CN118465261A

CN118465261A - ELISA kit for detecting physical titer of retrovirus vector

Info

Publication number: CN118465261A
Application number: CN202410912359.XA
Authority: CN
Inventors: 杨红; 朱建高; 杨文君
Original assignee: Carbiogene Therapeutics Co ltd
Current assignee: Carbiogene Therapeutics Co ltd
Priority date: 2024-07-09
Filing date: 2024-07-09
Publication date: 2024-08-09
Anticipated expiration: 2044-07-09

Abstract

The invention relates to the technical field of retroviral vector titer determination, in particular to a method for determining physical titer of a retrovirus by detecting envelope protein GALV-env of the retrovirus through an ELISA kit. The invention prepares an ELISA detection kit for retrovirus envelope protein GALV-env, which comprises a capture antibody, a detection antibody, a GALV-env protein standard substance, an ELISA plate, a diluent and a sealing liquid. The kit obtains the physical titer of the retrovirus by detecting the protein amount of GALV-env and converting the protein amount, and the kit has the advantages of simple and quick operation, high sensitivity and the like, and is different from the physical titer of the retrovirus represented by quantifying nucleic acid of a retrovirus genome.

Description

ELISA kit for detecting physical titer of retrovirus vector

Technical Field

The invention relates to the technical field of retroviral vector titer determination, in particular to a method for determining physical titer of a retrovirus by detecting envelope protein GALV-env of the retrovirus through an ELISA kit.

Background

The retroviral vector that may be used in the production of CAR-T cells is a replication-defective virus constructed to include gag-pol (MuLV-gag-pol) based on Moloney murine leukemia virus and the envelope protein (GALV-env) of gibbon ape leukemia virus. GALV-env is a membrane glycoprotein located on the surface of a viral particle, and can mediate the attachment of the viral particle on the surface of a host cell, so that the viral particle is fused with a host cell membrane surface receptor, and plays an important role in the process of infecting the host cell by the virus. Retroviruses are critical vectors for cell therapy and, due to their high complexity, require accurate and reliable analytical methods for monitoring during process development and mass production to ensure product quality and safety.

Functional titres of retroviruses are measured by the infection of target cells by the virus for a relatively long detection period of biologically active viral particles per unit volume. Whereas physical titres of retroviruses are defined by determining the genome of the retrovirus or the protein concentration of the retrovirus to define the total number of viral particles per unit volume, the detection period is relatively short and is relatively suitable for the detection of intermediate process stages of retrovirus production.

The method of determining the genome of a retrovirus is a nucleic acid quantification method of RT-qPCR, which can determine the total number of live virus and dead virus particles, and the detection process is affected by a plurality of experimental factors, and the detection result may have large variability, so that the accuracy of physical titer of the retrovirus is affected.

Whereas the method by determining the protein concentration of retrovirus is an ELISA method. However, it is not yet seen that the physical titer of the retrovirus is characterized by detecting GALV-env, so that the invention provides an ELISA detection kit for detecting the physical titer of the retrovirus, and the accuracy of detecting the physical titer of the retrovirus is improved by detecting the concentration of GALV-env to characterize the physical titer of the retrovirus.

Disclosure of Invention

The invention aims to provide an ELISA kit for detecting physical titer of a retrovirus vector.

In order to solve the technical problems, the invention specifically provides the following technical scheme:

an ELISA kit for detecting physical titer of a retrovirus vector comprises a capture antibody, a detection antibody and a GALV-env protein standard.

Preferably, the GALV-env protein standard is obtained by recombinant expression.

Preferably, the step of recombinant expression of the GALV-env protein standard comprises:

synthesizing GALV-env protein gene; inserting GALV-env protein genes into vectors, and constructing to obtain plasmids; transfecting the plasmid into CHO cells for GALV-env protein expression; and (3) obtaining a GALV-env protein crude extract, and purifying to obtain a GALV-env protein standard.

Preferably, the capture antibody is an anti-GALV-env rabbit monoclonal antibody F8, the F8 heavy chain amino acid sequence is shown in SEQ ID NO.5, the F8 light chain amino acid sequence is shown in SEQ ID NO.6, the F8 heavy chain DNA sequence is shown in SEQ ID NO.9, and the F8 light chain DNA sequence is shown in SEQ ID NO. 10.

Preferably, the detection antibody is C9-biotin, and is conjugated by anti-GALV-env rabbit monoclonal antibody C9 and biotin; the amino acid sequence of the C9 heavy chain is shown as SEQ ID NO.3, the amino acid sequence of the C9 light chain is shown as SEQ ID NO.4, the DNA sequence of the C9 heavy chain is shown as SEQ ID NO.7, and the DNA sequence of the C9 light chain is shown as SEQ ID NO. 8.

Preferably, C9 and F8 are obtained by taking GALV-env protein standard as antigen, immunizing by rabbits, taking serum for specific B cell enrichment, culturing and screening, and carrying out molecular cloning recombinant expression.

The detection kit also comprises an ELISA plate.

In an embodiment of the present invention, the preparation steps of the elisa plate include:

Diluting F8 to 1-5 mu g/mL with PBS to obtain F8 solution, adding 100-200 mu L/hole of F8 solution into a blank ELISA plate, coating at 2-8deg.C for 8-16h, discarding F8 solution, adding PBST, washing for 3-5 times, adding blocking solution at 100-300 mu L/hole, blocking at 30-40deg.C for 2-3h, discarding blocking solution, adding PBST, washing for 3-5 times, adding protective solution at 100-300 mu L/hole, blocking at 30-40deg.C for 10-60min, discarding protective solution, and air drying at room temperature to obtain ELISA plate.

Preferably, the protective solution is 1-2wt% glutarate PBS. The glutarate can play a role in bacteriostasis, so that the antibody can be protected, and the stability of the ELISA plate can be prolonged. When the kit prepared by the protective solution is used for detection, the kit has better precision.

The preparation method of the glutarate comprises the following steps:

Adding glutaric anhydride and dipropanolamine into a round-bottom flask, and uniformly mixing to obtain a mixed solution 1; then toluene sulfonic acid is added into the mixed solution 1 for reaction to obtain a reaction solution 1; adding 3-methoxybutanol into the reaction solution 1, and uniformly mixing to obtain a mixed solution 2; and (3) adding sodium bisulfate into the mixed solution 2 for reaction, filtering, washing and recrystallizing after the reaction is finished to obtain glutarate.

Preferably, glutaric anhydride and dipropanolamine are present in a molar ratio=10: 1-10.

Preferably, glutaric anhydride and 3-methoxybutanol are present in a molar ratio=10: 20-40.

Preferably, toluene sulfonic acid is added to the mixture 1 in an amount of 1 to 5wt%.

Preferably, sodium bisulfate is added to the mixture 2 in an amount of 1 to 5wt%.

More preferably, the glutarate is prepared by the following steps:

In a round bottom flask in molar mass ratio = 10: sequentially adding glutaric anhydride and dipropanolamine in a proportion of 1-10, uniformly mixing to obtain a mixed solution 1, adding 1-5wt% of p-toluenesulfonic acid into the mixed solution 1, reacting at a constant temperature of 100-160 ℃ for 3-5h to obtain a reaction solution 1, cooling to 40-60 ℃, and adding the molar mass ratio of glutaric anhydride and 3-methoxybutanol into the reaction solution 1=10: adding 3-methoxybutanol in a proportion of 20-40, uniformly mixing to obtain a mixed solution 2, adding 1-5wt% of sodium bisulfate into the mixed solution 2, heating and refluxing for 2-3 hours at 60-100 ℃, cooling to room temperature, performing reduced pressure suction filtration by a vacuum pump, washing with distilled water, recrystallizing, and drying in a vacuum drying oven to obtain glutarate.

The protective solution in one embodiment of the invention also contains 1-2wt% dibenzyl l-tartrate. The L-dibenzyl tartrate and glutarate synergistically protect the antibody, so that the stability of the ELISA plate is further improved. When the kit prepared by the protective solution is used for detection, the kit has better precision.

The invention also provides a method for detecting physical titer of the retroviral vector by using the kit, which comprises the following steps:

1) Diluting a sample to be detected to obtain a detection sample; the quality control product is GALV-env protein standard product with the volume of 50-100 pg/mL; diluting the GALV-env protein standard to obtain a standard; then adding the detection sample, the quality control product and the standard product into an ELISA plate, incubating, adding a washing buffer solution for washing for 3-5 times, adding a detection antibody, incubating at room temperature in a dark place, washing for 3-5 times, adding SA-HRP, incubating at room temperature in a dark place, washing for 3-5 times, adding a color development solution, adding a stop solution after developing at a dark place, and then putting into an ELISA reader for reading at 450 nm and 630 nm.

2) And (3) performing data processing by software matched with an enzyme-labeled instrument, taking the concentration of the standard substance as an abscissa and the OD _450nm-OD_630nm value as an ordinate, and establishing a standard curve and a regression equation of the GALV-env protein standard substance by four-parameter curve fitting.

3) And (3) evaluating the system applicability acceptance standard of the method, and if the detection result meets the system applicability acceptance standard, the experiment is effective.

4) The OD _450nm-OD_630nm value of the test sample should be within the standard curve range of the GALV-env protein standard.

The physical titer of the retrovirus in the sample to be tested is obtained by calculating the concentration of the GALV-env protein and then converting the concentration.

Preferably, the physical retroviral titres are derived by conversion of the GALV-env protein concentration as follows:

One retroviral particle contains 100-300 GALV-env protein molecules, the molecular weight of the carried-in GALV-env and the Avofiladelo constant, giving the mass A (ng/RPs) of each retroviral particle; ELISA measured GALV-env protein concentration B (ng/mL), B/A gave retroviral vector physical titres (RPs/mL).

Compared with the prior art, the invention has the following beneficial effects: the invention measures the protein concentration of the retroviral vector GALV-env by ELISA, and obtains the physical titer of the retroviral vector by conversion. The number of virus particles contained in a unit volume is calculated by calculating the molecular weight of GALV-env contained in one virus particle, and the relation between the number of virus particles and the functional titer is further clarified. In the preparation process of the ELISA plate, the protection liquid is applied, and contains glutarate and L-dibenzyl tartrate, which cooperate to protect antibodies, so that the stability of the ELISA plate is prolonged; the kit prepared by the protective solution has better application, and has better precision when being used for detection. The invention detects the retrovirus vector by ELISA method, which avoids the disadvantages of high requirements on environment, equipment and operation when RT-qPCR is used to detect the physical titer of the retrovirus.

Drawings

FIG. 1 is a standard curve of GALV-env protein standard.

FIG. 2 is an infrared spectrum of glutarate.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental methods in the following examples, unless otherwise specified, are conventional methods or according to the conditions recommended by the manufacturer. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The principle of ELISA method is based on solid phase sandwich ELISA technique. Coating the capture antibody on an ELISA plate, and washing to remove unbound capture antibody. Adding a detection sample and a capture antibody coated on an ELISA plate for binding, washing to remove unbound detection sample, adding the detection antibody, washing to remove unbound detection antibody, adding SA-HRP, washing to remove unbound SA-HRP, and obtaining a solid phase compound, wherein the amount of the solid phase compound is in a certain proportion with the amount of the detection sample in the sample. After adding the enzyme reaction substrate (TMB), the substrate is catalyzed by HRP in the solid phase complex to form colored products, and the amount of the products is directly related to the amount of the detection sample in the sample, so that qualitative or quantitative analysis can be performed according to the color depth. The result of immune reaction is indirectly amplified due to high catalytic efficiency of enzyme, so that the measuring method achieves high sensitivity.

Example 1: preparation of ELISA kit for physical titre of retrovirus

The ELISA detection kit for physical titer of retrovirus comprises: capture antibody, detection antibody, GALV-env protein standard, SA-HRP, wash buffer, chromogenic solution, stop solution, elisa plate, diluent, and blocking solution.

1. Preparation of GALV-env protein standards

The carboxyl end of the 1-489aa sequence on GALV-env is connected with 10 XHis tag to obtain the amino acid sequence of GALV-env protein, which is translated into DNA sequence, and then synthesized by Nanjing qingke science and technology Co-Ltd to obtain GALV-env protein gene. The DNA sequence and the corresponding amino acid sequence of the GALV-env protein gene are shown in SEQ ID NO. 1-2.

The GALV-env protein gene is constructed to obtain a plasmid containing the GALV-env protein gene by the prior art, then the plasmid is transfected into CHO cells to carry out GALV-env protein expression, a GALV-env protein crude extract is obtained, and the GALV-env protein standard substance is obtained by purifying through a Ni affinity chromatography column.

2. Preparation of Capture antibodies

Animal immunization was performed on New Zealand rabbits using GALV-env protein standard as an immunization antigen. In the first immunization, freund's complete adjuvant is adopted as an adjuvant, in the second and third immunization, freund's incomplete adjuvant is adopted as an adjuvant, and in the fourth immunization, namely, no adjuvant is added in the final immunization. At each immunization, the amount of GALV-env protein standard was 200 μg/min. After one week of final immunization, rabbit serum was taken and assayed for antibody titers by ELISA, and the blood of rabbits with the highest antibody titers was selected for PBMCs collection.

And (3) enrichment culture is carried out on PBMCs to obtain antigen positive B cells, the B cells are inoculated into a 96-well plate for B cell cloning, then ELISA is used for detecting the antibody titer of the culture supernatant of the B cells, and B cells with higher antibody titer of the culture supernatant are selected for extracting total RNA.

The total RNA is reverse transcribed into cDNA, and then the full length sequence of the light and heavy chain of the antibody is amplified from the cDNA, and the antibody expression plasmid is constructed by the prior art. And (3) transfecting the antibody expression plasmid into HEK293 cells for expression, collecting cell culture supernatant, and purifying by affinity chromatography to obtain the anti-GALV-env rabbit monoclonal antibody. anti-GALV-env rabbit monoclonal antibodies include F8, C9, wherein F8 is a capture antibody.

The antibody expression plasmids expressing F8 and C9 were sequenced to determine the sequences of F8 and C9. The amino acid sequence of the F8 heavy chain is shown as SEQ ID NO.5, the amino acid sequence of the F8 light chain is shown as SEQ ID NO.6, the DNA sequence of the F8 heavy chain is shown as SEQ ID NO.9, and the DNA sequence of the F8 light chain is shown as SEQ ID NO. 10. The amino acid sequence of the C9 heavy chain is shown as SEQ ID NO.3, the amino acid sequence of the C9 light chain is shown as SEQ ID NO.4, the DNA sequence of the C9 heavy chain is shown as SEQ ID NO.7, and the DNA sequence of the C9 light chain is shown as SEQ ID NO. 8.

3. Preparation of detection antibodies

1) The reagents used and the C9 antibody were returned to room temperature according to the instructions of Thermo Scientific ™ EZ-Link ™ Sulfo-NHS-LC-biotinylated kit.

2) A10 mM biotin reagent solution was prepared according to the instructions.

3) According to the calculation method of the kit instructions, the C9 antibody was concentrated to 2mg/mL with PBS, and 0.5mL of a solution was prepared. An appropriate amount of 10mM biotin reagent solution was then added.

4) The reaction was incubated at room temperature for 30 minutes to complete the coupling of biotin with C9 antibody, thereby obtaining C9-biotin.

5) The concentration of C9-biotin was measured by an ultraviolet-visible spectrophotometer, and the C9-biotin was diluted to 0.5. Mu.g/mL with a diluent to obtain a detection antibody.

The dilution was 2wt% BSA-PBST, and 2g BSA was contained per 100g PBST. BSA was purchased from sigma under the accession number WXBD5568V.

PBST is a PBS containing Tween 20, wherein the PBS contains 55mg Tween 20 per 100 g. Tween 20 was purchased from chinese medicine under the accession number 30189328.PBS was purchased from large tabogril under the product number PWL050.

4. Preparation of ELISA plate

The preparation steps of the ELISA plate 1 with the F8 coating and the ELISA plate 1 with the F8 coating comprise the following steps:

F8 was diluted to 2.5. Mu.g/mL with PBS, added to a blank ELISA plate at 100. Mu.L/well, and then coated overnight at 4℃for a period of not less than 8 hours to obtain ELISA plate a1. The liquid in the ELISA plate a1 was discarded, and the plate was dried on a piece of absorbent paper, then 300. Mu.L of PBST was added to each well, and the PBST was discarded, and the plate was dried, thereby completing one washing. Repeatedly washing with PBST for 4 times to obtain an ELISA plate a2, adding sealing liquid into the ELISA plate a2, sealing with a sealing plate membrane at 37 ℃ for 120min to obtain an ELISA plate a3. Discarding the sealing liquid in the ELISA plate a3, drying, and repeatedly washing with PBST for 4 times to obtain the F8 coated ELISA plate 1.

The blocking solution was 2wt% BSA-PBST.

5. Preparation of other reagents

SA-HRP was purchased from Jackson immunoResearch under the product number 016-030-084 at a concentration of 1mg/mL. The wash buffer was PBST. The color development liquid is 3,3', 5' -tetramethyl benzidine (TMB) single-component color development liquid, which is purchased from SolarBio with the product number PR1200. The stop solution was 0.5M H ₂SO₄.

Example 2: preparation of ELISA kit for physical titre of retrovirus

Example 2 differs from example 1 in that example 2 further treated the prepared elisa plate with a protective solution. The protection solution used was PBS containing 1wt% glutarate.

The preparation method of the glutarate comprises the following steps:

in a round bottom flask in molar mass ratio = 10:8, sequentially adding glutaric anhydride and dipropanolamine in proportion, uniformly mixing to obtain a mixed solution 1, then adding 5wt% of p-toluenesulfonic acid into the mixed solution 1, reacting for 3 hours at a constant temperature of 140 ℃ to obtain a reaction solution 1, cooling to 50 ℃, and adding the mixture into the reaction solution 1 according to the molar mass ratio of glutaric anhydride and 3-methoxybutanol of=10: 25, mixing uniformly to obtain a mixed solution 2, adding 5wt% of sodium bisulfate into the mixed solution 2, heating and refluxing for 2 hours at 70 ℃, cooling to room temperature, performing vacuum filtration by a vacuum pump, washing with distilled water, recrystallizing, and drying in a vacuum drying oven to obtain glutarate.

The ELISA plate is an F8 coated ELISA plate 2, and the preparation steps of the F8 coated ELISA plate 2 comprise:

F8 was diluted to 2.5. Mu.g/mL with PBS buffer, added to a blank ELISA plate at 100. Mu.L/well, and then coated overnight at 4℃for a period of not less than 8 hours to obtain ELISA plate a1. Discarding the liquid in the ELISA plate a1, drying the water-absorbing paper, repeatedly washing with PBST for 4 times to obtain an ELISA plate a2, adding a sealing liquid into the ELISA plate a2, sealing with a sealing plate membrane for sealing at 37 ℃ for 120min, and obtaining an ELISA plate a3. Discarding the sealing liquid in the ELISA plate a3, drying, and repeatedly washing with PBST for 4 times to obtain the ELISA plate a4. Adding protective liquid into the ELISA plate a4, sealing with a sealing plate membrane sealing plate at 37 ℃ for 30min to obtain an ELISA plate a5. And (5) discarding the protective solution in the ELISA plate a5, and airing at room temperature to obtain the F8 coated ELISA plate 2.

Example 3: preparation of ELISA kit for physical titre of retrovirus

Example 3 differs from example 2 in that the protection solution used in example 3 is PBS containing 2wt% glutarate.

Example 4: preparation of ELISA kit for physical titre of retrovirus

Example 4 differs from example 3 in that the protective solution used in example 4 also contains dibenzyl L-tartrate. The protective solution contained 1g L-dibenzyl tartrate and 2g glutarate relative to 100g of the protective solution, and the balance PBS.

Example 5: preparation of ELISA kit for physical titre of retrovirus

Example 5 differs from example 4 in that the protective liquid of example 5 contains dibenzyl 2g L-tartrate and glutaric acid ester 2g relative to 100g of the protective liquid.

Example 6: detection step of ELISA detection kit for physical titre of retrovirus

1) Diluting a sample to be detected by using a diluent to obtain a detection sample; the quality control product is GALV-env protein standard product with the volume of 50 pg/mL; gradient diluting the GALV-env protein standard substance with a diluent to obtain a standard substance; and then respectively sucking 100 mu L of standard substance, detection sample and positive quality control substance, adding the standard substance, the detection sample and the positive quality control substance into the ELISA plate, and standing and incubating for 120min at room temperature by using a sealing plate membrane sealing plate to obtain the ELISA plate 1. Then, the liquid in the ELISA plate 1 was discarded, the plate was dried by shaking, 300. Mu.L of the washing buffer was added to each well, the washing buffer was discarded, and the plate was dried by shaking, thereby completing one washing. And repeatedly washing for 4 times by using a washing buffer solution to obtain the ELISA plate 2. And adding 100 mu L/hole of detection antibody into the ELISA plate 2, sealing by using a sealing plate membrane, standing at room temperature, and incubating for 60min to obtain the ELISA plate 3. Discarding the liquid in the ELISA plate 3, drying, and repeatedly washing with a washing buffer solution for 4 times to obtain the ELISA plate 4. After diluting SA-HRP with a diluent for 5000 times, 50 mu L/hole is added into an ELISA plate 4, a sealing plate is used for sealing a plate, standing is carried out at room temperature for 30min, and an ELISA plate 5 is obtained. The liquid in the ELISA plate 5 is discarded, the plate is dried, and the ELISA plate 6 is obtained by repeated washing for 4 times with a washing buffer solution. And adding 100 mu L/hole of color developing solution into the ELISA plate 6, incubating for 10min at room temperature in a dark place, adding 100 mu L/hole of stop solution to obtain an ELISA plate 7, and then placing the ELISA plate 7 into an ELISA instrument to read readings at wavelengths of 450nm and 630 nm.

The standard substances were 0.00pg/mL (S7), 10.24pg/mL (S6), 25.6pg/mL (S5), 64pg/mL (S4), 160pg/mL (S3), 400pg/mL (S2), and 1000pg/mL (S1), respectively.

2) And (3) performing curve fitting on the readings by software matched with an enzyme-labeled instrument, taking the concentration of the standard substance as an abscissa and the OD _450nm-OD_630nm value as an ordinate, and establishing a standard curve and a regression equation of the GALV-env protein standard substance by four-parameter curve fitting.

3) The system applicability of the method ends the standard evaluation. The standard curve R ² is more than or equal to 0.980, the CV% of the OD _450nm-OD_630nm value compound holes of the standard products S1-S6 is less than or equal to 20.00%, and the recovery rate of the calculated concentration is between 70% and 130%. The OD _450nm-OD_630nm value of S1 should be equal to or greater than 1.000, and the OD _450nm-OD_630nm value of S7 should be equal to or less than 0.100. The recovery rate of the quality control product measurement result is 70% -130%, and CV% of the compound hole OD _450nm-OD_630nm value is less than or equal to 20.00%. CV% of the complex pore OD _450nm-OD_630nm value of the sample is less than or equal to 20.00%.

And if the detection result meets the system applicability acceptance standard, judging that the detection result is valid.

4) The OD _450nm-OD_630nm value of the detection sample is within the standard curve range of the GALV-env protein standard substance, and if the OD _450nm-OD_630nm value is out of the range, the dilution multiple of the detection sample is required to be adjusted, and then the detection is carried out again.

Calculation of GALV-env protein concentration:

And carrying the OD _450nm-OD_630nm value of the detection sample into a regression equation to obtain the concentration of the GALV-env protein in the detection sample, and then carrying out X dilution to obtain the concentration (pg/mL) of the GALV-env protein in the detection sample.

The physical titers of retroviruses were approximately calculated as follows:

(1) One retroviral particle contains 100-300 GALV-env protein molecules, and one retroviral particle is set to contain 200 GALV-env protein molecules;

(2) The mass of GALV-env in each retroviral particle (ng) = [200× (51.2×10 ³Da) / (6.03×10²³)]×10⁹= 1.71×10^-8 (ng);

(3) 1ng of GALV-env protein contains retroviral particle number (RPs) =1 ng/mass of GALV-env per retroviral particle (ng) =1/(1.71×10 ^-8)= 5.85×10⁷ RPs);

(4) Physical retroviral titre (RPs/mL) =1 ng GALV-env protein contains retroviral particle count (RPs) ×GALV-env protein concentration (ng/mL)

Note that: 51.2kDa (1 kDa=10 ³ g/mol) is the molecular weight of the GALV-env protein and 6.02X10- ²³ is the Avofiladellover constant.

Example 7: physical titer of retrovirus was detected by ELSA detection kit for GALV-env

ELISA experiments were performed as per the detection procedure of example 6 using the kit of example 1. Three batches of retroviral vectors (20231201, 20231101, 20231202) are used as samples to be tested, and the samples to be tested are obtained after dilution.

1) ELISA results of GALV-env protein standards and quality control are shown in Table 1, and regression equations (y=D+ (A-D)/[ 1+ (x/C)/(B ])) of the GALV-env protein standards are calculated as follows: y= 5.299+ (0.039-5.299)/[ 1+ (x/562.717) ] 1.116], the corresponding standard curve is shown in FIG. 1. ELISA results for the samples tested are shown in Table 2.

TABLE 1 ELISA results for GALV-env protein standards and quality controls

TABLE 2 ELISA results for the detection of samples

In combination with tables 1 and 2, the standard curve has an R ² value of 1.000, an OD _450nm-OD_630nm average value of S1 of 3.484, an OD _450nm-OD_630nm average value of S7 of 0.030, a CV% < 5.00% for the composite wells of OD _450nm-OD_630nm values of standards S1-S6, a recovery of the calculated concentration of 99% -109%, a CV% for the composite wells of OD _450nm-OD_630nm value of the quality control of 11.52%, a recovery of 109%, and a CV% < 3.00% for the composite wells of the test sample. The detection result meets the system applicability acceptance standard set by the method, and can be used for reporting the result of physical titer of the retrovirus vector.

2) The GALV-env protein concentration of the sample to be tested is obtained, and the physical titer of the sample to be tested can be obtained through conversion, and the result is shown in Table 3.

TABLE 3 ELISA results for GALV-env protein standards

Note that: physical retroviral titre (RPs/mL) =1 ng GALV-env protein contains retroviral particle count (RPs) ×GALV-env protein concentration (ng/mL)

Test example 1: infrared spectrum of glutarate

Infrared spectra of glutarates were measured using KBr pellet method. The results are shown in FIG. 2, which can be seen: the absorption peak near 3500cm ^-1 corresponds to the-OH stretching vibration peak; the absorption peak near 3000cm ^-1 is-CH ₃ stretching vibration peak; the absorption peak near 2900cm ^-1 is-CH ₂ stretching vibration peak; the absorption peak near 1700cm ^-1 is the stretching vibration peak of c=o bond; the absorption peak near 1300cm ^-1 corresponds to the C-O-C stretching vibration peak; an absorption peak near 1100cm ^-1 corresponds to the C-O stretching vibration peak; indicating that glutarate was obtained.

Test example 2: acceleration test of ELISA plate at 37 DEG C

Stability experiments were performed on the ELISA plates prepared in examples 1-5. Placing the ELISA plate into an aluminum foil bag, vacuumizing, and placing at 37 ℃ for 1 month acceleration test.

The GALV-env protein standard was diluted to 64pg/mL and used as a test sample. The stability of the ELISA plate before and after the acceleration test at 37℃was compared by ELISA test.

TABLE 4 accelerated test of ELISA plates at 37 ℃

Comparison of example 1 with examples 2 and 3 shows that the OD _450nm-OD_630nm values of the elisa plate of example 1 changed significantly before and after the 37 ℃ acceleration test, indicating that the stability of the elisa plates of examples 2 and 3 was superior to that of the elisa plate of example 1, probably because the antibodies coated on the elisa plate were protected after the treatment with the glutarate-containing protective solution. Glutarate can play a role in protecting an antibody by playing a role in bacteriostasis, so that the stability of the ELISA plate is prolonged. Wherein, the ELISA plate treated by the 2wt% glutarate protective solution has better stability.

In contrast, examples 3 and 4 and 5 showed small variation in OD _450nm-OD_630nm values before and after the accelerated test at 37℃in examples 4 and 5, which indicated that the enzyme-labeled plates of examples 4 and 5 had better stability, probably because the enzyme-labeled plates were further protected by treating the enzyme-labeled plates with a 2wt% glutarate-protecting solution after adding dibenzyl L-tartrate thereto. The dibenzyl L-tartrate may synergistically protect antibodies with glutarate. Wherein, the ELISA plate treated by the protective solution of 2wt% of glutarate and 2wt% of L-dibenzyl tartrate has better stability.

Test example 3: precision of the kit

The GALV-env protein standard was diluted to 25pg/mL and used as a test sample. ELISA assays were then performed as in example 6 using the kits of examples 1-5, respectively. Each kit was tested 5 times in parallel and RSD% was calculated between 5 times in parallel for each kit.

The results of ELISA are shown in Table 5.

TABLE 5 precision of the kit

Comparison of examples 1,2 and 3 shows that the RSD% value of example 1 is greater, which indicates that the precision of examples 2 and 3 is better, probably because the enzyme label plate is treated by the protection solution containing glutarate to protect the antibody, thereby ensuring the precision of detection of the kit. Wherein, the kit obtained by adopting 2wt% of glutarate protection solution treatment has better precision in detection.

Comparison of examples 3, 4 and 5 shows that the RSD% value of example 3 is larger, which indicates that the precision of examples 4 and 5 is better, probably because the enzyme-labeled plate is further protected by treating the enzyme-labeled plate with the protecting solution after adding dibenzyl L-tartrate to the protecting solution of 2wt% glutarate, thereby improving the precision of detection of the kit. Wherein, the kit obtained by adopting 2wt% of glutarate and 2wt% of protection solution of L-dibenzyl tartrate has better precision in detection.

The principles and embodiments of the present application have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present application and its core ideas. The foregoing is merely illustrative of the preferred embodiments of this application, and it is noted that there is objectively no limit to the specific structure disclosed herein, since numerous modifications, adaptations and variations can be made by those skilled in the art without departing from the principles of the application, and the above-described features can be combined in any suitable manner; such modifications, variations and combinations, or the direct application of the inventive concepts and aspects to other applications without modification, are contemplated as falling within the scope of the present application.

Claims

1. An ELISA kit for detecting physical titer of a retrovirus vector comprises a capture antibody, a detection antibody, a GALV-env protein standard substance and an ELISA plate, and is characterized in that the ELISA plate is treated by a protective solution, and the protective solution contains 1-2wt% of glutarate.

2. The kit according to claim 1, wherein the capture antibody is F8, the amino acid sequence of the F8 heavy chain is shown in SEQ ID NO.5, the amino acid sequence of the F8 light chain is shown in SEQ ID NO.6, the DNA sequence of the F8 heavy chain is shown in SEQ ID NO.9, and the DNA sequence of the F8 light chain is shown in SEQ ID NO. 10.

3. The kit of claim 1, wherein the detection antibody is C9-biotin; the C9-biotin is obtained by coupling C9 and biotin; the amino acid sequence of the C9 heavy chain is shown as SEQ ID NO.3, the amino acid sequence of the C9 light chain is shown as SEQ ID NO.4, the DNA sequence of the C9 heavy chain is shown as SEQ ID NO.7, and the DNA sequence of the C9 light chain is shown as SEQ ID NO. 8.

4. The kit of claim 1, wherein the step of treating the elisa plate with a protective solution comprises:

Diluting the capture antibody to 1-5 mu g/mL by using PBS to obtain a capture antibody solution, adding the capture antibody solution into a blank ELISA plate at 100-200 mu L/hole, coating at 2-8 ℃ for 8-16h, discarding the capture antibody solution, adding PBST, washing for 3-5 times, adding a blocking solution at 100-300 mu L/hole, blocking at 30-40 ℃ for 2-3h, discarding the blocking solution, adding PBST, washing for 3-5 times, adding a protection solution at 100-300 mu L/hole, blocking at 30-40 ℃ for 10-60min, discarding the protection solution, and airing at room temperature to obtain the ELISA plate.

5. A method for preparing glutarate, comprising the following steps:

6. The preparation method according to claim 5, wherein the glutaric anhydride and dipropanolamine are present in a molar ratio of 10: 1-10.

7. The preparation method according to claim 5, wherein the molar ratio of glutaric anhydride and 3-methoxybutanol is 10: 20-40.

8. A method for detecting physical titres of retroviral vectors using the kit of any one of claims 1 to 4 comprising the steps of:

1) Diluting a sample to be detected to obtain a detection sample; the quality control product is GALV-env protein standard product with the volume of 50-100 pg/mL; diluting the GALV-env protein standard to obtain a standard; then adding a detection sample, a quality control product and a standard product into an ELISA plate, incubating, washing, adding a detection antibody, incubating at room temperature in a dark place, washing, adding SA-HRP, incubating at room temperature in a dark place, washing, adding a color development liquid, developing at a dark place, adding a stop liquid, and putting into an ELISA meter for reading;

2) Performing data processing, and establishing a standard curve and a regression equation of the GALV-env protein standard substance;

3) Performing system applicability acceptance criteria evaluation of the method;

4) And calculating the concentration of GALV-env protein in the sample to be detected, and obtaining the physical titer of the retrovirus vector through conversion.

9. The method of claim 8, wherein the derivation of the physical titer of the retroviral vector by conversion is as follows:

One retroviral particle contains 100-300 GALV-env protein molecules, the molecular weight of the carried-in GALV-env and the Avofiladelo constant, resulting in the mass A, ng/RPs of each retroviral particle; ELISA measured GALV-env protein concentration B, ng/mL; B/A gives the physical titer of the retroviral vector, RPs/mL.

10. Use of a kit according to any one of claims 1 to 4 for detecting physical titres of retroviral vectors.