CN111272993A - Detection reagent box for biological product residues - Google Patents

Abstract

The invention relates to the technical field of immunology, in particular to a kit for detecting residues of biological products. The kit comprises a standard antigen, a negative and positive control solution, an anti-CHO HCP, an SPA, an ELISA plate coated by a bovine IgG protein antibody, a horseradish peroxidase (HRP) -labeled anti-CHO HCP, a bovine IgG protein antibody solution and an auxiliary reagent. The auxiliary reagent comprises a substrate solution A, a substrate solution B and a stop solution; the substrate liquid B is a solution of tetramethyl benzidine; the substrate solution A is a solution of carbamide peroxide. The kit for detecting the biological product residues provided by the invention carries out quantitative analysis on the four residues of CHO HCP, SPA and bovine IgG by using a double-antibody sandwich method ELISA; the operation time is short, and the detection technology can be mastered by technicians in a short-term training way; high-flux detection can be carried out, so that the efficiency of the quality control process is effectively improved; the sensitivity is high; the cost is low, expensive experimental equipment is not needed, and the enterprise detection cost can be greatly reduced.

Description

Detection reagent box for biological product residues

Technical Field

The invention relates to the technical field of immunology, in particular to a kit for detecting residues of biological products.

Background

The ELISA method is a common method for clinical detection, has been extended to a plurality of subject fields, and compared with the prior pure enzyme immunoassay technology, a plurality of derived forms have been developed, so that the detection method has the advantages of time and labor saving, strong specificity, high sensitivity and the like. The basis of ELISA is immobilization of antigen or antibody and enzyme labeling of antigen or antibody. The antigen or antibody combined on the surface of the solid phase carrier still keeps the immunological activity, and the enzyme-labeled antigen or antibody keeps the immunological activity and the enzyme activity. In the measurement, the specimen to be tested (the antibody or antigen to be measured therein) is reacted with the antigen or antibody on the surface of the solid carrier. The antigen-antibody complex formed on the solid phase carrier is separated from other substances in the liquid by washing. Then, an enzyme-labeled antigen or antibody is added thereto, and the resulting mixture is bound to a solid-phase carrier by reaction. At this time, the amount of enzyme on the solid phase is in a certain ratio to the amount of the substance to be detected in the specimen. After the substrate of enzyme reaction is added, the substrate is catalyzed by enzyme to become a colored product, and the amount of the product is directly related to the amount of the detected substance in the sample, so that qualitative or quantitative analysis can be carried out according to the color depth. The catalytic efficiency of the enzyme is high, so that the result of immune reaction is indirectly amplified, and the determination method achieves high sensitivity.

At present, all domestic enterprises which utilize eukaryotic expression or prokaryotic expression systems to produce medicinal recombinant proteins and other biomedical enterprises which produce therapeutic antibody vaccines use imported detection kits when detecting residual non-target proteins. The imported kit has high price and is one of the key factors for restricting the quality control cost. With the rising cost of human resources in China, antibody and drug manufacturers must try to control the cost of production in an effort to maximize profits. One of the important ways to save the expenses is to use the equipment and consumables provided by the local manufacturers in China as much as possible on the premise of ensuring the product quality and not using the production process. The quality control process of antibody and recombinant protein medicine is a link closely connected with the production process but relatively independent. The cost can be reduced by changing domestic quality control consumables, and the improvement of a large knife and broad axe in the production process of the product is not needed. At present, the 2010 version of Chinese pharmacopoeia only has a clear specified detection method for residual escherichia coli and Vero cell HCP, and the detection for the residual CHO HCP has no clear text specification. The Chinese pharmacopoeia has no regulation on the residue detection of SPA and bovine IgG. BSA is strictly limited and should not exceed 50 ng/dose. Therefore, the development of the method for detecting the residual quantity of CHO HCP, SPA and bovine IgG can fill the blank of the field and lay a foundation for the later establishment of related industrial standards.

Disclosure of Invention

Aiming at the technical problem, the invention provides a kit for detecting biological product residues, which comprises a standard antigen, a negative and positive control solution, an anti-CHO HCP, an SPA, an ELISA plate coated by a bovine IgG protein antibody, a horseradish peroxidase (HRP) -labeled anti-CHO HCP, a bovine IgG protein antibody solution and an auxiliary reagent.

As a preferred technical scheme, the horseradish peroxidase (HRP) labeled anti-CHO HCP, SPA, bovine IgG protein antibody is used at a dilution of 1: 2000.

As a preferred technical scheme, the auxiliary reagent comprises a substrate solution A and a substrate solution B; the substrate liquid B is a solution of tetramethyl benzidine; the substrate solution A is a solution of carbamide peroxide.

As a preferred technical scheme, the preparation method of the substrate liquid B comprises the following steps: 20mg of tetramethyl benzidine negative and positive control solution, 8-10 mL of absolute ethyl alcohol negative and positive control solution, adding double distilled water to 1 mL of negative and positive control solution, and filtering and sterilizing to obtain the product.

As a preferred technical scheme, the preparation method of the substrate liquid A comprises the following steps: dissolving NaHPO14.60g, citric acid 9.33g and carbamide peroxide 0.52g in triple distilled water, adjusting the final volume to 1000mL, and adjusting the pH value to 5.0-5.4 to obtain the product.

As a preferable technical scheme, the stop solution is a sulfuric acid solution, and the concentration of the stop solution is 1 mol/L.

As a preferred technical scheme, the preparation method of the ELISA plate coated by the bovine IgG protein antibody comprises the following steps:

diluting anti-CHO HCP, SPA and bovine IgG protein antibodies with diluent, adding 300 mu L of each hole of the ELISA plate, and incubating for 24 hours at 4 ℃; and removing liquid in the holes, washing the holes by PBST, drying the holes by beating, sealing the holes by using sealing liquid at 37 ℃, washing the holes by using PBST washing liquid for 3 times, washing the holes for 5 minutes each time, drying the holes by beating, and airing to obtain the ELISA plate coated by the anti-CHO HCP, SPA and bovine IgG protein antibodies.

As a preferred technical scheme, the preparation raw materials of the confining liquid comprise, by weight, 0.03-0.08 wt% of a nonionic surfactant, 15-25 wt% of bovine serum albumin, and the balance of a PBS buffer solution; the nonionic surfactant is polysorbate-20.

As a preferred technical scheme, the preparation of the diluent comprises the following steps:

0.2g KH was used₂PO₄、2.9g Na₂HPO₄·12H₂Preparing 0.01M PBS buffer solution by using O, 8g NaCl and 0.2g KCl, adding a nonionic surfactant and bovine serum albumin into the buffer solution for dissolving, and filtering by using a filter membrane with the aperture of 0.45 mu M to obtain a solution with the content of the nonionic surfactant of 0.05 wt% and the content of the bovine serum albumin of 0.5 wt%.

As a preferred technical scheme, the non-ionic surfactant in the diluent is polysorbate-20 and/or polysorbate-80.

The kit for detecting the biological product residues provided by the invention carries out quantitative analysis on the four residues of CHO HCP, SPA and bovine IgG by using a double-antibody sandwich method ELISA; the operation time is short, and the detection technology can be mastered by technicians in a short-term training way; high-flux detection can be carried out, so that the efficiency of the quality control process is effectively improved; the sensitivity is high; the cost is low, expensive experimental equipment is not needed, and the enterprise detection cost can be greatly reduced. The research and the production of a detection kit and an in-vitro diagnosis kit in the biopharmaceutical industry are promoted by the sale of the conventional scientific research kit, and the strategic transformation from clients in the scientific research field to industrial clients is realized; the cellular organelle protein residual kit is a global initiative concept, and the data of product detection can prove that residual protein belongs to which type of protein of cells, so that the reliability is extremely high, and the kit has great competitive advantage.

Detailed Description

The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

In addition, the indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the stated number clearly indicates that the singular form is intended.

The invention provides a kit for detecting residues of biological products, which comprises a standard antigen, a negative and positive control solution, an anti-CHO HCP, an SPA, an ELISA plate coated by a bovine IgG protein antibody, a Horse Radish Peroxidase (HRP) -labeled anti-CHOHCP, a bovine IgG protein antibody solution and an auxiliary reagent.

anti-CHO HCP in the present invention means CHO cell host protein; SPA refers to human staphylococcal protein A; wherein IgG is an immunoglobulin. The above raw materials are commercially available from general industries, for example, bovine IgG protein antibody is available from Emei technologies, Inc., anti-CHO HCP is available from Shanghai-Producer laboratories, Inc., and the like.

In some embodiments, the horseradish peroxidase (HRP) -labeled anti-CHO HCP, SPA, bovine IgG protein antibody is used at a 1:2000 dilution.

In some embodiments, the auxiliary reagent comprises substrate solution a, substrate solution B; the substrate liquid B is a solution of tetramethyl benzidine; the substrate solution A is a solution of carbamide peroxide.

In some embodiments, the method for preparing the substrate solution B comprises the following steps: 20mg of tetramethyl benzidine negative and positive control solution, 8-10 mL of absolute ethyl alcohol, adding double distilled water to 1 negative and positive control solution to 000mL, and filtering and sterilizing to obtain the product.

In some embodiments, the method for preparing the substrate liquid A comprises the following steps: dissolving NaHPO14.60g, citric acid 9.33g and carbamide peroxide 0.52g in triple distilled water, adjusting the final volume to 1000mL, and adjusting the pH value to 5.0-5.4 to obtain the product.

In some embodiments, the stop solution is a sulfuric acid solution with a concentration of 1 mol/L.

In some embodiments, the preparation method of the bovine IgG protein antibody-coated ELISA plate comprises the following steps:

diluting anti-CHO HCP, SPA and bovine IgG protein antibodies with diluent, adding 300 mu L of each hole of the ELISA plate, and incubating for 24 hours at 4 ℃; and removing liquid in the holes, washing the holes by PBST, drying the holes by beating, sealing the holes by using sealing liquid at 37 ℃, washing the holes by using PBST washing liquid for 3 times, washing the holes for 5 minutes each time, drying the holes by beating, and airing to obtain the ELISA plate coated by the anti-CHO HCP, SPA and bovine IgG protein antibodies.

The PBST and PBST solution in the invention refer to a solution prepared by introducing a surfactant into a PBS buffer solution, wherein the surfactant is Tween-20. The PBS buffer solution of the invention is prepared by 0.2g KH₂PO₄、2.9g Na₂HPO₄·12H₂O, 8g of NaCl and 0.2g of KCl, and adding water to a constant volume to prepare a 0.01M solution to obtain the PBS buffer solution.

In some embodiments, the preparation raw materials of the sealing liquid comprise, by weight, 0.03 to 0.08 wt% of a nonionic surfactant, 15 to 25 wt% of bovine serum albumin, and the balance of a PBS buffer solution; the nonionic surfactant is polysorbate-20.

Further, the preparation raw materials of the blocking solution comprise, by weight, 0.05 wt% of a nonionic surfactant, 20 wt% of bovine serum albumin, and the balance of a PBS buffer solution.

In some embodiments, the diluent formulation comprises the steps of:

0.2g KH was used₂PO₄、2.9g Na₂HPO₄·12H₂Preparing 0.01M PBS buffer solution by using O, 8g NaCl and 0.2g KCl, adding a nonionic surfactant and bovine serum albumin into the buffer solution for dissolving, and filtering by using a filter membrane with the aperture of 0.45 mu M to obtain a solution with the content of the nonionic surfactant of 0.05 wt% and the content of the bovine serum albumin of 0.5 wt%.

In some embodiments, the non-ionic surfactant in the diluent is polysorbate-20 and/or polysorbate-80.

Further, the non-ionic surfactant in the diluent is a mixture of polysorbate-20 and polysorbate-80.

Further, the mass ratio of the polysorbate-20 to the polysorbate-80 is 3: 1.

the CHO HCP project produced in the existing market is mainly the host cell total protein product, while the CHOHCP product developed by the invention has comprehensive lines, has the host cell total protein product, and also has the organelle protein product obtained after the organelle is separated and developed by completely original technical thought, the high-purity nucleus and mitochondria can be separated, and the product lines comprise: 4 types of products including total protein residue, mitochondrial protein residue, nuclear protein residue and supernatant protein residue; moreover, the product inspection result can prove that the residual protein belongs to which type of protein in the cell, the method can be suitable for the protein residual quality verification of various medicines, and the data reliability is higher.

Examples

Example 1: a kit for detecting biological product residues comprises a standard antigen, a negative and positive control solution, an anti-CHO HCP, an SPA, an ELISA plate coated by a bovine IgG protein antibody, a horseradish peroxidase (HRP) -labeled anti-CHO HCP, a bovine IgG protein antibody solution and an auxiliary reagent. Wherein, the horseradish peroxidase (HRP) labeled anti-CHO HCP, SPA, bovine IgG protein antibody is diluted according to 1: 2000. The auxiliary reagent comprises a substrate solution A, a substrate solution B and a stop solution; the substrate liquid B is a solution of tetramethyl benzidine; the substrate solution A is a solution of carbamide peroxide.

The preparation method of the substrate solution B comprises the following steps: 20mg of tetramethyl benzidine, 10mL of absolute ethanol, and 1000mL of double distilled water, and filtering and sterilizing to obtain the finished product. The preparation method of the substrate liquid A comprises the following steps: 14.60g of NaHPO, 9.33g of citric acid and 0.52g of carbamide peroxide are dissolved in triple distilled water, the final volume is 1000mL, and the pH is adjusted to 5.2 to obtain the compound. The stop solution is a sulfuric acid solution, and the concentration of the stop solution is 1 mol/L.

The preparation method of the ELISA plate coated by the bovine IgG protein antibody comprises the following steps:

diluting anti-CHO HCP, SPA and bovine IgG protein antibodies with diluent, adding 300 mu L of each hole of the ELISA plate, and incubating for 24 hours at 4 ℃; and removing liquid in the holes, washing the holes by PBST, drying the holes by beating, sealing the holes by using sealing liquid at 37 ℃, washing the holes by using PBST washing liquid for 3 times, washing the holes for 5 minutes each time, drying the holes by beating, and airing to obtain the ELISA plate coated by the anti-CHO HCP, SPA and bovine IgG protein antibodies. Wherein, the preparation raw materials of the confining liquid comprise, by weight, 0.05 wt% of nonionic surfactant, 20 wt% of bovine serum albumin, and the balance of PHS buffer solution; the nonionic surfactant is polysorbate-20; is prepared by dissolving bovine serum albumin and nonionic surfactant in PBS buffer solution, and filtering with filter membrane with pore diameter of 0.45 μm. Wherein the diluent preparation comprises the following steps:

0.2g KH was used₂PO₄、2.9g Na₂HPO₄·12H₂Preparing 0.01M PBS buffer solution by using O, 8g NaCl and 0.2g KCl, adding a nonionic surfactant and bovine serum albumin into the buffer solution for dissolving, and filtering by using a filter membrane with the aperture of 0.45 mu M to obtain a solution with the content of the nonionic surfactant of 0.05 wt% and the content of the bovine serum albumin of 0.5 wt%. Wherein the non-ionic surfactant in the diluent is a mixture of polysorbate-20 and polysorbate-80, and the mass ratio of the polysorbate-20 to the polysorbate-80 is 3: 1.

the kit for detecting the residues of the biological products provided by the embodiment quantitatively analyzes the residues of CHO HCP, SPA and bovine IgG in industrial production; the operation time is short, and the detection technology can be mastered by technicians in a short-term training way; high-flux detection can be carried out, so that the efficiency of the quality control process is effectively improved; the sensitivity is high; the cost is low, expensive experimental equipment is not needed, and the enterprise detection cost can be greatly reduced. Moreover, the reagent in the kit has good stability, and the reagent is basically not changed when placed in an environment at 37 ℃ for a week, so that the detection sensitivity is still good.

Example 2: a kit for detecting biological product residues comprises a standard antigen, a negative and positive control solution, an anti-CHO HCP, an SPA, an ELISA plate coated by a bovine IgG protein antibody, a horseradish peroxidase (HRP) -labeled anti-CHO HCP, a bovine IgG protein antibody solution and an auxiliary reagent. Wherein, the horseradish peroxidase (HRP) labeled anti-CHO HCP, SPA, bovine IgG protein antibody is diluted according to 1: 2000. The auxiliary reagent comprises a substrate solution A, a substrate solution B and a stop solution; the substrate liquid B is a solution of tetramethyl benzidine; the substrate solution A is a solution of carbamide peroxide.

The preparation method of the substrate solution B comprises the following steps: 20mg of tetramethyl benzidine negative and positive control solution, 10mL of absolute ethyl alcohol, adding double distilled water to 1000mL, and filtering and sterilizing to obtain the product. The preparation method of the substrate liquid A comprises the following steps: 14.60g of NaHPO, 9.33g of citric acid and 0.52g of carbamide peroxide are dissolved in triple distilled water, the final volume is 1000mL, and the pH is adjusted to 5.2 to obtain the compound. The stop solution is a sulfuric acid solution, and the concentration of the stop solution is 1 mol/L.

The preparation method of the ELISA plate coated by the bovine IgG protein antibody comprises the following steps:

diluting anti-CHO HCP, SPA and bovine IgG protein antibodies with diluent, adding 300 mu L of each hole of the ELISA plate, and incubating for 24 hours at 4 ℃; and removing liquid in the holes, washing the holes by PBST, drying the holes by beating, sealing the holes by using sealing liquid at 37 ℃, washing the holes by using PBST washing liquid for 3 times, washing the holes for 5 minutes each time, drying the holes by beating, and airing to obtain the ELISA plate coated by the anti-CHO HCP, SPA and bovine IgG protein antibodies. Wherein, the preparation raw materials of the confining liquid comprise, by weight, 0.05 wt% of nonionic surfactant, 20 wt% of bovine serum albumin, and the balance of PHS buffer solution; the nonionic surfactant is polysorbate-20; is prepared by dissolving bovine serum albumin and nonionic surfactant in PBS buffer solution, and filtering with filter membrane with pore diameter of 0.45 μm. Wherein the diluent preparation comprises the following steps:

0.2g KH was used₂PO₄、2.9g Na₂HPO₄·12H₂Preparing 0.01M PBS buffer solution by using O, 8g NaCl and 0.2g KCl, adding a nonionic surfactant and bovine serum albumin into the buffer solution for dissolving, and filtering by using a filter membrane with the aperture of 0.45 mu M to obtain a solution with the content of the nonionic surfactant of 0.05 wt% and the content of the bovine serum albumin of 0.5 wt%. Wherein the non-ionic surfactant in the diluent is polysorbate-20.

The kit for detecting the residues of the biological products provided by the embodiment quantitatively analyzes the residues of CHO HCP, SPA and bovine IgG in industrial production; the operation time is short, and the detection technology can be mastered by technicians in a short-term training way; high-flux detection can be carried out, so that the efficiency of the quality control process is effectively improved; the sensitivity is high. However, after the kit is placed at 37 ℃ for a week, the detection sensitivity is reduced, the stability is poor, and the detection efficiency is low.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art may modify or change the technical content of the above disclosure into equivalent embodiments with equivalent changes, but all those simple modifications, equivalent changes and modifications made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the present invention.

Claims (10)

1. A kit for detecting biological product residues is characterized by comprising a standard antigen, a negative and positive control solution, an anti-CHO HCP, an SPA, an ELISA plate coated by a bovine IgG protein antibody, a horseradish peroxidase-labeled anti-CHO HCP, a bovine IgG protein antibody solution and an auxiliary reagent.

2. The kit for bioproduct residue detection of claim 1 wherein the horseradish peroxidase labeled anti-CHO HCP, SPA, bovine IgG protein antibody is used at a 1:2000 dilution.

3. The kit for bioproduct residue detection of claim 1 wherein the auxiliary reagent comprises substrate solution a, substrate solution B; the substrate liquid B is a solution of tetramethyl benzidine; the substrate solution A is a solution of carbamide peroxide.

4. The kit for detecting a biological product residue according to claim 3, wherein the method for preparing the substrate solution B comprises the steps of: 20mg of tetramethyl benzidine negative and positive control solution, 8-10 mL of absolute ethyl alcohol negative and positive control solution, adding double distilled water to 1000mL, and filtering and sterilizing to obtain the finished product.

5. The kit for detecting a biological product residue according to claim 3, wherein the method for preparing the substrate liquid A comprises the steps of: 14.60g of NaHPO negative and positive control solution, 9.33g of citric acid and 0.52g of carbamide peroxide are dissolved in triple distilled water, the final volume is 1000mL, and the pH value is adjusted to 5.0-5.4 to obtain the product.

6. The kit for detecting biological product residues according to claim 3, wherein the auxiliary reagent further comprises a stop solution, and the stop solution is a sulfuric acid solution with a concentration of 1 mol/L.

7. The kit for detecting biological product residues as claimed in any one of claims 1 to 6, wherein the preparation method of the ELISA plate coated with the bovine IgG protein antibody comprises the following steps:

diluting anti-CHO HCP, SPA and bovine IgG protein antibodies with diluent, adding 300 mu L of each hole of the ELISA plate, and incubating for 24 hours at 4 ℃; and removing liquid in the holes, washing the holes by PBST, drying the holes by beating, sealing the holes by using sealing liquid at 37 ℃, washing the holes by using PBST washing liquid for 3 times, washing the holes for 5 minutes each time, drying the holes by beating, and airing to obtain the ELISA plate coated by the anti-CHO HCP, SPA and bovine IgG protein antibodies.

8. The kit for detecting the residue in the biological product according to claim 7, wherein the blocking solution is prepared from the following raw materials, by weight, 0.03 to 0.08% of the nonionic surfactant, 15 to 25% of bovine serum albumin, and the balance of the PBS buffer solution; the nonionic surfactant is polysorbate-20.

9. The kit for the detection of a residue of a biological product according to claim 7 or 8, wherein the diluent is formulated to comprise the steps of:

0.2g KH was used₂PO₄、2.9g Na₂HPO₄·12H₂Preparing 0.01M PBS buffer solution by using O, 8g NaCl and 0.2g KCl, adding a nonionic surfactant and bovine serum albumin into the buffer solution for dissolving, and filtering by using a filter membrane with the aperture of 0.45 mu M to obtain a solution with the content of the nonionic surfactant of 0.05 wt% and the content of the bovine serum albumin of 0.5 wt%.

10. The kit for bioproduct residue detection according to claim 9, wherein the nonionic surfactant in the diluent is polysorbate-20 and/or polysorbate-80.