Aug 12, 2020
Dot Blots Analysis in the Separation of Anti-HIV Antibodies.
- 1University of the West Indies St. Augustine
- University of the West Indies
- angel.vaillant@sta.uwi.edu
Protocol Citation: Angel A Justiz-Vaillant 2020. Dot Blots Analysis in the Separation of Anti-HIV Antibodies.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjm4kk8w
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
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Created: August 12, 2020
Last Modified: August 12, 2020
Protocol Integer ID: 40348
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Abstract
Dot blot frequently is underutilized or is not frequently used because it cannot assess the molecular weight of macromolecules, but it is simple to perform and is useful when a particular biomolecule needs to be identified. In this protocol dot blot analysis is used for detection of anti-HIV antibody [1], and it is also available for determination or separation of any protein. It can be very useful in the immunodiagnosis of infectious diseases caused by viruses [1] and bacteria [2] and in addition for the determination of specific DNA sequences by using probes [3].
References
1. Justiz Vaillant AA, McFarlane-Anderson N, Akpaka PE, Smikle MP, Ramirez N, et al. (2013) Use of Dot Blots Analysis in the Separation of Anti-HIV Antibodies in Animals. J Chromat Separation Techniq 4: 181. doi:10.4172/2157- 7064.1000181
2. Cardona-Castro N, Gotuzzo E, Rodriguez M, Guerra H. Clinical application of a dot blot test for diagnosis of enteric fever due to Salmonella enterica serovar typhi in patients with typhoid fever from Colombia and Peru.Clin Diagn Lab Immunol. 2000;7(2):312-313. doi:10.1128/cdli.7.2.312-313.2000
3. Clément G, Benhattar J. A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples.J Clin Pathol. 2005;58(2):155-158. doi:10.1136/jcp.2004.021147
Materials
MATERIALS
3,3′,5,5′-TetramethylbenzidineSigma AldrichCatalog #54827-17-7
Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum 500 mL
Stemcell TechnologiesCatalog #7905
Nitrocellulose Membrane, Precut, 0.45 µm, 7 x 8.5 cmBioRad SciencesCatalog #1620145
BioDot SF apparatus (Bio-Rad Laboratories Richmond CA USA).
Peroxidase-labeled HIV proteins; Murex Diagnostics Norcross USA)
Two (2) µl of 1:2 dilutions of chicken IgY or 2 µl of serum from rats and cats is dotted onto nitrocellulose paper. This animal specimens tested positive for the presence of anti-HIV antibodies by ELISA, after being immunized with HIV proteins.
Place the nitrocellulose membrane in a BioDot SF apparatus (Bio-Rad Laboratories, Richmond, CA, USA).
The membrane is blocked with 5 μL/well of fetal bovine serum with 1%Tris buffer saline.
Then 5 μL of a commercial conjugate (peroxidase-labeled HIV proteins; Murex Diagnostics, Norcross, USA) is added and allow to drain by gravity.
Finally, 5 μL of the substrate 3,3’,5,5’-tetramethylbenzidine (Sigma Chemical Co., St Louis, MO, USA) is added and the mixture is incubated for 20 min.
The reaction is stopped by washing the wells with distilled water under a vacuum.
The membrane is left to dry.
Visualization of color development.