What Other Vaccines Use mRNA And The Application Of Homogeneous Technology In mRNA Vaccine And Drug Development.

An mRNA vaccine is an introduction of mRNAencoding a disease-specific antigen into the body to produce an antigen usingthe protein synthesis mechanism of the host cell, thereby triggering an immuneresponse. The mRNA sequences of specific antigens can usually be constructedaccording to different diseases and transported to cells wrapped by novel lipidnanocarrier particles, and then the human ribosome is used to translate themRNA sequences to produce the antigenic protein of the disease, which issecreted and recognized by the autoimmune system to generate an immuneresponse, thus achieving disease prevention in the face of the continuing newcoronavirus epidemic, which works as follows.

Have you thought about what other vaccinesuse mrna? With the global epidemic of COVID-19, countries are devoting theirefforts to researching vaccines to stop the epidemic of COVID-19. mRNA vaccinehas played a leading role as a novel vaccine when the epidemic of COVID-19hits. Scientists study what other vaccines use mrna. With the development oftechnology, mRNA technology has also played a very good therapeutic role in thefield of tumor treatment in recent years. Tackling solid tumors is an importantdirection for the future development of CAR-T cell therapy, and mRNA technologyis one of the powerful helpers.

This paper provides some solutions ofhomogeneous technology in mRNA vaccine development, including dsRNA residuemeasurement, DNA residue measurement and neutralizing antibody measurement todetermine efficacy.

Detection of dsRNA residues

The kit is based on HTRF technology(homogeneous time-resolved fluorescence) for simple and rapid quantification ofviral double-stranded RNA (dsRNA) in cell lysates and provides a simplealternative to quantitative PCR (qPCR).

Principle of HTRF dsRNA detection

The dsRNA is detected by sandwich methodusing Eu (fluorescence energy donor) labeled anti-dsRNA antibody and d2(acceptor) labeled anti-dsRNA antibody. when the fluorescence energy donor andacceptor are in close proximity to each other due to dsRNA, the donor isexcited with a light source (laser or flash), which triggers fluorescenceresonance energy transfer (FRET), which in turn emits a fluorescent signal at aspecific wavelength (665 nm). The signal intensity is proportional to theconcentration of dsRNA.

Advantages of HTRF technology

①Simple operation: empty plate spiking, noneed to wash and coat the plate.

②Saving sample: only 10 μl is required.

③High sensitivity and low experimentalbackground.

④High throughput: suitable for 96/384/1536well plates.

Operation procedure

Two-step method: first add 10μl of sampleto the empty plate, then add 5μl of each detection reagent, incubate overnightto read the plate using the enzyme marker.

B. Measurement of neutralizing antibody todetermine drug efficacy

1. Luminescence Assays

Firefly luciferase (Luciferase) cancatalyze the reaction of luciferin and ATP to generate luciferin-AMP(intermediate) and pyrophosphate (PPi) in the presence of magnesium ions. Theoxidation of the intermediate is then catalyzed to produce AMP, CO2 and theexcited state of oxidized luciferin. The excited state of oxidized fluoresceinemits light when it returns to the ground state. luminescence reaction ofLuciferase does not require additional excitation light, as long as Luciferaseand substrate are in contact to generate fluorescence, the emitted photons canbe measured by light sensitive elements, avoiding background fluorescenceinterference due to excitation of background material.

When the reporter gene is embedded in thevirus, the luminescence signal can be detected after the virus infects the hostcell. The addition of neutralizing antibodies to the virus can block the virusfrom invading the host cell, and the luminescence intensity will be reduced todetermine the efficacy of the neutralizing antibodies.

Advantages of PerkinElmer Luminescenceproducts

①Simple operation and high sensitivity.

②Transportation at room temperature 2-8°Cand storage at 4°C.

③Does not contain DTT.

Operation procedure

Add lyophilized buffer to the lyophilizedsubstrate and mix thoroughly for 5min, add equal amount to the cells andincubate for 10-20min to read the plate. The sensitivity and half-life resultsare shown below.

(This is the Britelite plus kit used in thestudy on the evaluation of the neutralizing antibody activity of the newcoronavirus by the Chinese Academy of Inspection)

New Coronavirus SARS-CoV-2 S1/ACE2 antibodyresearch and development in HTRF and AlphaLISA technology have relevantfinished product detection kits are available.

2、HTRFSARS-CoV-2S1/ACE2 Assay

The HTRF S1/ACE2 binding kit is used tomeasure the interaction between S1 and ACE2. Using HTRF (homogeneoustime-resolved fluorescence) technology, the assay enables: high throughput,simple and rapid screening of compounds and inhibitors.

ACE2 is labeled with Eu (HTRF donor) and S1is labeled with d2 (HTRF acceptor). When the donor and the acceptor are inclose proximity due to S1 and ACE2 binding, the excitation of the donortriggers a fluorescence resonance energy transfer (FRET) to the acceptor, whichemits a signal at 665 nm. This specific signal is proportional to the degree ofS1/ACE2 interaction. Therefore, blocking S1/ACE2 interaction by compounds orantibodies leads to a decrease in HTRF signal.

3. SARS-CoV-2 S1/ACE2 AlphaLISA Assay

Donor microbeads coated with streptavidincapture biotinylated S1 protein and acceptor microbeads couple to human ACE2protein. The donor and acceptor microbeads are approached by S1 binding toACE2. Under 680 nm laser irradiation, the photosensitizer on the donormicrobeads converts oxygen from the surrounding environment to a single linearstate after diffusion into the acceptor microbeads, causing a chemiluminescentreaction that generates a 615 nm signal. This analysis facilitates thescreening of S1/ACE2 binding inhibitors. Pharmaceutical companies aredeveloping what other vaccines use mrna.

Advantages of AlphaLISA technology

①Simple operation: no plate washing andcoating required.

②High sensitivity and wide linear range:cascade amplification effect of chemiluminescence and microbeads.

③Low experimental background.

DNA residue detection

LANCE is TR-FRET (time-resolvedfluorescence resonance energy transfer) homogeneous technology. It has theadvantages of homogeneous phase technology without washing and separationthroughout the process, and is used to analyze and quantify DNA contaminants inbuffer solutions and cell culture media. Well, what other vaccines use mrna?

Anti-DNA antibody (donor) labeled with Euand anti-DNA antibody (acceptor) labeled with ULight. The donor and acceptorare placed in close proximity to each other, and upon excitation at 320 or 340nm, a fluorescence resonance energy transfer is triggered and the acceptoremits a fluorescence wavelength of 665 nm. The signal intensity is proportionalto the number of antigen-antibody complexes formed.