 rpm for 15 minutes. The 
serum samples were transferred to two micro centrifuge 
tubes. One tube for each participant was taken to the 
immunology laboratory of  the BIHS General Hospital 
for detection of  seromarkers for HBV by ICT methods 
and second was preserved for ELISA test to be done at 
the Immunology Laboratory, Bangladesh University of  
Health Sciences (BUHS).

Laboratory Methods
Screening of  HBsAg was done by ICT in immunology 
laboratory of  the BIHS General Hospital Ltd. Kits used 
for ICT were from Excel. ELISA 4th generation kits 
(Enzo diagnostics Inc, USA) were used as gold standard 
for comparative evaluation1. 

Test Procedure
Procedure of  HBsAg detection (Sandwich ELISA, 
Wantai Biologicals, China)
A microtiter plate coated with known quantity of  
monoclonal antibody (solid-phase) is taken. Serum 
sample is added to the plate that may or may not contain 
HBsAg. HBsAg if  present in the serum, bind to solid 
phase antibody in the plate during incubation (at 37°c). 
The plate is then washed at the end of  incubation to 

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remove unbound antigen and any other non-specific 
binding. After soaking the plate on a paper towel, enzyme-
linked antihuman antibodies (conjugate) which are also 
specific to HBsAg antigen are added and then incubated 
at 37°c. The plate is washed with working buffer solution 
to remove unbound conjugates. Substrate is added so that 
enzyme act on the substrate to produce a colored reaction 
product. Finally, blocking reagent is added to stop the 
enzymatic reaction and absorbance is measured using 
specific wavelength by Spectrophotometric plate Reader. 

Procedure for HBsAg (ICT)
• Test cassette, serum or plasma specimen, and /or 

controls were allowed to equilibrate to room temperature 
(15-20 c) prior to testing.

• The pouch was brought to room temperature before 
opening it. The test cassette was removed from the sealed 
pouch and used it as soon as possible.

• For Serum or Plasma specimen.
• The dropper was hold vertically and  3 drops of  

serum or plasma (approximately 120 ul) was transferred 
to the specimen well of  test cassette and the timer was 
started.

• After waiting for few minutes a colored line appeared. 
The result was read at 15-30 minutes.
 
Interpretation
Positive
Two distinct colored lines appear. One colored line 
should be in the control region(C) and another colored 
line should be in the test region (T).

Negative
One colored line appears in the control region(C). No 
apparent colored line appears in the test region (T).

Data Collection
Variables recorded at the time of  enrollment of  study 
subjects include participants’ demographics, personal 
history, vital signs and clinical findings. Data relating 
to clinical information were taken from pre-designed 
questionnaire sheets of  donors kept in the Blood bank of  
the BIHS General Hospital. Donors consent was obtained 
in the questionnaire sheets by the Blood bank personnel 
before donation of  blood and laboratory reports.

Data Analysis 
Data were analyzed with the help of  the software SPSS 
(Statistical Package for Social Sciences) version 23 and 
Microsoft Excel 2016. The results were expressed as 
mean ±SD (standard deviation).  The p-value <0.05 was 
considered as statistically significant.

RESULTS
Total of  240 patients were included in this study. Mean 
(±SD) age was 27.45±7.63 years. Age distribution of  the 
donors was shown in table I. Majority (76.3%) of  the 
donors were within the age group of  19-30 years, 16.3% 

Among the total 240 patients, the male was 225 (93.75%) 
and the female was 15 (6.25%). Among the donors, male 
donors were more predominant (93.75%) than females 
(6.25%) (Figure 1).

Table 1: Distribution of  patients on the basis of  age groups
Age 
(yrs)

Frequency Percentage 
(%)

Mean±SD

19-30 183 76.3 27.45±7.63
31-40 39 16.3
41-50 9 3.8
>50 9 3.8
Total 240 100.0

Results were expressed as number (percent) and mean±SD as 
appropriate

Figure 1: Gender distribution of  the patients

Table 2 shows that all the specimens were also screened 
for HBsAg by immunochromatography (ICT) method. 
Out of  240 samples, 6 (2.5%) samples were positive and 
234 (97.5%) samples were negative.

Table 2:  Distribution of  seromarkers status analyzed by 
Immunochromatography (ICT) (n= 240) 
Seromarkers ICT P 

valuePositive Negative
No % No %

HBsAg 6 2.5% 234 97.5% -
Results were expressed as number and percent

Table 3 showed that target variables hepatitis B surface 
antigen (HBsAg) were determined by Enzyme linked 
immunosorbent assay (ELISA). Out of  240 samples, 
240 sample (100%) were negative. The 6 samples that 
were positive by ICT method were also negative by 
ELISA method.

of  the donors was within the age group of  31-40 years, 
and 3.8% of  age group 41-50 years and >50 years.

Table 3:  Distribution of  seromarkers status by ELISA 
(n=240) 
Seromarkers ELISA P 

valuePositive Negative
No % No %

HBsAg 0 0% 240 100% -
Results were expressed as number and percent

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DISCUSSION
Screening for common blood borne infection like HBV is 
crucial to ensure the safety of  transfusion. The selection 
of  screening tests depends upon a number of  factors. 
Among them, evaluation of  test performance, measured 
by sensitivity and specificity, is the most important factor 
while kit cost, equipment used, expertise, consumables and 
disposables are also taken into account during selection of  
analytical process (Saadia et al. 2016). This is for the first 
time through a structured protocol; ICT was compared 
with gold standard ELISA method using donors’ blood 
for HBV seromarker HBsAg. Almost all donors were 
male (93.75%) which is consistent with other reports more 
specifically by Khan et al where 96.8% of  the blood donor 
population was male as males are the predominant donor 
both in developed and developing countries (Sajed et al. 
2014; Khan et al. 2011) Mean (±SD) age of  the donors 
was 27.45±7.63 and 76.3% (183 out of  240) of  them were 
between the age group 19-30 years. This demonstrated the 
fact that donors belong to the younger age groups which 
is consistent to the study done by Sajed (Sajed et al. 2014). 
In their study 80% donors belonged to the thirties years 
of  age. The present study compared ICT and ELISA for 
hepatitis B surface antigen HBsAg. Test result for HBsAg 
was found negative for all 240 samples in both ICT and 
ELISA method. 
Reference centers or central blood banks found to be 
widely using most sensitive test methods (ELISA, CMIA 
and PCR) as quantitative immunoassay globally (Hayde et 
al 2012; Clement et al.2002). Rapid test, ICT is intended 
for qualitative detection of  HBsAg in serum (Torlesse et 
al. 1997). ELISA, CMIA and other advanced methods 
are laboratory based, time consuming and require trained 
laboratory personnel. Chemiluminescence based assays 
are usually used for screening of  blood donors in high 
volume blood banks owing to automation facilities, higher 
testing throughput and objective interpretation of  results, 
however, expensive instrumentation is required for them 
thus limiting their use in resource limited settings (Saadia 
et al. 2016).  
Rapid test enables early detection at sites where laboratory 
facilities or trained manpower are not available or there 
is issue of  accessibility. Most rapid tests are based on 
immunochromatographic principles (Plitt et al. 2007). The 
rapid tests reduce the potential loss of  follow up of  a case 
when test results are on demand right away (Sato et al. 1996; 
Raj et al.2001). ICT showed potentially good findings in 
the present study. No false positive test was observed by 
this method since it provided negative results of  all 240 
donors’ sample for both HBsAg and anti-HCV that was 
also revealed to be negative by ELISA. Using ICT for both 
infections, ICT and CMIA were equally sensitive to ELISA 
as all the 240 samples showed negative reaction in both 
ICT and ELISA though 1 (1.25%) sample was positive for 
HBsAg. Our results showed comparable performances of  
the three techniques with almost 99% agreement of  results.  
In evaluating both the seromarkers, specificity and negative 
predictive value of  ICT were 100% that was consistent 

with a study done in Pakistan where ICT and ELISA were 
compared for detection of  HBsAg in healthy individual 
from Karachi that showed comparable sensitivity and 
specificity of  ICT kits with ELISA technique (Shamsul 
et al 2001). It is again consistent with another study done 
by Herring (Herring et al. 2006) where evaluation of  nine 
rapid syphilis ICT kits reported 93-98% specificity. A meta-
analysis reported the sensitivity of  different ICT devices 
ranging from 85-100% and specificity 98-100% (Jafari et 
al. 2013). An Indian study reported 100% specificity and 
93.4% sensitivity of  rapid kits when detecting HBsAg 
(Kaur et al. 2000).  ICT is suitable for use in remote and 
developing regions since they are simple to perform, can 
be transported, stored and performed at room temperature 
and microscopic and electrical equipment not needed. 
Moreover, these are cheaper and quicker as compared to 
other diagnostic procedures (Herring et al. 2006).
Findings of  the present study is also consistent with study 
conducted in Iran where 6 rapid strips/devices were 
compared with gold standard method (Khadem et al 2007). 
In another study from Seoul for detecting HBsAg, rapid 
technique showed 97% sensitivity and 100% specificity 
(Irwig et al. 2002). In our study, overall specificity results 
for both HBsAg was high i.e. 97-100%. These results are 
different to an study conducted in Lahore, Pakistan by 
Khan (Khan et al. 2010) who demonstrated 93% to 100% 
specificity for HBsAg by ICT method but the sensitivity was 
50% for both HBsAg. In the present study sensitivity was 
100% for HBsAg which is higher than the just mentioned 
study. The present study was carried out as a pilot basis 
to compare two methods ICT with ELISA for HBsAg. 
Although the number of  samples tested was limited yet we 
could infer that the two methods had performed equally 
well and in limited resource settings, the ICT could be used 
as an alternative for HBV seromarker screening.  
A total number of  240 HBsAg tests were done in ICT 
method and found 6 Positive (2.5%) of  them. Then we 
done correspondence test to confirm these by ELISA 
method and found 100% negative results. We know, 
ELISA method is more specific and sensitive than that of  
ICT. We conclude that, those 6 positive results found in 
ICT method was false positive. 

CONCLUSION
This study shows ICT method were able to determine 
HBsAg negative samples reasonably well that was detected 
negative by ELISA. The rapid test was not only compatible 
with currently established and advanced diagnostic 
methods but also cheaper. It can be recommended that 
ELISA comparable rapid devices may be allowed to be 
used for initial screening of  hepatitis B, in remote areas 
where cost is an issue. The present study concludes that 
our findings demonstrate comparable performances 
of  ICT and ELISA for screening of  HBV seromarker. 
We suggest using the ICT assay in situation i) where the 
laboratory cannot afford to have a more advanced system 
for blood donors ii) as a backup and iii) in life-threatening 
situations where time saving may be life-saving. 

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Financial Support & Sponsorship
None 

Conflicts of  Interest
The author hereby declares that there are no conflicts of  
interest concerning this paper.

Informed Consent
Informed consent was obtained from all individuals 
included in this study.

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