Abstract:
Hepatitis B infection is a disease of the liver caused by Hepatitis B Virus (HBV), a
double-stranded DNA virus coated with an envelope containing Hepatitis B surface
antigens (HBsAg). HBsAg levels in blood are high as long as the viral particles continue
to exist in the liver cells. Hence they are the most important markers used in screening for
the presence of Hepatitis B infection in many of the diagnostic test kits in the market. The
currently available ELISA diagnostic kits for HBV are both imported and expensive.
The main objective of this study was to develop a cost-effective Enzyme-linked
Immunosorbent Assay (ELISA) kit for detection of HBsAg in plasma and serum using
polyclonal antibodies produced in KEMRI.
The capture polyclonal antibodies were obtained from KEMRI Production department
where they had been raised in guinea pigs inoculated with locally prepared HBsAg. The
Sandwich-based ELISA system was prepared by coating capture antibodies on 96-well
microplates and blocking the void spaces using the Bovine Serum Albumin and Tween
20 (BSAT) blocking buffer after which samples were applied. The Horse Radish
Peroxide (HRP)-linked ovine HBsAg detection antibodies were incubated in the wells
and washed with Tween 20 Wash Buffer to remove the unbound antibodies. The 3, 3’, 5,
5’ tetramethylbenzidine (TMB)-hydrogen peroxide substrate was incubated for a 30
minutes and absorbance values read using an ELISA plate reader. The reagents used in
preparation of this kit were optimized using the ELISA checkerboard technique.
The developed kit was assessed and found to have diagnostic sensitivity of 96.1%,
diagnostic specificity of 100%, positive predictive value of 100% and negative predictive
value of 95.7% with Hepanostika Ultra HBsAg kit as a gold standard. The analytical
xv
sensitivity of the kit was found to be 4.62ng/ml and no analytical non-specificities were
noted in samples positive for HIV and HCV respectively. The kit showed desirable
repeatability profile with the coefficient of variance of intra-run repeatability of 1.38%
and inter-run repeatability of 5.3% against the limit of 20%. The overall inter-observer
variation agreement, Kappa statistic, was found to be 1 (one) signifying perfect
correlation while the Pearson correlation coefficient (r2) to determine correlation between
values measured by different assays were 0.917, 0.939 and 1.00 against three established
kits.
The ELISA kit developed in this study will be tested further in the field for a period of
one year before it is applied for registration at the National Public Health Laboratory
Services, Nairobi. It will also be validated against a number of mutagenic cysteine
variants and subtypes on the “a” determinants of HBsAg.
