Cloning, expression and purification of toxocara canis recombinant antigens (rTES-26, rTES-32, rTES-120) and development of serodiagnostic test for toxocariasis
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Date
2009
Authors
Mohamad, Suharni
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Abstract
Routine serodiagnosis of human toxocariasis is based on indirect IgG-ELISA kits which
employ native Toxocara canis excretory-secretory (TES) antigen. However, these assays
lacked specificity especially when used in tropical countries where multiparasitism are
prevalent. In an effort to improve the diagnostic test for this infection, we have developed an
IgG4-ELISA assay which uses three recombinant antigens.
In this study, recombinant T. canis DNA which encode for rTES-26, rTES-32 and
rTES-120 were produced by cloning of open-reading frames (ORF) of the respective genes
via reverse-transcriptase-PCR (RT-PCR) using mRNA extracted from a culture of T. canis
second stage larvae into PCR2.1 TOPO vector. Sequence analysis revealed that TOPO/TES-
32 and TOPO/TES-120 were 100% similar to the reported sequences in the GenBank,
however, TOPO/TES-26 gene fragment had four mutations. After all mutations in
TOPO/TES-26 gene fragments had been corrected, TES-26 and TES-32 were subsequently
subcloned into a GST-tagged, while TES-120 was subcloned into a His-tagged prokaryotic
expression vector; and all constructs were expressed in E. coli BL21(DE3) expression host.
The recombinant proteins were subsequently purified under native condition by
affinity chromatography using GST and His-Trap resins since these recombinant proteins
were abundantly expressed in soluble form. The site-specific protease, Factor Xa, was used
to remove GST tag in the TES-26 and TES-32 GST-tagged fusion proteins. Western blot
analysis revealed that these recombinant antigens were immunologically reactive and
specific. Sera from patients infected with toxocariasis had IgG4 antibodies that recognized
these recombinant antigens, while sera from individuals with other infections and from
healthy normals did not.
When the three recombinant antigens were tested in ELISAs specific for
immunoglobulin IgM and IgE classes, and IgG subclasses (IgG1-IgG4), the results clearly
showed that only IgG4 assay displayed good specificity. The diagnostic utility of each
purified recombinant antigen and rTES-30USM (previously produced in our laboratory) was
further evaluated by IgG4-ELISA assay using 242 serum samples which included 30 sera
from patients with clinical, haematological and serological evidence of toxocariasis. Both
rTES-26 and rTES-32 IgG4-ELISAs demonstrated sensitivity of 80.0%; while rTES-120
IgG4-ELISA showed sensitivity of 93.3.0%, which is similar to that previously reported for
rTES-30USM IgG4-ELISA. The sensitivity of rTES-120/rTES-30USM IgG4-ELISA was
found to be significantly higher than rTES-26/rTES-32 IgG4-ELISA (p<0.001). However,
the mean O.Ds of the 30 toxocariasis samples among the IgG4 assays using the four
recombinant antigens were shown not to be significantly different (p>0.05). At p<0.05,
there was marginally no significant difference between the specificities of rTES-26 and
rTES-120 (p=0.059), rTES-26 and rTES-30USM or between rTES-30USM and rTES-120.
In the final assay, rTES-32 was excluded since it was not better than rTES-26 in
terms of sensitivity or specificity. Instead rTES-30USM was included due to its high
sensitivity and the fact that a 100% detection of toxocariasis cases was achieved when results
of IgG4 assays using rTES-30USM and rTES-120 were combined.
In summary, a final assay which is sensitive (80% to 93.3%) and specific (92.0%-
96.2%) for detection of toxocariasis was successfully developed using three adjacent wells,
each separately coated with rTES-26, rTES-30USM and rTES-120. This study is novel in
several ways namely it is the first report of the use of multiple recombinant antigens for
serodiagnosis of toxocariasis; the use of rTES-26 (and rTES-32) in Toxocara serodiagnosis;
the use of IgG4 assay for rTES-120 and rTES-26 and the use of GST tag in the expression
and purification of Toxocara recombinant proteins. These test maybe a significant
improvement over commercially available tests for diagnosis of toxocariasis and may be use
especially in countries co-endemic with other soil-transmitted helminthes.
Description
PhD
Keywords
Biological Science , Cloning , Recombinant antigens , Toxocariasis