Dot Blots Analysis in the Separation of Anti-HIV Antibodies. v1

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:10.4172/21577064.1000181 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 DOI dx.doi. org/10.17504/protocols.io. bjm4kk8w 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 CREATED Aug 12, 2020 LAST MODIFIED Aug 12, 2020 PROTOCOL INTEGER ID 40348 1 08/12/2020 Cita tion : Angel A Justiz-Vaillant (08/12/2020). Dot Blots Analysis in the Separation of Anti-HIV Antibodies. https://dx.doi. org/10.17504/protocols.io. bjm4kk8w This is an open access protocol distributed under the terms of the Crea tive Com m ons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited DISCLAIMER: MATERIALS TEXT MATERIALS 3,3′,5,5′-Tetramethylbenzidine Sigma Aldr ich Catalog #54827-17-7 Dulbecco's Phosphate Buffered Saline with 2% Fetal Bovine Serum 500 mL Stemcell Technologies Catalog #7905 Nitrocellulose Membrane, Precut, 0.45 μm, 7 x 8.5 cm BioRad Sciences Catalog #1620145 BioDot SF apparatus (Bio-Rad Laboratories Richmond CA USA). Contr ibuted by users Peroxidase-labeled HIV proteins; Murex Diagnostics Norcross USA) Contr ibuted by users 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. 1 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. 2 Place the nitrocellulose membrane in a BioDot SF apparatus (Bio-Rad Laboratories, Richmond, CA, USA). 3 The membrane is blocked with 5 μL/well of fetal bovine serum with 1%Tris buffer saline. 4 Then 5 μL of a commercial conjugate (peroxidase-labeled HIV proteins; Murex Diagnostics, Norcross, USA) is added and allow to drain by gravity. 5 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. 6 The reaction is stopped by washing the wells with distilled water under a vacuum. 7 The membrane is left to dry. 2 08/12/2020 Cita tion : Angel A Justiz-Vaillant (08/12/2020). Dot Blots Analysis in the Separation of Anti-HIV Antibodies. https://dx.doi. org/10.17504/protocols.io. bjm4kk8w This is an open access protocol distributed under the terms of the Crea tive Com m ons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited 8 Visualization of color development. 3 08/12/2020 Cita tion : Angel A Justiz-Vaillant (08/12/2020). Dot Blots Analysis in the Separation of Anti-HIV Antibodies. https://dx.doi. org/10.17504/protocols.io. bjm4kk8w This is an open access protocol distributed under the terms of the Crea tive Com m ons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited