An ELISA using a recombinant chimera of protective antigen and lethal factor for serodiagnosis of cutaneous anthrax in India (original) (raw)
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Clinical and Vaccine Immunology, 2012
Anthrax, caused by Bacillus anthracis , is primarily a zoonotic disease. Being a public health problem also in several developing countries, its early diagnosis is very important in human cases. In this study, we describe the use of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-lethal factor (anti-LF) IgG in human serum samples. A panel of 203 human serum samples consisting of 50 samples from patients with confirmed cutaneous anthrax, 93 samples from healthy controls from areas of India where anthrax is nonendemic, 44 samples from controls from an area of India where anthrax is endemic, and 16 patients with a disease confirmed not to be anthrax were evaluated with an anti-LF ELISA. The combined mean anti-LF ELISA titer for the three control groups was 0.136 ELISA unit (EU), with a 95% confidence interval (CI) of 0.120 to 0.151 EU. The observed sensitivity and specificity of the ELISA were 100% (95% CI, 92.89 to 100%) and 97.39% (95% CI, 93.44 to 99.28%)...
Emerging Infectious Diseases, 2002
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 97.6%. 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.
Defence Life Science Journal
Anthrax, a zoonotic disease caused by Bacillus anthracis is important for biowarfare as well as public health point of view. The virulence factors of B. anthracis are encoded by the two plasmids, pXO1 and pXO2. Protective antigen (PA), an 83 kDa protein encoded by pXO1 along with lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa), makes the anthrax toxin responsible for causing the disease. Current detection and diagnostic systems for anthrax are mostly based on PA, a potential biomarker of B. anthracis. The objective of the present study was to produce and purify the PA for development of a sandwich ELISA for its detection. In this study, pYS5 plasmid containing the full PA gene was transformed into an 8 proteases deficient Bacillus anthracis host BH480. The PA was produced under shake flask conditions and purified using the gel filtration chromatography. The reactivity of PA with rabbit and mouse anti-PA antibodies was confirmed by Western blotting. The antibodies were purifi...
Clinical and Vaccine Immunology, 2006
Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.
Journal of Applied Microbiology, 2018
Aims: The aim of this work was to identify a protein which can be used for specific detection of antibodies against Bacillus cereus biovar anthracis (Bcbva), an anthrax-causing pathogen that so far has been described in African rainforest areas. Methods and Results: Culture supernatants of Bcbva and classic Bacillus anthracis (Ba) were analysed by gel electrophoresis, and a 35-kDa protein secreted only by Bcbva and not Ba was detected. The protein was identified as pXO2-60 by mass spectrometry. Sequence analysis showed that Ba is unable to secrete this protein due to a premature stop codon in the sequence for the signal peptide. Immunization of five outbred mice with sterile bacterial culture supernatants of Bcbva revealed an immune response in ELISA against pXO2-60 (three mice positive, one borderline) and the protective antigen (PA; four mice). When supernatants of classic Ba were injected into mice or human sera from anthrax patients were analysed, only antibodies against PA were detected. Conclusions: In combination with PA, the pXO2-60 protein can be used for the detection of antibodies specific against Bcbva and discriminating from Ba. Significance and Impact of the Study: After further validation, serological assays based on pXO2-60 can be used to perform seroprevalence studies to determine the epidemiology of B. cereus bv anthracis in affected countries and assess its impact on the human population.
Defence Science Journal, 2016
Anthrax, caused by Bacillus anthracis is known to occur globally since antiquity. Besides being an important biothreat agent, it is an important public health importance pathogen also in countries like India. B. anthracis secretes three distinct toxins, namely protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the central moiety of the anthrax toxin complex and therefore has been a molecule of choice for vaccine development. PA has four different domains with different functions. In this study, the major domains of PA were cloned and expressed in bacterial system. The purified recombinant proteins were used to determine the humoral immune response by ELISA using 43 human cutaneous anthrax serum samples. The maximum immunoreactivity was observed with the whole PA protein followed by domain 2, 4 and 1. The study corroborated that in addition to full PA, individual domain 2 and 4 can also be good target for vaccine development as well as for serodiagnostic assays ...
Journal of Immunological Methods, 2014
Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10 −6 μM (0.551 ng/ml) for PA83 and 2.51 × 10 −5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n = 7), inhalation (n = 2), and gastrointestinal (n = 1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex.
PLOS ONE, 2021
Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacteriumBacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) ofB.anthracisare mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen ofB.anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Usingin silicoanalyses, we screened coding s...
Comparative evaluation of protective antigen produced from Bacillus anthracis & Escherichia coli
The Indian journal of medical research, 2003
Anthrax has been reported from almost every country and India is endemic for this disease. There is considerable under reporting of the disease because of lack of microbiological facilities and diagnostic reagents. In India only conventional methods which have limitations, are being used to diagnose the disease. Hence the aim of this study was to isolate and purify protective antigen (PA) using different protocols and to use this PA for detection of anti-PA antibodies from sera samples. Protective antigen was isolated and purified from the Sterne strain of Bacillus anthracis. B. anthracis lacking pXO1 and pXO2 transformed with pYS5 (B. anthracis pYS5) and recombinant Escherichia coli transformed with pQE30 containing PA gene using hydroxyapatite (HA), Q-sepharose fast protein liquid chromatography (FPLC) and nickel-nitrilotriacetic acid (Ni-NTA) chromatographic methods, respectively. A mixture of PA and edema factor (EF) was injected subcutaneously into rabbits to test the biologica...