Limits...
Cloning, Expression and Purification of L. Donovani Specific Antigen for Serodiagnosis of Visceral Leishmaniasis.

Kumar D, Tiwary P, Dube A, Chakravarty J, Rai M, Sundar S - J Mol Biomark Diagn (2013)

Bottom Line: The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39.For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39.At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen.

View Article: PubMed Central - PubMed

Affiliation: Infectious Disease Research Laboratory, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, U.P, India.

ABSTRACT

Background: Rapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients.

Methods: The protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals.

Results: The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen.

Conclusion: Though the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.

No MeSH data available.


Related in: MedlinePlus

Sequencing result of L. donovani BHUP1 clone sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4007064&req=5

Figure 2: Sequencing result of L. donovani BHUP1 clone sequence.

Mentions: Using the primers, a 1959 base pair hsp70 gene fragment flanked by BamHI and EcoRI restriction sites was amplified (Figure 1A). The amplified product was gel purified and cloned in the pTZ57R/T vector (Figure 1B). Recombinant clones were selected by colony PCR using same primers. Clones were confirmed by BamHI/EcoRI digestion, and by sequence analysis (Figure 2). The amplified hsp70 gene fragment was 99% identical with L. donovani HSP70 in GenBank. The hsp70 gene fragment was sub cloned into pET28a+ (Figure 1C) and recombinant protein was purified (Figure 3).


Cloning, Expression and Purification of L. Donovani Specific Antigen for Serodiagnosis of Visceral Leishmaniasis.

Kumar D, Tiwary P, Dube A, Chakravarty J, Rai M, Sundar S - J Mol Biomark Diagn (2013)

Sequencing result of L. donovani BHUP1 clone sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007064&req=5

Figure 2: Sequencing result of L. donovani BHUP1 clone sequence.
Mentions: Using the primers, a 1959 base pair hsp70 gene fragment flanked by BamHI and EcoRI restriction sites was amplified (Figure 1A). The amplified product was gel purified and cloned in the pTZ57R/T vector (Figure 1B). Recombinant clones were selected by colony PCR using same primers. Clones were confirmed by BamHI/EcoRI digestion, and by sequence analysis (Figure 2). The amplified hsp70 gene fragment was 99% identical with L. donovani HSP70 in GenBank. The hsp70 gene fragment was sub cloned into pET28a+ (Figure 1C) and recombinant protein was purified (Figure 3).

Bottom Line: The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39.For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39.At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen.

View Article: PubMed Central - PubMed

Affiliation: Infectious Disease Research Laboratory, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, U.P, India.

ABSTRACT

Background: Rapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients.

Methods: The protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals.

Results: The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen.

Conclusion: Though the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.

No MeSH data available.


Related in: MedlinePlus