Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

Erica D Bruce ,Carolyn M Greene ,Han Li,Vera Semenova ,David S Stephens,Sandra Romero-Steiner,Lilah M Besser ,Scott E Johnson,Brian D Plikaytis,Robert F Benson,Alison Freeman ,Janet M Pruckler ,Sandra Martin ,Stephanie Schwartz,Daniel S Schmidt,George M Carlone,Trudy O Messmer ,Leta O Helsel,Jim Sejvar,Anne Schuchat,Elizabeth A Mothershed ,Patricia Holder ,Melinda A Bronsdon ,Karen Stamey,Jairam R Lingappa,Mary Bajani-Ari,David A Ashford,Evelene Steward‐Clark ,W L Thacker ,Cheryl M Elie,Bradley A Perkins,Thomas H Taylor,Peter Dull,Conrad P Quinn,Molly Kellum,Kelly J Wallace,Rosa L Moreno ,John Walls

doi:10.3201/eid0810.020380

Erica D Bruce , Carolyn M Greene + Show 36 more

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https://doi.org/10.3201/eid0810.020380

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Abstract

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Highlights

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax
In response to the anthrax emergency of 2001, we report the accelerated development and qualification of a quantitative enzyme-linked immunosorbent assay (ELISA) for detection of anti-protective antigen (PA) specific immunoglobulin (Ig) G in human serum and the development of a competitive inhibition assay to enhance diagnostic specificity
Mass value assignment to the standard serum was done by differential adsorption, homologous enzyme-linked immunoassay (EIA), and heterologous ELISA (Semenova VA, et al, manuscript in preparation), with U.S Food and Drug Administration (FDA) 1983 Haemophilus influenzae type b (Hib) reference serum [6]

Summary

Introduction

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The occurrence of human anthrax in the country and the public perception of the disease changed dramatically in the fall of 2001, with the first successful bioterrorist anthrax attack on the U.S civilian population This event necessitated the simultaneous development and application of qualified laboratory assays— including serologic assays—to evaluate patients suspected of having anthrax. The Centers for Disease Control and Prevention (CDC) had, before the attacks, instituted the development of anthrax serologic assays— enzyme-linked immunosorbent assays (ELISAs)—for use in anthrax vaccine studies in humans and to provide a standard human reference serum. In response to the anthrax emergency of 2001, we report the accelerated development and qualification of a quantitative ELISA for detection of anti-protective antigen (PA) specific immunoglobulin (Ig) G in human serum and the development of a competitive inhibition assay to enhance diagnostic specificity. The assays were applied to diagnosis of cutaneous and inhalational anthrax to evaluate serologic responses in persons considered at risk from anthrax spore exposure and enhance anthrax serologic tests with standardized techniques for distribution to public health and clinical laboratories

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