HEPES buffer: the 'invisible assistant' in rabies virus detection

4-Hydroxyethylpiperazine ethanesulfonic acid, abbreviated as HEPES in English, has a CAS number of 7365-45-9. It appears as a white crystalline powder and has a stable pH range between 6.8-8.2. As a biological buffering agent, regulating pH is its most common use and is highly favored by major manufacturers in the field of skincare. However, its crucial role in rabies virus detection is little known, and we will provide a detailed introduction below.

The dilemma of traditional rabies virus detection methods

When rabies virus reproduces in infected cells, it usually does not cause cytopathic effects (CPE), and it is also difficult to form plaques under conventional culture conditions. Although many scholars at home and abroad have successfully established rabies virus plaque methods on chicken embryo cells and BHK-21 cells, these methods have obvious drawbacks. Some have extremely strict requirements for experimental conditions, requiring precise control of various factors such as temperature, humidity, gas composition, etc; Some operation steps are complicated and require professional technicians to undergo long-term training to master them proficiently, which greatly limits the promotion and application of these methods.

Breakthrough application of HEPES in rabies virus detection

Researchers accidentally discovered that HEPES can enhance the pathogenic effect of rabies virus on infected cells. Based on this characteristic, researchers have innovatively established a simple and fast HEPES plaque etching method for rabies virus.

HEPES significantly promotes the formation of corrosion spots

In the experiment, infected cells were cultured at 37 ℃ for 3-5 days, and then one group of infected cells was treated with HEPES. After processing, obvious cellular lesions can be clearly observed at the bottom of the covering. Subsequently, crystal violet staining was used, and the corrosion spots were clearly visible. In sharp contrast, the infected cell group without HEPES treatment showed neither cytopathic effects nor plaque formation. This experimental result strongly demonstrates that HEPES has a significant promoting effect on the plaque formation of rabies virus on BHK cells.

HEPES etching method has high sensitivity

The HEPES plaque assay is not only easy to operate, but also performs well in the titration of viral infection titers. Experimental data shows that there is a clear titer relationship between the number of plaques formed after viral infection and virus dilution. Taking the CTN-BHK strain of rabies virus as an example, the infection titers were determined using HEPES plaque assay and mouse assay, respectively. After four consecutive measurements, it was found that the number of plaques measured by the HEPES plaque method ranged from 1.0 × 10 ° to 4.0 × 10 * PFU/ml, while the infection titers measured by the mouse method were between 6.0-6.8log or 1.0 × 10 ° to 6.3 × 10/ml. This result fully demonstrates the consistency in sensitivity between the rapid HEPES plaque method and the mouse method.

HEPES etching method has outstanding advantages

When studying the infection titer titration of rabies virus CTN-181 and CTN-BNK strains, it was found that the use of HEPES can effectively induce and enhance the pathogenic effect of rabies virus on infected cells. The specific operation method is as follows: after virus infection of cells, cultivate them at 37 ℃ for 3-5 days, add 50-100mmol/L HEPES dropwise onto the cover of methyl cellulose semi-solid medium, stain with crystal violet solution after 24 hours, and dry at room temperature to directly calculate the number of corrosion spots with the naked eye. Compared with previous literature reports on etching methods, this method has many advantages such as speed, simplicity, economy, sensitivity, and ease of mastery.

The HEPES produced by Desheng Company reaches the reagent level with a purity greater than 99%. With excellent product performance, we have established cooperative relationships with numerous manufacturers. For the convenience of customer experience, the company can provide free samples for trial. Desheng will always adhere to its original intention and provide customers with high-quality products. If you have any questions or needs, please click on the official website to learn more details or contact me directly!