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Development of indigenous detection tool for Entamoeba histolytica from stool sample

Primary Information

Domain

Healthcare

Project No.

7814

Sanction and Project Initiation

Sanction No: F. No.: 3-18/2015-TS-TS.I

Sanction Date: 29/11/2016

Project Initiation date: 08/02/2017

Project Duration: 36

Partner Ministry/Agency/Industry

Ministry of Health and Family Welfare

 

Role of partner:

 

Support from partner:Received the financial support.

Principal Investigator

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Prof. Sudip K. Ghosh
Indian Institute of Technology Kharagpur, Kharagpur

Host Institute

Co-PIs

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Prof. T.K. Maiti
Indian Institute of Technology Kharagpur, Kharagpur

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Prof. Ananta K. Ghosh
Indian Institute of Technology Kharagpur, Kharagpur

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Prof. A.S. Ghosh
Indian Institute of Technology Kharagpur, Kharagpur

 

Scope and Objectives

Amebiasis is a diarrheal disease caused by the protozoan parasite, Entamoeba histolytica. It is one of the most common parasitic infections, affecting about 50 million people across the globe and resulting in 10,000 to 40,000 deaths per annum. The diagnosis of E. histolytica infection has traditionally involved microscopic examination of stool specimens which is highly unreliable on account of its less sensitivity and false-positive results. Newer diagnostic tests include ELISA based kits for detection of antigens in fecal samples which makes it more sensitive. However, these kits suffer from cross-reactivity issues as it fails to differentiate the other Entamoeba species. Diagnosis of E.histolytica infection would be improved by the identification of unique antigenic biomarkers that would not cross-react with other organisms. The purpose of this project is to design a sandwich ELISA based antigen detection kit exploiting highly antigenic and novel surface glycoproteins of E.histolytica that do not cross react with other intestinal pathogens.

Objectives
1. Expression and purification of recombinant novel Entamoeba histolytica glycoproteins for the production of polyclonal or monoclonal antibody against surface glycoproteins.
2. Testing of antigen specificity of the produced antibody against specific antigen.
3. Scheming and standardization of sandwich based ELISA for detecting E. histolytica from axenic culture, xenic culture and patients stool. 4. Screening of patients stool from diverse population for evaluation of reproducibility of the designed method

Deliverables

A sandwich ELISA based antigen detection kit, exploiting a set of highly antigenic and novel surface glycoproteins of E. histolytica as diagnostic markers for the detection of the pathogen from patients stool.

 

Scientific Output

To meet the above objectives, the work done is as follows:
1. Expression of EhS1, EhS6 and EhS17 and purification of EhS1 and EhS17.
2. Generation of antiEhS1 antibody.
3. Purification of the antibody
4. Preliminary testing of antigen specificity of the EhS1 and ES6 by ELISA and Western Blot.

 

Results and outcome till date

1. Expression of EhS1, EhS6 and EhS17 and purification of EhS1 and EhS17.
2. Generation of antiEhS1 antibody.
3. Purification of the antibody
4. Preliminary testing of antigen specificity of the EhS1 and ES6 by ELISA and Western Blot.

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Project image

 

Societal benefit and impact anticipated

Simple and cost effective: The kit will be designed in a manner so that it can be used conveniently in the remotest villages of India where there is a minimum molecular facility.

Next steps

1. Testing of anti-EhS1 antibody against total protein of E. histolytica and cross-checking the result with other Entamoeba species.
2. Antibody generation against EhS6 and EhS17 proteins.
3. Protein expression studies for other surface glycoproteins
4. Detection of EhS1, EhS6 and EhS17 by ELISA and Western Blotting with respective antibodies from E. histolytica whole cell lysates and stool samples.

Publications and reports

None yet

Patents

None yet

Scholars and Project Staff

At present Existing one research scholar is working on the project. One RA just left and the advertisement for the post is done.

Challenges faced

So far general difficulties in protein expression and purification was faced and solved. Otherwise not much problems were faced to be mentioned.

Other information

Conclusion:
1. EhS1, EhS6 and EhS17 have been expressed in recombinant forms.
2. EhS1, Eh S6 and EhS17 have been purified in bulk amounts.
3. AntiEhS1 antibody has been generated and purified.
4. AntiEhS1, anti EhS6 antibody were found to be positive against Native EhS1 and EhS6 antigen in E. histolytica.

Financial Information

  • Total sanction: Rs. 147.55 lakhs

  • Amount received: Rs. 108.8 lakhs

  • Amount utilised for Equipment: Rs. 13.45 lakhs

  • Amount utilised for Manpower: Rs. 5.24 lakhs

  • Amount utilised for Consumables: Rs. 16.94 lakhs

  • Amount utilised for Contingency: Rs. 1.85 lakhs

  • Amount utilised for Travel: Rs. 0.11 lakhs

  • Amount utilised for Other Expenses: 0

  • Amount utilised for Overheads: Rs. 18.13 lakhs

Equipment and facilities

 

1. Table top Refrigerated Centrifuge.
2. -80 degree freezer
3. 4 degree refrigerator
4. pH Meter, Vortex, pipetteman.