Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens

G. Halli R. Rajasekariah, Jeffrey R. Ryan, Scott R. Hillier, Lisa P. Yi, John M. Stiteler, Liwang Cui, Anthony M. Smithyman, Samuel K. Martin

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 μg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref - ve sera and higher absorbance with Ref + ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref + ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 μg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n = 22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

Original languageEnglish (US)
Pages (from-to)105-119
Number of pages15
JournalJournal of Immunological Methods
Volume252
Issue number1-2
DOIs
StatePublished - Jun 1 2001

Fingerprint

Visceral Leishmaniasis
Serologic Tests
Enzyme-Linked Immunosorbent Assay
Antigens
Serum
Caseins
Glyoxal
Monoclonal Antibodies
Antibodies
Antigen-Antibody Reactions
Leishmania donovani
Sensitivity and Specificity
Northern Africa
In Vitro Techniques
Serum-Free Culture Media
Polyacrylamide Gel Electrophoresis
Buffers
Temperature

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

Cite this

Rajasekariah, G. H. R., Ryan, J. R., Hillier, S. R., Yi, L. P., Stiteler, J. M., Cui, L., ... Martin, S. K. (2001). Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens. Journal of Immunological Methods, 252(1-2), 105-119. https://doi.org/10.1016/S0022-1759(01)00341-6
Rajasekariah, G. Halli R. ; Ryan, Jeffrey R. ; Hillier, Scott R. ; Yi, Lisa P. ; Stiteler, John M. ; Cui, Liwang ; Smithyman, Anthony M. ; Martin, Samuel K. / Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens. In: Journal of Immunological Methods. 2001 ; Vol. 252, No. 1-2. pp. 105-119.
@article{6bf5f6b87c834eaa9bd003a8c1149f05,
title = "Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens",
abstract = "An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 μg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5{\%} casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5{\%} casein, generated lowest absorbance with Ref - ve sera and higher absorbance with Ref + ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref + ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 μg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5{\%} casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n = 22 endemic sera from North Africa were evaluated and resulted with 100{\%} specificity and sensitivity, 99.99{\%} PPV and 95.45{\%} NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.",
author = "Rajasekariah, {G. Halli R.} and Ryan, {Jeffrey R.} and Hillier, {Scott R.} and Yi, {Lisa P.} and Stiteler, {John M.} and Liwang Cui and Smithyman, {Anthony M.} and Martin, {Samuel K.}",
year = "2001",
month = "6",
day = "1",
doi = "10.1016/S0022-1759(01)00341-6",
language = "English (US)",
volume = "252",
pages = "105--119",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",
number = "1-2",

}

Rajasekariah, GHR, Ryan, JR, Hillier, SR, Yi, LP, Stiteler, JM, Cui, L, Smithyman, AM & Martin, SK 2001, 'Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens', Journal of Immunological Methods, vol. 252, no. 1-2, pp. 105-119. https://doi.org/10.1016/S0022-1759(01)00341-6

Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens. / Rajasekariah, G. Halli R.; Ryan, Jeffrey R.; Hillier, Scott R.; Yi, Lisa P.; Stiteler, John M.; Cui, Liwang; Smithyman, Anthony M.; Martin, Samuel K.

In: Journal of Immunological Methods, Vol. 252, No. 1-2, 01.06.2001, p. 105-119.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Optimisation of an ELISA for the serodiagnosis of visceral leishmaniasis using in vitro derived promastigote antigens

AU - Rajasekariah, G. Halli R.

AU - Ryan, Jeffrey R.

AU - Hillier, Scott R.

AU - Yi, Lisa P.

AU - Stiteler, John M.

AU - Cui, Liwang

AU - Smithyman, Anthony M.

AU - Martin, Samuel K.

PY - 2001/6/1

Y1 - 2001/6/1

N2 - An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 μg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref - ve sera and higher absorbance with Ref + ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref + ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 μg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n = 22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

AB - An antibody detection ELISA was developed for diagnosis of visceral leishmaniasis. Antigens released by Leishmania donovani promastigotes into a protein-free medium were used. SDS-PAGE analysis has indicated that Ld-ESM contain several protein antigens. Titration and chequer-board analyses were performed to optimise the assay protocol. Optimal results were obtained when antigen (50 μg/ml) was coated with PBS-methyl glyoxal buffer, and wells blocked with 0.5% casein. A serum dilution of 1:500 in antigen-coated wells, blocked with 0.5% casein, generated lowest absorbance with Ref - ve sera and higher absorbance with Ref + ve sera. All steps of the ELISA were performed at room temperature. The S/N ratio, the differential absorbance between the negative sample vs. the test or Ref + ve sample, was used to quantify the specific antigen and antibody reactions. An anti-human monoclonal antibody conjugated with HRP (MAb-conjugate) outperformed a commercially available anti-human polyclonal antibody conjugate (PAb-conjugate). The MAb-conjugate gave minimal background reactions with endemic sera. Optimised final assay steps mentioned below were used to evaluate sera samples from field trials. ELISA wells were coated with 50 μg/ml Ld-ESM mixed in PBS-methyl glyoxal overnight, and after removing the antigen, blocked with 0.5% casein for 1 h at RT. Patient sera along with control sera, diluted to 1:500 in PBS/T, were reacted for 1 h at RT. After washing the plate with PBS/T, wells were reacted with MAb-conjugate for 40 min at RT, and after washing, binding of antibodies was visualized by using TMB as a chromogen substrate. The relative specific binding was quantified by the S/N ratio. A batch of n = 22 endemic sera from North Africa were evaluated and resulted with 100% specificity and sensitivity, 99.99% PPV and 95.45% NPV. The specificity and sensitivity of this assay will be further evaluated in planned retrospective and prospective multi-site trials.

UR - http://www.scopus.com/inward/record.url?scp=0035371663&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035371663&partnerID=8YFLogxK

U2 - 10.1016/S0022-1759(01)00341-6

DO - 10.1016/S0022-1759(01)00341-6

M3 - Article

C2 - 11334970

AN - SCOPUS:0035371663

VL - 252

SP - 105

EP - 119

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

IS - 1-2

ER -