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Research article
Ruichang Chen
Shandong Agricultuial University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9439-1396
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production.
Results: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99x107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37% and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays.
Conclusions: Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.
Keywords
Goat Klebsiella pneumonia, Polyclonal antibodies, Indirect ELISA detection method, Optimal working conditions, Clinical veterinary application
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CURRENT STATUS: Published
View the published article at BMC Veterinary Research.
Version 2
Posted 08 Dec, 2020
No community comments so far
Editorial decision: Minor revision
On 02 Jan, 2021
Review #2 received
Received 01 Jan, 2021
Reviewer #2 agreed
On 11 Dec, 2020
Review #1 received
Received 11 Dec, 2020
Reviewers invited
Invitations sent on 06 Dec, 2020
Reviewer #1 agreed
On 06 Dec, 2020
Editor assigned
On 25 Nov, 2020
Submission checks complete
On 25 Nov, 2020
Editor invited
On 25 Nov, 2020
No comments provided
Editorial decision: Major revision
On 22 Oct, 2020
Review #2 received
Received 17 Oct, 2020
Review #1 received
Received 12 Oct, 2020
Reviewer #2 agreed
On 22 Sep, 2020
Reviewers invited
Invitations sent on 15 Sep, 2020
Reviewer #1 agreed
On 15 Sep, 2020
Editor assigned
On 30 Aug, 2020
Submission checks complete
On 29 Aug, 2020
Editor invited
On 29 Aug, 2020
Metrics
Comments: 0
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Subject Areas
Large Animal Medicine
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A new preprint design is coming!
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See the published version of this preprint at BMC Veterinary Research.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
Ruichang Chen
Shandong Agricultuial University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9439-1396
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at BMC Veterinary Research.
Version 2
Posted 08 Dec, 2020
No community comments so far
Editorial decision: Minor revision
On 02 Jan, 2021
Review #2 received
Received 01 Jan, 2021
Reviewer #2 agreed
On 11 Dec, 2020
Review #1 received
Received 11 Dec, 2020
Reviewers invited
Invitations sent on 06 Dec, 2020
Reviewer #1 agreed
On 06 Dec, 2020
Editor assigned
On 25 Nov, 2020
Submission checks complete
On 25 Nov, 2020
Editor invited
On 25 Nov, 2020
No comments provided
Editorial decision: Major revision
On 22 Oct, 2020
Review #2 received
Received 17 Oct, 2020
Review #1 received
Received 12 Oct, 2020
Reviewer #2 agreed
On 22 Sep, 2020
Reviewers invited
Invitations sent on 15 Sep, 2020
Reviewer #1 agreed
On 15 Sep, 2020
Editor assigned
On 30 Aug, 2020
Submission checks complete
On 29 Aug, 2020
Editor invited
On 29 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Large Animal Medicine
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at BMC Veterinary Research.
Background: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production.
Results: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99x107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37% and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays.
Conclusions: Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.
Figure 1
Figure 2
Figure 3
Figure 4
This is a list of supplementary files associated with this preprint. Click to download.
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