A method for evaluating virus adsorption capacity of purification fillers

Abstract

The present invention discloses a method for evaluating the virus adsorption capacity of a purified filler, wherein the filler is mixed with a virus liquid to adsorb the virus onto the filler; after cleaning the filler, animal serum containing antiviral antibodies is added to react with the antigen on the filler; after cleaning the filler, an anti-animal serum enzyme-labeled secondary antibody is added to react with the antigen on the filler; after cleaning the filler, a display liquid is added to perform a color reaction; after the color reaction is completed, the supernatant is taken after centrifugation and added to a 96-well plate, and an ELISA is used to calculate the absorbance OD of each well at a wavelength of 450nm, and the antigen adsorption capacity of the filler is evaluated by the OD value. Compared with the prior art, the present invention provides a basis for screening fillers for virus purification by fixing the antigen on the filler and applying the indirect ELISA method to detect the antigen fixing capacity of different fillers, and has the advantages of low cost, low technical threshold and fast reaction.

Description

Technical Field

[0001] The invention relates to a field, in particular to a method for evaluating the virus adsorption capacity of a purification filler. Background Art

[0002] It has become a trend to purify the semi-finished products after the production of veterinary vaccines to effectively increase the virus content per unit volume, reduce impurity proteins, and reduce vaccine side effects. Viral vectors have become a common method in scientific research. Viral vectors need to go through packaging, concentration, purification and other steps, among which the concentration and purification steps are more cumbersome and time-consuming. The commonly used method of virus concentration and purification is ion exchange column chromatography purification, which requires the use of various adsorption fillers. The ability of the filler to adsorb viruses determines the effect of virus purification.

[0003] However, there is currently no effective evaluation method for various adsorption-type fillers. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies in the prior art and provide a method for evaluating the virus adsorption capacity of a purification filler.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] A method for evaluating the virus adsorption capacity of a purification filler comprises the following steps:

[0007] S1, mixing the filler with the virus liquid to allow the virus to adsorb onto the filler;

[0008] S2, after cleaning the filler, adding animal serum containing antiviral antibodies to react with the antigen on the filler;

[0009] S3, after cleaning the filler, add anti-animal serum enzyme-labeled secondary antibody to react with the antigen on the filler;

[0010] S4, after cleaning the filler, adding a display liquid to perform a color development reaction;

[0011] S5. After the color development reaction is completed, the supernatant is centrifuged and added to a 96-well plate. The absorbance OD of each well is calculated using an ELISA reader at a wavelength of 450 nm. The antigen adsorption capacity of the filler is evaluated by the OD value.

[0012] Furthermore, the step S1 specifically includes: taking 1 mL of filler and 10 mL of virus solution and mixing them in a 15 mL centrifuge tube, and reacting them on a shaking table at 37° C. and 50 rpm for 1 hour to allow the virus to be adsorbed onto the filler.

[0013] Furthermore, the specific steps of cleaning the filler are: centrifuging the mixed solution containing the filler at 37°C, 300rpm, and 1min, discarding the supernatant, taking 10mL of 0.1M sodium chloride and adding it to a 15mL centrifuge tube, shaking for 10min, and centrifuging at 37°C, 300rpm, and 1min to discard the 0.1M sodium chloride solution, and repeating 1-2 times each time to clean the filler.

[0014] Furthermore, the step S3 specifically includes: adding 5 mL of antiviral positive animal serum into a 15 mL centrifuge tube, and reacting on a shaking table at 37° C. and 50 rpm for 1 hour.

[0015] Furthermore, the step S4 specifically includes: adding 5 mL of enzyme-labeled secondary antibody to a 15 mL centrifuge tube, and reacting on a shaking table at 37° C. and 50 rpm for 1 hour.

[0016] Furthermore, the step S5 specifically includes: adding 5 mL of TMB substrate display solution to a 15 mL centrifuge tube, reacting on a shaker at 37° C. in the dark for 15 minutes, centrifuging at 300 rpm for 1 minute after the color reaction, taking 50 μL of the supernatant and adding 50 μL of the stop solution to each well of a 96-well plate.

[0017] Compared with the prior art, the present invention fixes the antigen on the filler and uses the indirect ELISA method to detect the antigen fixing ability of different fillers, thereby providing a basis for screening fillers for virus purification. It has the advantages of low cost, low technical threshold and rapid response. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0019] Example 1

[0020] 1. Adsorption: Take 1 mL of strong anion adsorption filler and 10 mL of pseudorabies virus solution filtered through a 2 μm filter membrane in a 15 mL centrifuge tube and mix them upside down 10 times. Shake at 37°C and 50 rpm for 1 hour and place it horizontally to allow the virus to be fully adsorbed on the filler.

[0021] 2. Washing: Centrifuge at 37°C, 300 rpm, 1 min and discard the supernatant. Take 10 mL of 0.1 M sodium chloride and add it to a 15 mL centrifuge tube, place it on a shaker for 10 min, and centrifuge at 37°C, 300 rpm, 1 min to discard the 0.1 M sodium chloride solution (to wash the filler).

[0022] 3. Repeat step 2 twice.

[0023] 4. Add primary antibody: Add 5 mL of antiviral positive animal serum to a 15 mL centrifuge tube and place on a horizontal shaker at 37°C and 50 rpm for 1 hour (add primary antibody to react with the antigen on the filler).

[0024] 5. Cleaning: Repeat steps 2 and 3.

[0025] 6 Add secondary antibody: Add 5 mL of enzyme-labeled secondary antibody to a 15 mL centrifuge tube and shake it horizontally at 37°C and 50 rpm for 1 hour (add secondary antibody).

[0026] 7. Cleaning: Repeat steps 2 and 3.

[0027] 8 Color development: Add 5 mL of TMB substrate display solution to a 15 mL centrifuge tube and place horizontally on a 37°C light-proof shaker.

[0028] The reaction was allowed to develop color for 15 minutes. After the color development reaction, the plate was centrifuged at 300 rpm for 1 minute, and 50 μL of the supernatant was added to each well of a 96-well plate. The absorbance (OD) value of each well was calculated using an ELISA reader at a wavelength of 450 nm.

[0029] Example 2

[0030] 1. Adsorption: Take 1 mL of strong anion adsorption filler and 10 mL of pseudorabies virus cell culture medium semi-finished product filtered by 2 μm filter membrane, mix them upside down 10 times in a 15 mL centrifuge tube, shake at 37°C and 50 rpm for 1 hour, and place it horizontally to allow the virus to be fully adsorbed to the filler.

[0031] 2. Washing: Centrifuge at 37°C, 300 rpm, 1 min and discard the supernatant. Take 10 mL of 0.1 M sodium chloride and add it to a 15 mL centrifuge tube, place it on a shaker for 10 min, and centrifuge at 37°C, 300 rpm, 1 min to discard the 0.1 M sodium chloride solution (to wash the filler).

[0032] 3. Repeat step 2 twice.

[0033] 4. Add primary antibody: Add 5 mL of antiviral positive animal serum to a 15 mL centrifuge tube and place on a horizontal shaker at 37°C and 50 rpm for 1 hour (add primary antibody to react with the antigen on the filler).

[0034] 5. Cleaning: Repeat steps 2 and 3.

[0035] 6 Add secondary antibody: Add 5 mL of enzyme-labeled secondary antibody to a 15 mL centrifuge tube and shake it horizontally at 37°C and 50 rpm for 1 hour (add secondary antibody).

[0036] 7. Cleaning: Repeat steps 2 and 3.

[0037] 8 Color development: Add 5 mL of TMB substrate display solution to a 15 mL centrifuge tube and place horizontally on a 37°C light-proof shaker.

[0038] The reaction was allowed to develop color for 15 minutes. After the color development reaction, the plate was centrifuged at 300 rpm for 1 minute, and 50 μL of the supernatant was added to each well of a 96-well plate. The absorbance (OD) value of each well was calculated using an ELISA reader at a wavelength of 450 nm.

[0039] Example 3

[0040] 1. Adsorption: Take 1 mL of strong cationic adsorption filler and 10 mL of pseudorabies virus solution filtered through a 2 μm filter membrane in a 15 mL centrifuge tube and mix them upside down 10 times. Incubate the tube at 37°C and 50 rpm for 1 hour and place it horizontally to allow the virus to be fully adsorbed onto the filler.

[0041] 2. Washing: Centrifuge at 37°C, 300 rpm, 1 min and discard the supernatant. Take 10 mL of 0.1 M sodium chloride and add it to a 15 mL centrifuge tube, place it on a shaker for 10 min, and centrifuge at 37°C, 300 rpm, 1 min to discard the 0.1 M sodium chloride solution (to wash the filler).

[0042] 3. Repeat step 2 twice.

[0043] 4. Add primary antibody: Add 5 mL of antiviral positive animal serum to a 15 mL centrifuge tube and place on a horizontal shaker at 37°C and 50 rpm for 1 hour (add primary antibody to react with the antigen on the filler).

[0044] 5. Cleaning: Repeat steps 2 and 3.

[0045] 6 Add secondary antibody: Add 5 mL of enzyme-labeled secondary antibody to a 15 mL centrifuge tube and shake it horizontally at 37°C and 50 rpm for 1 hour (add secondary antibody).

[0046] 7. Cleaning: Repeat steps 2 and 3.

[0047] 8 Color development: Add 5 mL of TMB substrate display solution to a 15 mL centrifuge tube and place horizontally on a 37°C light-proof shaker.

[0048] The reaction was allowed to develop color for 15 minutes. After the color development reaction, the plate was centrifuged at 300 rpm for 1 minute, and 50 μL of the supernatant was added to each well of a 96-well plate. The absorbance (OD) value of each well was calculated using an ELISA reader at a wavelength of 450 nm.

[0049] Example 4

[0050] 1. Adsorption: Take 1 mL of strong cationic adsorption filler and 10 mL of pseudorabies virus cell culture medium semi-finished product filtered through a 2 μm filter membrane in a 15 mL centrifuge tube and mix them upside down 10 times. Shake at 37°C and 50 rpm for 1 hour, and place it horizontally to allow the virus to be fully adsorbed on the filler.

[0051] 2. Washing: Centrifuge at 37°C, 300 rpm, 1 min and discard the supernatant. Take 10 mL of 0.1 M sodium chloride and add it to a 15 mL centrifuge tube, place it on a shaker for 10 min, and centrifuge at 37°C, 300 rpm, 1 min to discard the 0.1 M sodium chloride solution (to wash the filler).

[0052] 3. Repeat step 2 twice.

[0053] 4. Add primary antibody: Add 5 mL of antiviral positive animal serum to a 15 mL centrifuge tube and place on a horizontal shaker at 37°C and 50 rpm for 1 hour (add primary antibody to react with the antigen on the filler).

[0054] 5. Cleaning: Repeat steps 2 and 3.

[0055] 6 Add secondary antibody: Add 5 mL of enzyme-labeled secondary antibody to a 15 mL centrifuge tube and shake it horizontally at 37°C and 50 rpm for 1 hour (add secondary antibody).

[0056] 7. Cleaning: Repeat steps 2 and 3.

[0057] 8 Color development: Add 5 mL of TMB substrate display solution to a 15 mL centrifuge tube and place horizontally on a 37°C light-proof shaker.

[0058] The reaction was allowed to develop color for 15 minutes. After the color development reaction, the plate was centrifuged at 300 rpm for 1 minute, and 50 μL of the supernatant was added to each well of a 96-well plate. The absorbance (OD) value of each well was calculated using an ELISA reader at a wavelength of 450 nm.

[0059] Comparative Example 1

[0060] Take 1mL of strong cationic adsorption filler and 5mL of TMB substrate display solution, place horizontally on a 37℃ dark shaking table, and react for 15min. After the color reaction, centrifuge at 300rpm for 1min, take 50μL of supernatant and add it to a 96-well plate, add 50μL of stop solution to each well. Use an ELISA reader to calculate the absorbance (OD) value of each well at a wavelength of 450nm.

[0061] Comparative Example 2

[0062] Take 1mL of strong anion adsorption filler and 5mL of TMB substrate display solution, place horizontally on a 37℃ dark shaking table, and react for 15min. After the color reaction, centrifuge at 300rpm for 1min, take 50μL of supernatant and add it to a 96-well plate, add 50μL of stop solution to each well. Use an ELISA reader to calculate the absorbance (OD) value of each well at a wavelength of 450nm.

[0063] The OD values ​​calculated in Example 1 to Example 4 are shown in Table 1.

[0064] Table 1

[0065] project OD value Strong anion adsorption filler + pseudorabies virus 1.7 Strong anion adsorption filler + cell culture medium 0.5 Strong cationic adsorption filler + pseudorabies virus 0.6 Strong cationic adsorption filler + cell culture medium 0.4 Strong cationic adsorption filler directly reacts with TMB substrate display solution 0 Strong anion adsorption filler directly reacts with TMB substrate display solution 0

[0066] It can be seen from Table 1 that the strong anion adsorption filler has a good adsorption effect on pseudorabies virus.

[0067] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for evaluating the virus adsorption capacity of a purification filler, characterized in that: The following steps are involved: S1. Mix the filler with the virus solution to allow the virus to adsorb onto the filler; mix 1 mL of filler with 10 mL of virus solution in a 15 mL centrifuge tube and shake at 37°C and 50 rpm for 1 hour to allow the virus to adsorb onto the filler; S2. After cleaning the filler, add animal serum containing antiviral antibodies to react with the antigen on the filler; add 5 mL of antiviral positive animal serum to a 15 mL centrifuge tube and react at 37°C and 50 rpm on a shaker for 1 hour; S3, centrifuge the mixed solution containing the filler at 37°C, 300rpm, 1min, discard the supernatant, take 10mL of 0.1M sodium chloride and add it to a 15mL centrifuge tube, shake for 10min, centrifuge at 37°C, 300rpm, 1min, discard the 0.1M sodium chloride solution, and repeat 1-2 times each time to wash the filler; add anti-animal serum enzyme-labeled secondary antibody to react with the antigen on the filler; add 5mL of enzyme-labeled secondary antibody to the 15mL centrifuge tube, shake at 37°C, 50rpm for 1h; S4, after cleaning the filler, add a display solution to perform a color development reaction; S5. After the color development reaction is completed, the supernatant is centrifuged and added to a 96-well plate. The absorbance OD of each well is calculated using an ELISA reader at a wavelength of 450 nm. The antigen adsorption capacity of the filler is evaluated by the OD value.

2. The method for evaluating the virus adsorption capacity of a purification filler according to claim 1, characterized in that: The step S4 specifically includes: adding 5 mL of TMB substrate display solution to a 15 mL centrifuge tube, reacting and coloring at 37° C. in a dark shaking incubator for 15 min, centrifuging at 300 rpm for 1 min after the color development reaction, taking 50 μL of supernatant and adding it to a 96-well plate, and adding 50 μL of stop solution to each well.