Madiha Sana (2013-VA-957)
Indigenous Elisa Kit For Toxoplasma Gondii: Optimization Of Antibody Detection Elisa Of Sag 1 Protein As An Antigen In Mouse Model - 2015. - 39p.;
Toxoplasma is an apicomplexan intracellular parasite which is the cause of toxoplasmosis
in man and animals. It occurs by the ingestion of oocyst from feces of cats or by eating raw meat
in which cysts are present. It is the one of the major cause of encephalitis and abortion in
immuno-compromised animals and humans. As it is difficult to screen out infected live animals
from field, it is important to vaccine animals as well as humans for toxoplasma to prevent its
transmission from animals to humans and from humans to their off springs. Cloning of surface
antigen genes plays an important role in development of vaccine and for serology of T. gondii.
Enzyme linked immuno-sorbant assay proves to be a significant tool to estimate the humoral
response elicited against expressed recombinant protein in mice.
The recombinant protein of SAG1 was collected from Molecular Parasitology
Laboratory, University of Veterinary and Animal Sciences, Lahore. In the previous studies,
SAG1 sequence was cloned in the expression plasmid and successfully expressed in prokaryotic
expression system. In the current study, rSAG1 was quantified by using BCA protein assay
through BioWORLD protein quantification kit. In another experiment, the Swiss mice were
immunized with 15 μg rSAG1 protein 3 times with 2 weeks intervals. Two groups of mice were
formed with five mice in each group. Sera were collected after 2 weeks of each inoculation. For
performing ELISA, four different experiments were performed with different concentrations i.e.
5μg/ml, 250μg/ml and 500μg/ml with two different dilutions; 1/50 and 1/20. The O.D. values of
concentrations 5μg/ml and 250 μg/ml with two dilution series of 1/20 and1/50 were not observed
significant while the antigen coating concentration of 500 μg/ml with 1/50 dilution showed 1:160
titre and with 1/20 dilution showed 1: 1280 titre after the 3rd shot. The O.D values with 500
CHAPTER 6
SUMMARY
SUMMARY
36
μg/ml concentration with 1/20 dilution after the 3rd shot were observed significant in the
inoculated group as compared to the O.D values of un-inoculated negative group. It is suggested
to carry out ELISA with purified rSAG-1 protein and to optimize ELISA to test toxoplasma
infected mice.
Department of Parasitology
2433-T
Indigenous Elisa Kit For Toxoplasma Gondii: Optimization Of Antibody Detection Elisa Of Sag 1 Protein As An Antigen In Mouse Model - 2015. - 39p.;
Toxoplasma is an apicomplexan intracellular parasite which is the cause of toxoplasmosis
in man and animals. It occurs by the ingestion of oocyst from feces of cats or by eating raw meat
in which cysts are present. It is the one of the major cause of encephalitis and abortion in
immuno-compromised animals and humans. As it is difficult to screen out infected live animals
from field, it is important to vaccine animals as well as humans for toxoplasma to prevent its
transmission from animals to humans and from humans to their off springs. Cloning of surface
antigen genes plays an important role in development of vaccine and for serology of T. gondii.
Enzyme linked immuno-sorbant assay proves to be a significant tool to estimate the humoral
response elicited against expressed recombinant protein in mice.
The recombinant protein of SAG1 was collected from Molecular Parasitology
Laboratory, University of Veterinary and Animal Sciences, Lahore. In the previous studies,
SAG1 sequence was cloned in the expression plasmid and successfully expressed in prokaryotic
expression system. In the current study, rSAG1 was quantified by using BCA protein assay
through BioWORLD protein quantification kit. In another experiment, the Swiss mice were
immunized with 15 μg rSAG1 protein 3 times with 2 weeks intervals. Two groups of mice were
formed with five mice in each group. Sera were collected after 2 weeks of each inoculation. For
performing ELISA, four different experiments were performed with different concentrations i.e.
5μg/ml, 250μg/ml and 500μg/ml with two different dilutions; 1/50 and 1/20. The O.D. values of
concentrations 5μg/ml and 250 μg/ml with two dilution series of 1/20 and1/50 were not observed
significant while the antigen coating concentration of 500 μg/ml with 1/50 dilution showed 1:160
titre and with 1/20 dilution showed 1: 1280 titre after the 3rd shot. The O.D values with 500
CHAPTER 6
SUMMARY
SUMMARY
36
μg/ml concentration with 1/20 dilution after the 3rd shot were observed significant in the
inoculated group as compared to the O.D values of un-inoculated negative group. It is suggested
to carry out ELISA with purified rSAG-1 protein and to optimize ELISA to test toxoplasma
infected mice.
Department of Parasitology
2433-T