Aug 11, 2020
Protocol for study of the in vitro effect of purified serum anti-anti-anti-SpA antibodies (Ab-3) on cultures of Staphylococcus aureus.
- Angel A Justiz-Vaillant1,
- Patrick E. Akpaka2
- 1University of the West Indies St. Augustine;
- 2University of the West Indies. St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant, Patrick E. Akpaka 2020. Protocol for study of the in vitro effect of purified serum anti-anti-anti-SpA antibodies (Ab-3) on cultures of Staphylococcus aureus.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjkekkte
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 11, 2020
Last Modified: August 11, 2020
Protocol Integer ID: 40294
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
This test shows the ability of Ab-3 antibody to inhibit the growth of S. aureus in vitro. The amounts of samples tested were 18 samples from chicks fed with hyper-immune eggs and therefore have the Ab-3, and six samples from chicks fed non-hyperimmune eggs and defenetively do not have the Ab-3. Inhibition of the growth of the bacteria plated out in blood agar was observed only when the Ab-3 was added to said culture media, sugesting that the anti-anti-anti-SpA (Ab-3) bound to the S. aureus and inhibited their ability to proliferate in culture. On the other hand, bacterial growth was observed when blanks or sera of chicks fed non-hyper-immune eggs were used because these samples lacked the protective Ab-3 [1].
Reference
Justiz Vaillant AA, Akpaka PE, Smikle M, McFarlane-Anderson N (2012) In vitro Inhibition of Staphylococcus aureus isolates by anti-antiIdiotypic antibodies to Staphylococca protein (SpA). J Vaccines Vaccin 3:127. doi:10.4172/2157-7560.1000127.
Materials
MATERIALS
Blood agarOxoid Microbiology Products - Thermo Fischer
Culti-Loops™ Staphylococcus aureus subsp. aureus ATCC™ 33592™Thermo FisherCatalog #R4607020
Safety warnings
The risk associated with a particular microbe can be consulted in the following website of the CDC. (http://www.cdc.gov/biosafety/publications/BiologicalRiskAssessmentWorksheet.pdf)
The investigation of the neutralizing ability of purified anti-SpA antibodies is carried out as follows: One ml of brain heart infusion (BHI) broths is placed in 10 sterile test tubes.
To each tube is added an equal volume of 10 µl of purified anti-anti-anti-SpA (Ab-3) antibodies in a concentration of 1.25-µg/µl.
All samples are tested in duplicate.
An inoculum of an ATCC Staphylococcus aureus strain (ATCC #33592) is prepared to 0.5 commercially prepared McFarland scale standard (1=300 x 10^6/ml bacteria concentration). Then, ten µl of this inoculum is added serially from tubes 1 to 10 (prepared for each sample) and the dilutions were reported in this study as a bacterial concentration of 0.1, 0.01, and 0.001.
The absorbance value of each tube at 600 nm is measured.
A sample from each tube is plated out on Blood agar and incubated overnight at 35oC.
Inhibition of the bacterial growth is observed in individual and pooled sera of chicks fed with anti-SpA hyperimmune eggs up to 28 days, 15 days, and eight days period.