Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers

C. M. Carpenter, E. R. Hall, R. Randall, Robin McKenzie, F. Cassels, N. Diaz, N. Thomas, P. Bedford, M. Darsley, C. Gewert, C. Howard, R. B. Sack, David Allen Sack, H. S. Chang, G. Gomes, Louis Bourgeois

Research output: Contribution to journalArticle

Abstract

In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 × 108 or 4 × 109 cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 × 109 cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p <0.001) and 0.82 (p <0.001), respectively after challenge and 0.78 (p <0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p <0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

Original languageEnglish (US)
Pages (from-to)3709-3718
Number of pages10
JournalVaccine
Volume24
Issue number18
DOIs
StatePublished - May 1 2006

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mucosal immunity
Enzyme-Linked Immunospot Assay
Enterotoxigenic Escherichia coli
Mucosal Immunity
enterotoxigenic Escherichia coli
Histocompatibility Antigens Class II
volunteers
Volunteers
lymphocytes
Lymphocytes
antigens
antibodies
Antibodies
assays
Antigens
Vaccination
vaccination
mouth
dosage
Fluorescence

Keywords

  • ALS
  • ELISPOT
  • Enterotoxigenic E. coli

ASJC Scopus subject areas

  • Immunology
  • Microbiology
  • Virology
  • Infectious Diseases
  • Public Health, Environmental and Occupational Health
  • veterinary(all)

Cite this

Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers. / Carpenter, C. M.; Hall, E. R.; Randall, R.; McKenzie, Robin; Cassels, F.; Diaz, N.; Thomas, N.; Bedford, P.; Darsley, M.; Gewert, C.; Howard, C.; Sack, R. B.; Sack, David Allen; Chang, H. S.; Gomes, G.; Bourgeois, Louis.

In: Vaccine, Vol. 24, No. 18, 01.05.2006, p. 3709-3718.

Research output: Contribution to journalArticle

Carpenter, C. M. ; Hall, E. R. ; Randall, R. ; McKenzie, Robin ; Cassels, F. ; Diaz, N. ; Thomas, N. ; Bedford, P. ; Darsley, M. ; Gewert, C. ; Howard, C. ; Sack, R. B. ; Sack, David Allen ; Chang, H. S. ; Gomes, G. ; Bourgeois, Louis. / Comparison of the antibody in lymphocyte supernatant (ALS) and ELISPOT assays for detection of mucosal immune responses to antigens of enterotoxigenic Escherichia coli in challenged and vaccinated volunteers. In: Vaccine. 2006 ; Vol. 24, No. 18. pp. 3709-3718.
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abstract = "In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 × 108 or 4 × 109 cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 × 109 cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95{\%} of the subjects. A similar proportion (75{\%}) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95{\%}, 94{\%} and 78{\%}, respectively and 83{\%} for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p <0.001) and 0.82 (p <0.001), respectively after challenge and 0.78 (p <0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p <0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.",
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AU - McKenzie, Robin

AU - Cassels, F.

AU - Diaz, N.

AU - Thomas, N.

AU - Bedford, P.

AU - Darsley, M.

AU - Gewert, C.

AU - Howard, C.

AU - Sack, R. B.

AU - Sack, David Allen

AU - Chang, H. S.

AU - Gomes, G.

AU - Bourgeois, Louis

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N2 - In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 × 108 or 4 × 109 cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 × 109 cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p <0.001) and 0.82 (p <0.001), respectively after challenge and 0.78 (p <0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p <0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

AB - In the present study we compared the ELISPOT and antibody in lymphocyte supernatants (ALS) assays as surrogate measures of mucosal immunity. In separate studies, 20 inpatient volunteers received oral doses of 6 × 108 or 4 × 109 cfu of ETEC strain E24377A (LT+, ST+, CS1+, CS3+) and 20 subjects received 1 (n = 9) or 2 (n = 11) oral doses of the attenuated ETEC vaccine, PTL-003 expressing CFA/II (CS1+ and CS3+) (2 × 109 cfu/dose). Peripheral blood mononuclear cells (PBMCs) from all subjects were assayed for anti-colonization factor or toxin-specific IgA antibody responses using the ALS and ELISPOT procedures. ALS responses were measured using a standard ELISA, as well as by time-resolved fluorescence (TRF). Following challenge with E24377A, significant anti-CS3, CS1 and LT ALS responses were detected in the lymphocyte supernatants of 75-95% of the subjects. A similar proportion (75%) of subjects mounted an ALS response to CFA/II antigen after vaccination with the PTL-003 vaccine. Inter-assay comparisons between ALS and ELISPOT methods also revealed a high degree of correlation in both immunization groups. ALS sensitivity versus the ELISPOT assay for LT, CS3 and CS1-specific responses following challenge were 95%, 94% and 78%, respectively and 83% for the ALS response to CFA/II antigen after vaccination with PTL-003. Correlation coefficients for the LT and CS3 antigens were 0.94 (p <0.001) and 0.82 (p <0.001), respectively after challenge and 0.78 (p <0.001) after vaccination. The association between ALS and ELISPOT for the CS1 antigen was however, significant only when ALS supernatants were tested by TRF (r = 0.91, p <0.001). These results demonstrate the value and flexibility of the ALS assay as an alternative to ELISPOT for the measurement of mucosal immune responses to ETEC antigens, particularly when the complexities of ELISPOT may make it impractical to perform.

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