scholarly journals Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen

2002 ◽  
Vol 8 (10) ◽  
pp. 1103-1110 ◽  
Author(s):  
Conrad P. Quinn ◽  
Vera A. Semenova ◽  
Cheryl M. Elie ◽  
Sandra Romero-Steiner ◽  
Carolyn Greene ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Protective Antigen ◽  
Human Immunoglobulin ◽  
Anthrax Toxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Immunoglobulin G

scholarly journals Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

2004 ◽  
Vol 11 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Raymond E. Biagini ◽  
Deborah L. Sammons ◽  
Jerome P. Smith ◽  
Barbara A. MacKenzie ◽  
Cynthia A. F. Striley ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Immunoglobulin G ◽  
Dynamic Range ◽  
Protective Antigen ◽  
Competitive Inhibition ◽  
Lethal Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Minimum Detectable Concentration ◽  
Diagnostic Specificity ◽  
High Positive Correlation

ABSTRACT Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r 2 = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.

scholarly journals Covalent Polymyxin B Conjugate with Human Immunoglobulin G as an Antiendotoxin Reagent

1998 ◽  
Vol 42 (3) ◽  
pp. 583-588 ◽  
Author(s):  
Joseph J. Drabick ◽  
Apurba K. Bhattacharjee ◽  
David L. Hoover ◽  
George E. Siber ◽  
Vivian E. Morales ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Lipid A ◽  
Polymyxin B ◽  
Human Immunoglobulin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Neutralizing Activity ◽  
Human Immunoglobulin G ◽  
Dose Dependent

ABSTRACT Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmacokinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 μg of bound functional PMB per 100 μg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli,Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 μg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.

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