Tris-Based Immunoblot Wash Buffer (TBST)

doi:10.1101/pdb.rec100800

Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis

ISRN Urology ◽

10.5402/2012/618247 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5

Author(s):

Brittany Fitzpatrick ◽

Catherine Schuler ◽

Robert E. Leggett ◽

Robert M. Levin

Keyword(s):

Quantitative Analysis ◽

Western Blotting ◽

Optical Density ◽

Tissue Concentration ◽

Detrusor Muscle ◽

Pvdf Membrane ◽

Sds Page ◽

Wash Buffer ◽

Rabbit Bladder ◽

Plate Analysis

Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

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Technical viability of the YF MAC-HD ELISA kit for use in yellow fever-endemic regions

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009417 ◽

2021 ◽

Vol 15 (6) ◽

pp. e0009417

Author(s):

Christin H. Goodman ◽

Maurice Demanou ◽

Mick Mulders ◽

Jairo Mendez-Rico ◽

Alison Jane Basile

Keyword(s):

South America ◽

Yellow Fever ◽

Accurate Diagnosis ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Elisa Kit ◽

Wash Buffer ◽

Antibody Capture ◽

Vaccination Campaigns ◽

Arboviral Disease

Yellow fever (YF), an arboviral disease, affects an estimated 200,000 people and causes 30,000 deaths per year and recently has caused major epidemics in Africa and South America. Timely and accurate diagnosis of YF is critical for managing outbreaks and implementing vaccination campaigns. A YF immunoglobulin M (IgM) antibody-capture (MAC) enzyme-linked immunosorbent assay (ELISA) kit, the YF MAC-HD, was successfully introduced starting in 2018 to laboratories in Africa and South America. The YF MAC-HD kit can be performed in 3.5 hours, test up to 24 samples, and includes all reagents necessary to perform the test, except for water used to dilute wash buffer. In 2018 and 2019, a total of 56 laboratory personnel from 39 countries in Africa and South America were trained to use the kit during workshops, followed by take-home YF IgM proficiency testing (PT) exercises. Participants received either a 10- or 20-sample YF PT panel and performed testing using the YF MAC-HD kit. All countries obtained 90% or higher correct results. These results verified the technical viability and transferability of YF MAC-HD kit use for laboratories in YF-endemic countries.

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