Baculovirus Immunostaining Rapid Titration Kit

The rapid assay kit for baculovirus titer using immunostaining specifically recognizes the GP64 protein expressed on the surface of infected cells via a baculovirus GP64 antibody, enabling rapid quantification of baculovirus titers. The entire process takes only 2–3 days, whereas traditional plaque assays and the tissue culture infectious dose 50 (TCID50) method typically require 5–8 days, are labor-intensive, highly dependent on the technician's experience, and involve subjective interpretation of results. Compared with qPCR, which can complete testing within a few hours, qPCR quantifies viral nucleic acids and cannot distinguish between infectious and non-infectious virus particles; in contrast, this product directly detects the GP64 protein, measuring the titer of infectious units (IFU/mL), yielding results that more closely reflect actual infection efficiency and providing a more reliable basis for precise MOI calculation.

In addition, this kit employs a newly developed coating material that not only effectively prevents viral spread through the culture medium but also exhibits significantly lower viscosity compared to traditional methylcellulose. Moreover, it remains liquefied at 37°C, thereby reducing the adverse effects of overheated, overly viscous coatings on cells. The kit is easy to use, simple to remove, and helps minimize human error. With high batch-to-batch precision and excellent reproducibility, this product is suitable for determining the titer of AcMNPV-type baculoviruses, providing rapid and accurate technical support for virus quantification and large-scale production in baculovirus expression systems.

The components and specifications of the baculovirus rapid titer assay kit are as follows. By following the example experimental procedure, you can perform100tests.

1.Reagent preparation:

1)4%Paraformaldehyde solution: Weigh out4gParaformaldehyde was placed in a glass beaker, and then added…50 mLIn distilled water, use a magnetic stirrer in a fume hood to raise the temperature toSixty℃ (not exceedingSixty-five℃), add while stirringThirty~FiftyμLof/'s4NSodium hydroxide is used to neutralize the solution, withpHObtained from a test strippHValue, ultimatelypHThe value should bepH 6~8, once the solution has cooled, add50 mL 0.2 MSodium phosphate solution(pH 7.4)Dilution, finalpHThe value should beseven~8, adjust as necessary. Use0.45 μmMembrane filtration, followed by aliquoting and storage at –Twenty℃, before use from–Twenty℃ Remove and allow to return to room temperature.

2)PBSTSolution:Weigh outNa2HPO4 1.42 g,KH2PO4 0.24g,KCl 0.2g,NaCl 8ginto the beaker, add800mLultrapure water, and after all has dissolved, add0.5 mL Tween 20, stir well, and make up to volume with ultrapure water to1L

3)1×goat serum: dilute10×goat serum withPBSTsolution by a factor of10times

4)1×mouseGP64antibody: dilute1000×mouseGP64antibody with1×goat serum dilutedby a factor of1000

5)1×goat anti-mouse antibody-HRP: dilute1000×goat anti-mouse antibody-HRPwith1×goat serum dilutedby a factor of1000

2, baculovirus infection:

1)in96well cell culture plate, one row (12wells) is inoculated with cells in the early logarithmic growth phaseSf9Cells (per well6.5~7×10⁴cells)

2)Incubate the culture plate at28℃ for >2 hoursor overnight

3)Take50 μLof the virus sample and prepare a series of diluted virus samples using culture medium as the diluent. Adjust the dilution range as needed, ensuring thorough mixing between each dilution step. This instruction uses virus sample dilutions of10⁴,10⁵and10⁶as examples; this dilution range is suitable for virus samples with titers ranging from2×10⁶to1×10⁹ IFU/mLfor the virus sample

4)Carefully aspirate the culture medium, taking care not to scrape the cells at the bottom of the wells

5)Add25μLof the virus dilutionsample(10⁴dilutedsampletake3wells,10⁵and10⁶dilutedsampleseach take4wells), add25 μL to the negative control wellsAdd the culture medium, gently shake the plate to evenly distribute the virus

6)Seal the culture plate with sealing film,28℃Incubate1hr

7)Prepare in advance15minPut10×the overlay together withSf9cells completelyin the culture medium at37℃ water bath to preheat

8)Aspirate the inoculum, avoiding scraping the cells at the bottom of the wells, and dilute the10×overlay layer withSf9cell completeculture medium to1×overlay layer,add50 μLof1×overlay layer to each well,seal with sealing film, and incubate at28℃ for43-48hr

3, baculovirus titer detection:

1)add carefully150μLof4%paraformaldehyde (which needs to be brought back to room temperature in advanceand kept at room temperature) into each well containing the overlay, and incubate at room temperaturefor 30min

2)discard the liquid in the wells, and gently tap the96well plateon a paper towel, then add200μLPBSTsolution, wash3~4times, each time incubate on the shaker at low speed for5min

3)Add to each well50μLof1×goat serum, incubate at room temperature on a shaker at low speed for20min

4)Discard the liquid in the wells, place the96well plategently tap it on paper towels,no washing required, add to each well25μL of 1×mouseGP64antibody,37℃ incubation25min

5)Discard the liquid in the wells, gently tap the96well plateon paper towels, and add 200μLPBSTPBSTsolution, wash2times, each time incubate on the shaker at low speed for5min

6)Add to each well50μL of 1×goat anti-mouse antibody-HRP,incubate at37℃ for

25min7)Discard the liquid in the wells, take the96Pat gently with a tissue, then add to each pore.200μLPBSTSolution, washing3Next, incubate the plate on the shaker at low speed each time.5 minutes

8)Add to each wellFiftyμLChromogenic substrate (to be prepared in advance)Restore to room temperature), incubate at room temperature1.5-3 hours. After adding the chromogenic substrate, the fastest10 minutesThis allows for a preliminary estimation of the viral titer, though with relatively low accuracy.

9)Infection foci in each well (blue‑purple cell aggregates) were counted under a microscope.

10)Containing5~25The virus titer is calculated based on the dilution of a single infection focus: Virus titer (IFU/mL)=Average number of lesions per well × dilution factor ×40

It is not recommended to mix different batches.