Development of Enzyme Linked Immunosorbent Assay for humoral immuneresponse and infection monitoring of anthrax

Immune assays were taken into consideration to diagnose and quantify metabolites such as antigen and antibody. Enzyme-Linked Immunosorbent Assays (ELISAs), which are used to detect antigens and antibodies, generated several periods of infectious and vaccination conditions. There is an extensive range of commercial infectious disease ELISA kits useful for the detection of human and animal IgG, IgA, IgM antibodies and microorganism antigens. Anthrax is one of the serious infectious diseases caused by rod-shaped, gram-positive bacteria known as Bacillus anthracis. Subunit or attenuated vaccines applied against anthrax disease increase the antibody against the Protective Antigen (PA) which has a critical role as a toxin of B. anthracis. Herein, the ELISA was developed using PA domain 4 and anthrax Lethal Factor to detect IgG antibody in serum. Besides, the level of anti-LF antibodies were determined as a complementary test to measure variance in antibody titers associated with vaccination or infection that leads to detection of anthrax in livestock. The results show that we developed high-quality ELISA kit that can be used to test immunogenicity of vaccines and infections in mice. We tried to develop the Anti- PA4 ELISA kit and conduct the validation studies to evaluate the fluctuation level of the antibody in the anthrax vaccine and distinction between disease and vaccination in mice.

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Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles

Clinical and Vaccine Immunology ◽

10.1128/cvi.00412-08 ◽

2009 ◽

Vol 16 (3) ◽

pp. 408-413 ◽

Cited By ~ 74

Author(s):

Shixing Tang ◽

Mahtab Moayeri ◽

Zhaochun Chen ◽

Harri Harma ◽

Jiangqin Zhao ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Laboratory Research ◽

Enzyme Linked Immunosorbent Assay ◽

Plasma Samples ◽

Serum Samples ◽

Detection Range ◽

Edema Factor ◽

Anthrax Lethal Factor ◽

Europium Nanoparticles

ABSTRACT We developed a europium nanoparticle-based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen (PA). The ENIA exhibited a linear dose-dependent pattern within the detection range of 0.01 to 100 ng/ml and was approximately 100-fold more sensitive than enzyme-linked immunosorbent assay (ELISA). False-positive results were not observed with serum samples from healthy adults, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor or edema factor alone. For the detection of plasma samples spiked with PA, the detection sensitivities for ENIA and ELISA were 100% (11/11 samples) and 36.4% (4/11 samples), respectively. The assay exhibited a linear but qualitative correlation between the PA injected and the PA detected in murine blood (r = 0.97731; P < 0.0001). Anthrax PA was also detected in the circulation of mice infected with spores from a toxigenic Sterne-like strain of Bacillus anthracis, but only in the later stages of infection. These results indicate that the universal labeling technology based on europium nanoparticles and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research.

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Pneumococcal Type 22F Polysaccharide Absorption Improves the Specificity of a Pneumococcal-Polysaccharide Enzyme-Linked Immunosorbent Assay

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.8.2.266-272.2001 ◽

2001 ◽

Vol 8 (2) ◽

pp. 266-272 ◽

Cited By ~ 210

Author(s):

Nelydia F. Concepcion ◽

Carl E. Frasch

Keyword(s):

Correlation Coefficient ◽

Immunosorbent Assay ◽

Pneumococcal Vaccination ◽

Correlation Coefficients ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibody ◽

Functional Activities ◽

Antibody Titers ◽

The Common ◽

Elisa Inhibition

ABSTRACT The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.

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Characterization of the Native Form of Anthrax Lethal Factor for Use in the Toxin Neutralization Assay

Clinical and Vaccine Immunology ◽

10.1128/cvi.00046-13 ◽

2013 ◽

Vol 20 (7) ◽

pp. 986-997 ◽

Cited By ~ 2

Author(s):

Hang Lu ◽

Jason Catania ◽

Katalin Baranji ◽

Jie Feng ◽

Mili Gu ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Neutralization Assay ◽

Dominant Factor ◽

Anthrax Lethal Toxin ◽

Serum Samples ◽

Native Form ◽

Anthrax Lethal Factor ◽

Methionine Residues ◽

Anthrax Vaccines

ABSTRACTThe cell-based anthrax toxin neutralization assay (TNA) is used to determine functional antibody titers of sera from animals and humans immunized with anthrax vaccines. The anthrax lethal toxin is a critical reagent of the TNA composed of protective antigen (PA) and lethal factor (LF), which are neutralization targets of serum antibodies. Cytotoxic potency of recombinant LF (rLF) lots can vary substantially, causing a challenge in producing a renewable supply of this reagent for validated TNAs. To address this issue, we characterized a more potent rLF variant (rLF-A) with the exact native LF amino acid sequence that lacks the additional N-terminal histidine and methionine residues present on the commonly used form of rLF (rLF-HMA) as a consequence of the expression vector. rLF-A can be used at 4 to 6 ng/ml (in contrast to 40 ng/ml rLF-HMA) with 50 ng/ml recombinant PA (rPA) to achieve 95 to 99% cytotoxicity. In the presence of 50 ng/ml rPA, both rLF-A and rLF-HMA allowed for similar potencies (50% effective dilution) among immune sera in the TNA. rPA, but not rLF, was the dominant factor in determining potency of serum samples containing anti-PA antibodies only or an excess of anti-PA relative to anti-rLF antibodies. Such anti-PA content is reflected in immune sera derived from most anthrax vaccines in development. These results support that 7- to 10-fold less rLF-A can be used in place of rLF-HMA without changing TNA serum dilution curve parameters, thus extending the use of a single rLF lot and a consistent, renewable supply.

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Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus

Molecular Microbiology ◽

10.1111/j.1365-2958.1995.tb02375.x ◽

2006 ◽

Vol 15 (4) ◽

pp. 661-666 ◽

Cited By ~ 99

Author(s):

Jill C. Milne ◽

Steven R. Blanket ◽

Philip C. Hanna ◽

R. John Collier

Keyword(s):

Heterologous Protein ◽

Protective Antigen ◽

Lethal Factor ◽

Binding Domain ◽

Antigen Binding ◽

Carboxy Terminus ◽

Anthrax Lethal Factor

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Protective Immune Responses to the 42-Kilodalton (kDa) Region of Plasmodium yoelii Merozoite Surface Protein 1 Are Induced by the C-Terminal 19-kDa Region but Not by the Adjacent 33-kDa Region

Infection and Immunity ◽

10.1128/iai.70.2.820-825.2002 ◽

2002 ◽

Vol 70 (2) ◽

pp. 820-825 ◽

Cited By ~ 30

Author(s):

Niklas Ahlborg ◽

Irene T. Ling ◽

Wendy Howard ◽

Anthony A. Holder ◽

Eleanor M. Riley

Keyword(s):

Gene Segment ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Plasmodium Yoelii ◽

Antibody Responses ◽

Enzyme Linked Immunosorbent Assay ◽

Igg Antibody ◽

Merozoite Surface Protein 1 ◽

Antibody Titers ◽

Processing Product

ABSTRACT Vaccination of mice with the 42-kDa region of Plasmodium yoelii merozoite surface protein 1 (MSP142) or its 19-kDa C-terminal processing product (MSP119) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP133) of MSP142 also induces protection, the gene segment encoding MSP133 was expressed as a glutathione S-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP133 and subsequently challenged with the lethal P. yoelii YM blood stage parasite. GST-MSP133 failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP119 were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP133 had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP119. The finding that P. yoelii MSP133 elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based on Plasmodium falciparum or Plasmodium vivax MSP142.

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Association of Bacillus anthracis Capsule with Lethal Toxin during Experimental Infection

Infection and Immunity ◽

10.1128/iai.00764-08 ◽

2008 ◽

Vol 77 (2) ◽

pp. 749-755 ◽

Cited By ~ 28

Author(s):

J. W. Ezzell ◽

T. G. Abshire ◽

R. Panchal ◽

D. Chabot ◽

S. Bavari ◽

...

Keyword(s):

Bacillus Anthracis ◽

Protective Antigen ◽

Lethal Factor ◽

Lethal Toxin ◽

Size Exclusion ◽

Terminal Phase ◽

Enzyme Linked Immunosorbent Assay ◽

Animal Blood

ABSTRACT Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA63) and the residual 20-kDa fragment (PA20) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA63 was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-γ-d-glutamic acid (γ-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/γ-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including γ-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for γ-DPGA in anthrax pathogenesis.

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A fragment of anthrax lethal factor delivers proteins to the cytosol without requiring protective antigen

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1131930100 ◽

2003 ◽

Vol 100 (11) ◽

pp. 6652-6657 ◽

Cited By ~ 24

Author(s):

N. Kushner ◽

D. Zhang ◽

N. Touzjian ◽

M. Essex ◽

J. Lieberman ◽

...

Keyword(s):

Protective Antigen ◽

Lethal Factor ◽

Anthrax Lethal Factor

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A Modified Enzyme-Linked Immunosorbent Assay for Measurement of Antibody Responses to Meningococcal C Polysaccharide That Correlate with Bactericidal Responses

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.4.479-485.1998 ◽

1998 ◽

Vol 5 (4) ◽

pp. 479-485 ◽

Cited By ~ 78

Author(s):

Dan M. Granoff ◽

Susan E. Maslanka ◽

George M. Carlone ◽

Brian D. Plikaytis ◽

George F. Santos ◽

...

Keyword(s):

Immunosorbent Assay ◽

Geometric Mean ◽

Correlation Coefficients ◽

Antibody Responses ◽

Enzyme Linked Immunosorbent Assay ◽

High Avidity ◽

Igg Antibody ◽

Standard Assay ◽

Bactericidal Antibody ◽

Antibody Titers

ABSTRACT The standardized enzyme-linked immunosorbent assay (ELISA) for measurement of serum immunoglobulin G (IgG) antibody responses to meningococcal C polysaccharide has been modified to employ assay conditions that ensure specificity and favor detection primarily of high-avidity antibodies. The modified and standard assays were used to measure IgG antibody concentrations in sera of toddlers vaccinated with meningococcal polysaccharide vaccine or a meningococcal C conjugate vaccine. The results were compared to the respective complement-mediated bactericidal antibody titers. In sera obtained after one or two doses of vaccine, the correlation coefficients, r, for the results of the standard assay and bactericidal antibody titers were 0.45 and 0.29, compared to 0.85 and 0.87, respectively, for the modified assay. With the standard assay, there were no significant differences between the geometric mean antibody responses of the two vaccine groups. In contrast, with the modified assay, 5- to 20-fold higher postvaccination antibody concentrations were measured in the conjugate than in the polysaccharide group. Importantly, the results of the modified assay, but not the standard ELISA, paralleled the respective geometric mean bactericidal antibody titers. Thus, by employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.

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Anti-S. typhi Vi IgG levels in children with and without typhoid vaccinations

Paediatrica Indonesiana ◽

10.14238/pi54.5.2014.284-8 ◽

2014 ◽

Vol 54 (5) ◽

pp. 284

Author(s):

Sriandayani Sriandayani ◽

Tonny H. Rampengan ◽

Hesti Lestari ◽

Novie Rampengan

Keyword(s):

Typhoid Fever ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Igg Antibody ◽

Protective Antibody ◽

Significant Difference ◽

Antibody Titers ◽

The Mean ◽

And Gender ◽

Antibody Levels

Background Typhoid fever is endemic to Indonesia, with an annual incidence of 13/10,000 people. Vaccination has been shown to be an effective method to prevent typhoid fever. Of several vaccine types, the polysaccharide Vi vaccine is the most commonly used typhoid vaccine in developing countries. Results of previous studies remain inconclusive on the necessity of revaccination every 3 years.Objective To compare the mean serum anrioody titers of anti-S. typhi Vi IgG and the proportion of children with protective antibody levels between children with and without typhoid Vi vaccination.Methods We conducted a cross-secrional study at Tuminring District, 11anado from June to September 2012. Data was analyzed using independent T-test and Fisher's test. Serum anti-S. typhi Vi IgG levels were measured by enzyme-linked immunosorbent assay (ELISA) method.Results Seventy-six subjects were divided into two groups: 38 children who had received the typhoid Vi vaccination more than 3 years prior to this study and 38 children who never had typhoid vaccinations as a control group. No statistically significant difference in age and gender was found between the two groups. The mean serum anti-Vi IgG level was 0.55 ug/mL (SD 0.58; 95%CI 0.36 to 0.74) in the vaccinated group, significantly higher than that of the control group [0.31 ug/mL (SD 0.12); 950/£1 0.17 to 0.44; P􀂥0.0381. The proportion of children with protective antiNi antioody level was higher in the vaccinated group (23.7%) than in the control group (10.5%), howevet; this difference was not statistically significant (P=0.128).Conclusion The mean serum anti-S. typhi Vi IgG antibody level in children who had been vaccinated more than 3 years prior to the study is higher than in children who had never received typhoid vaccinations. Nevertheless, the mean antibody titers are generally non-protective in ooth groups. Also, the proportion of children with protective antibody levels is not significantly different between the two groups.

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