Preparation method and application of universal detection kit for E.coli host residual protein

Abstract

The invention relates to the technical field of quality control of biological products, in particular to a preparation method and application of a universal detection kit for E.coli host residual protein. According to the preparation method of the universal detection kit for the E.coli host residual protein, provided by the invention, the detection accuracy is high, the limit of quantitation sensitivity reaches 1.56 ng / mL, the antibody coverage rate is relatively high, and no cross reaction with Pichia yeast with relatively close genetic relationship exists. In addition, according to the method, the antigen and the detection antibody are mixed for incubation, namely, a one-step method is adopted, the steps are few, the consumed time is short, and the error rate is low according to standard substance concentration calculation. The method provided by the invention can be used for rapidly, efficiently and accurately calculating the residual protein content of the E.coli host, and is applied to quality control of biological products.

Description

Technical Field

[0001] The present invention relates to the technical field of biological product quality control, and in particular to a preparation method and application of a universal detection kit for residual protein in an E. coli host. Background Art

[0002] Host cell protein (HCP) residues are the main impurities involved in the production process of biological products. The main risk of HCP in biological products is its immunogenicity. HCP, a complex mixture with multiple physiological, chemical and immunological properties, may trigger an immune response in the human body and induce the body to produce corresponding HCP antibodies; some HCPs have adjuvant effects, which can cause inflammatory responses in the human body, or combine with antibody drugs and other ingredients to cause immunogenicity to the drug, produce antibodies against the drug, and reduce the efficacy of the drug. Therefore, almost all HCPs have clinical safety risks as foreign protein drugs.

[0003] The HCP detection in the prior art has a long detection cycle, a long preparation time, and relatively complicated preparation work, and is greatly affected by the environment, so there are certain requirements for the detection environment. Some existing ELISA detection kits provide coated plates and configured reagents in the kits, thereby improving the complex operations and high error rates in the existing experimental detection operations, but the problem of linear range accuracy of the detection has not been solved; some kits have good specificity, high sensitivity, and high detection accuracy, but the problem of cumbersome preliminary preparation work has not been solved.

[0004] Therefore, in order to solve the above problems, the present application provides a preparation method and application of a universal detection kit for residual protein in E. coli host. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a universal detection kit for residual protein in E. coli host to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for preparing a universal detection kit for residual protein in an E. coli host comprises the following steps:

[0008] S1. Preparation of Escherichia coli antigen;

[0009] S11. Cultivate Escherichia coli and obtain the cells by centrifugation;

[0010] S12. The bacteria in step S11 are homogenized under high pressure to obtain Escherichia coli antigens;

[0011] S2. Preparation of sheep anti-E. coli antigen antibodies;

[0012] S21. Select goats of appropriate age for immunization;

[0013] S22. After the immunization, the antiserum was collected and purified to obtain antibodies against residual proteins of the E. coli host;

[0014] S3. E. coli host residual protein antibody labeling, using horseradish peroxidase labeling, the E. coli host residual protein antibody prepared in step S22 is labeled;

[0015] S4. Establishment of ELISA method for detecting residual protein in Escherichia coli host;

[0016] S41. Coating: dilute the purified antibody in step S22 to 5 μg / mL to 25 μg / mL with carbonate buffer or phosphate buffer, and coat at 4°C overnight;

[0017] S42. Blocking: drying to remove the coating solution, washing three times with a washing solution composed of phosphate buffer containing 0.01% to 0.1% Tween 20, blocking with a blocking solution composed of 1 to 10 wt% sucrose, 1 to 8 wt% trehalose and 0.1 to 6 wt% fish collagen at 25 ° C to 37 ° C for 0.5 h to 2 h, and after the blocking, washing three times with a washing solution;

[0018] S43. Combining antigen and detection antibody mixture: the E. coli antigen prepared in step S12 is used as a calibrator or a sample to be tested and a host residual protein labeled antibody diluted with a diluent containing 2% to 8wt% sucrose, 3% to 8wt% trehalose and 0.5% to 3wt% fish collagen at a volume of 1:50 to 1:400 at a ratio of 1:1 to 1:3, and then added to the corresponding wells and incubated for 1h to 2h;

[0019] S44. Dried to remove the well solution in step S43, washed with a detergent, added TMB colorimetric substrate to each well, and incubated at room temperature in the dark for 15 to 35 minutes;

[0020] S45. Add 30 μL to 150 μL of stop solution to each well to terminate the reaction, and measure the OD450 value with a microplate reader;

[0021] S46. Use the four-parameter fitting equation to draw a calibration curve, and calculate the concentration of the sample to be tested based on the calibration curve and the OD450 value of the sample to be tested.

[0022] Furthermore, in the step S41, the host residual protein antibody purified in the step S22 is coated with 0.02-0.2 M carbonate buffer.

[0023] Moreover, the selected concentration of the calibrator in step S43 is 0-400 ng / mL.

[0024] Furthermore, in the step S43, a diluent containing 3% to 6wt% sucrose, 3.5% to 7wt% trehalose and 1% to 2wt% fish collagen is added to dilute the E. coli host residual protein labeled antibody.

[0025] Moreover, the blocking solution used in step S42 contains 3-8 wt% sucrose, 2-6 wt% trehalose and 0.5-5 wt% fish collagen.

[0026] Moreover, the one-step method of mixed incubation of antigen and detection antibody only takes 2 hours to complete the test.

[0027] Moreover, it was shown that the antibody coverage reached more than 80%.

[0028] Furthermore, this method has no cross-reactivity with the closely related Pichia yeast.

[0029] Moreover, its detection accuracy is high, the sensitivity of the quantitative limit reaches 1.56ng / mL, the antibody coverage rate is high, and there is no cross-reaction with the closely related Pichia yeast. In addition, this method uses a one-step method of mixed incubation of antigen and detection antibody, which has simple steps and takes a short time. The sample concentration is calculated by fixing the standard concentration, and the error rate is low.

[0030] The second aspect of the present invention provides an application of a method for preparing a universal E. coli HCP detection kit, including application of the E. coli HCP ELISA detection kit in a rapid and simple method for detecting the content of E. coli HCP.

[0031] The advantages and positive effects of the present invention are:

[0032] 1. The universal detection kit for residual protein of E. coli host prepared by the preparation method of the present invention has high detection accuracy. The detection uses a one-step method of mixed incubation of antigen and detection antibody. The limit of quantification can reach 1.56 ng / mL, and the sample concentration is calculated with a fixed standard concentration, with a low error rate, which is convenient for the smooth implementation of the experiment and obtains more reliable and accurate data.

[0033] 2. The preparation method of the universal detection kit for residual protein of E. coli host described in the present invention can achieve an antibody coverage rate of more than 80%, and has no cross reaction with the closely related Pichia yeast.

[0034] 3. The present invention can omit the step of coating the plate the day before, and the kit contains prepared reagents that can be directly used in the experiment, saving the entire operation time. In addition, the one-step method of mixed incubation of antigen and detection antibody is adopted, and the test can be completed in only 2 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a graph showing the antibody coverage results of the E. coli HCP ELISA detection kit of the present invention.

[0036] Figure 2 It is a four-parameter equation diagram of the E. coli HCP ELISA detection kit of the present invention.

[0037] Figure 3 This is a diagram showing the specificity verification results of the E. coli HCP ELISA detection kit of the present invention.

[0038] Figure 4 The figure is a comparative flow chart of the preparation steps of the conventional kit of the present invention and the E. coli HCP ELISA detection kit. DETAILED DESCRIPTION

[0039] The following is a clear and comprehensive description of the technical embodiments of the present invention in conjunction with the drawings of the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] It should be noted that the relational terms used in this article, such as first and second, etc., are only used to distinguish between operations, and do not require that the operations must be performed in this relationship or order.

[0041] Example 1

[0042] Determination of sheep anti-E.coli HCP antibody coverage

[0043] Using E.coli HCP antigen and goat anti-E.coli HCP antibody as materials, immunoaffinity chromatography-liquid chromatography-mass spectrometry (AAE-MS) was used to verify the coverage of goat anti-E.coli HCP antibody.

[0044] According to the final LC-MS analysis data, the protein number was retrieved and the coverage of goat anti-E. coli HCP polyclonal antibody was 82%. Figure 1 shown.

[0045] Example 2

[0046] Establishment and application of E.coli HCP ELISA detection method

[0047] 1. Establishment of E.coli HCP ELISA detection method The prepared E.coli HCP was quantified and the standard in the E.coli HCP residual detection kit was prepared. The preparation method is as follows: the prepared E.coli HCP antigen was diluted with ST1-ST7 and NTC with diluent to prepare E.coli HCP standard, the OD450 value was detected by sandwich ELISA method, and a four-parameter fitting curve was drawn.

[0048] The establishment process is as follows:

[0049] (1) Coating: Dilute the purified sheep anti-E. coli HCP antibody to 12 μg / mL or 24 μg / mL with coating buffer and coat at 4°C overnight;

[0050] (2) Blocking: Spin dry to remove the coating solution, wash three times with washing solution, add blocking solution, and incubate at 37°C for 1 hour;

[0051] (3) Binding antigen and detection antibody: dilute the E. coli HCP standard to 100 ng / mL and 0 ng / mL with dilution buffer for later use, dilute the HCP labeled antibody freshly prepared in Example 3 at a ratio of 1:100 with dilution buffer for later use, mix the two at a volume ratio of 1:1 for later use, pour off the blocking solution, wash with washing solution 3 times, spin dry, and turn it upside down on filter paper and pat dry;

[0052] (4) Pour off the liquid, wash with detergent 6 times, spin dry, and pat dry on filter paper. Add TMB chromogenic substrate and incubate for 30 minutes;

[0053] (5) Add 50 μL of stop solution to each well to terminate the reaction, and measure the OD450 value using an ELISA reader.

[0054] According to the test data, the coating antibody concentration was determined to be 12 μg / mL, and the optimal dilution ratio of the HCP-labeled antibody was 1:100.

[0055] 2. Correlation of the calibration curve of the E.coli HCP ELISA kit. Based on the above-determined optimal working concentrations of the coating antibody and the detection antibody, the E.coli HCP calibrator was diluted in 7 concentration gradients at a certain ratio (2 times) while keeping the coating antibody concentration and the detection antibody concentration unchanged. Each concentration was repeated to minimize the interference of experimental operation errors. The ELISA test was performed on the same strip according to the above-mentioned antibody pairing method to obtain the detection reaction value, and the calibration curve was drawn based on this. The calibration concentration and the corresponding reaction value are shown in Table 1.

[0056] Table 1

[0057]

[0058] The calibration value of the calibrator and the measured OD value are used as the X and Y axes for four-parameter fitting to draw a calibration curve, such as Figure 2 The calibrator was diluted to 100 ng / mL, and then diluted downward by two times until 1.56 ng / mL. No antigen was added to the last point. The antigen concentration was used as the horizontal axis and the OD450 value obtained by the test was used as the vertical axis. The above data were fitted by four parameters, and the fitting degree satisfied R 2 The requirement of greater than 0.99 indicates that this method has good linearity in the E. coli HCP concentration range of 0-100 ng / mL.

[0059] Example 3

[0060] E.coli HCP ELISA Kit Performance Verification

[0061] 1. The organism that is most likely to cross-react with E. coli in nature is Pichia yeast, so in the specificity verification stage, the currently established double antibody sandwich ELISA detection method was used to detect Pichia yeast at three concentrations of 400ng / mL, 100ng / mL and 25ng / mL to verify the specificity. The blank value was used as a control reference, and each concentration was tested in two groups. The experimental results are as follows Figure 3 As shown, the detection values ​​of the three concentrations of Pichia yeast were all lower than the blank value, which confirmed that the double antibody sandwich ELISA method established in this experiment had no cross reaction with Pichia yeast.

[0062] 2. Analysis of the quantitative lower limit of E. coli HCP ELISA kit The lowest concentration of the calibration curve (1.5625 ng / mL) was selected as the quantitative limit. The calibrator was mixed with an equal volume of the sample to be tested so that the concentration of the calibrator was 1.5625 ng / mL at the lowest point of the test. The recovery rate was determined and the RSD was calculated. The results are shown in Table 2.

[0063] Table 2

[0064]

[0065] According to the experimental results, the recovery rate of the minimum detection limit ranges from 99.84% to 117.76%, meeting the standard of 70% to 130%, and the recovery rate is qualified; the calculated value of RSD is 8.41%, meeting the requirement of RSD less than 15%, so the quantitative limit of this method is 1.5625 ng / mL.

[0066] 3. Repeatability analysis of E.coli HCP ELISA kit

[0067] The ELISA method and kit established in Example 1 were used, and three calibrators of high, medium and low concentrations were selected. Six independent samples were prepared for each sample, which were mixed with the sample to be tested at the same concentration and validated by spike recovery. RSD was calculated. The results are shown in Table 3.

[0068] Table 3

[0069]

[0070] The test results show that when the high concentration (50ng / mL) is added, the RDS (n=6) of the HCP detection amount of the calibration standard in six samples is 13.10%; when the medium concentration (6.25ng / mL) is added, the RDS (n=6) of the HCP detection amount of the calibration standard in six samples is 9.03%; when the low concentration (1.56ng / mL) is added, the RDS (n=6) of the HCP detection amount of the calibration standard in six samples is 13.40%. The RSD values ​​detected in the three concentrations are all less than 15%, so the repeatability is qualified.

[0071] 4. Precision analysis of E.coli HCP ELISA kit

[0072] Using the ELISA method and kit established in Example 1, another experimenter selected three calibrators of high, medium and low concentrations, and prepared six independent samples for each sample, which were mixed with the sample to be tested at the same concentration, and spike recovery verification was performed to calculate CV. The results are shown in Table 4.

[0073] Table 4

[0074]

[0075] The test results show that when the high concentration (50ng / mL) is spiked, the RDS (n=6) of the intermediate precision of the six samples is 11.62%; when the medium concentration (6.25ng / mL) is spiked, the RDS (n=6) of the intermediate precision of the six samples is 10.13%; when the low concentration (1.56ng / mL) is spiked, the RDS (n=6) of the intermediate precision of the six samples is 13.85%. The RDS (n=12) of the intermediate precision and repeatability of the high concentration in 12 samples is 11.62%, the RDS (n=12) of the intermediate precision and repeatability of the medium concentration in 12 samples is 10.13%, and the RDS (n=12) of the intermediate precision and repeatability of the low concentration in 12 samples is 13.85%, all of which meet the standard of RSD less than 15%, and the intermediate precision is qualified.

[0076] 5. Accuracy analysis of E.coli HCP ELISA kit

[0077] The ELISA method and kit established in Example 1 were used for detection on the same ELISA strip. The recovery rate of the HCP calibrator was mixed into the sample to be tested in equal volumes at high, medium and low concentrations. The concentration of the sample to be tested was calculated based on the calibration curve obtained in the experiment and the OD of the sample to be tested, and compared with the theoretical concentration. At the same time, the spike recovery experiment was performed with the quality control product to calculate the back-calculated concentration value and spike recovery rate of each sample. The spike recovery results are shown in Table 5.

[0078] Table 5

[0079]

[0080] The results show that the recovery rate ranges from 84.12% to 108.8% at high concentrations, from 91.92% to 97.12% at medium concentrations, and from 96% to 104% at low concentrations, all of which meet the range requirement of 70% to 130%; in addition, the RSD (n=3) of the recovery rate at high concentrations is 14.51%, the RSD (n=3) of the recovery rate at medium concentrations is 2.81%, and the RSD of the recovery rate at low concentrations is 4.15%, all of which meet the standard of RSD less than 15%, so the accuracy of this method is qualified.

[0081] In summary, the method was validated and proved to have good linearity when the standard curve was fitted using the four-parameter equation within the range of 0-100 ng / ml. 2 >0.99; the sensitivity detection limit of quantification is 1.56ng / ml; the spiked recoveries at high, medium and low calibration concentrations are all between 80% and 120%, and the RSDs are all less than 15%; the repeatability and intermediate precision test RSDs are all less than 15%; and there is no cross-reaction between this method and Pichi yeast.

[0082] Figure 4 The flowchart is a comparison of the preparation steps of the conventional kit of the present invention and the E. coli HCP ELISA detection kit. The method of the present kit takes about 2 hours, and the experimental operation has 7 steps; the method of the conventional kit takes about 3 hours, and the experimental operation has 9 steps.

[0083] Finally, it should be noted that the above are only some preferred embodiments of the present invention, and the present invention is not limited thereto. Although the present invention is described in detail with reference to the above embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the above embodiments, or to replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc., as long as it is within the spirit and principle of the present invention, shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a universal detection kit for residual protein in E. coli host, characterized in that: The steps include: S1. Preparation of Escherichia coli antigen; S11. Cultivate Escherichia coli and obtain the cells by centrifugation; S12. The bacteria in step S11 are homogenized under high pressure to obtain Escherichia coli antigens; S2. Preparation of sheep anti-E. coli antigen antibodies; S21. Select goats of appropriate age for immunization; S22. After the immunization, the antiserum was collected and purified to obtain antibodies against residual proteins of the E. coli host; S3. E. coli host residual protein antibody labeling, using horseradish peroxidase labeling, the E. coli host residual protein antibody prepared in step S22 is labeled; S4. Establishment of ELISA method for detecting residual host proteins of Escherichia coli; S41. Coating: dilute the purified antibody in step S22 to 5 μg / mL to 25 μg / mL with carbonate buffer or phosphate buffer, and coat at 4°C overnight; S42. Blocking: drying to remove the coating solution, washing three times with a washing solution composed of phosphate buffer containing 0.01% to 0.1% Tween 20, blocking with a blocking solution composed of 1 to 10 wt% sucrose, 1 to 8 wt% trehalose and 0.1 to 6 wt% fish collagen at 25 ° C to 37 ° C for 0.5 h to 2 h, and after the blocking, washing three times with a washing solution; S43. Combining antigen and detection antibody mixture: the E. coli antigen prepared in step S12 is used as a calibrator or a sample to be tested and a host residual protein labeled antibody diluted with a diluent containing 2% to 8wt% sucrose, 3% to 8wt% trehalose and 0.5% to 3wt% fish collagen at a volume of 1:50 to 1:400 at a ratio of 1:1 to 1:3, and then added to the corresponding wells and incubated for 1h to 2h; S44. Dried to remove the well solution in step S43, washed with a detergent, added TMB colorimetric substrate to each well, and incubated at room temperature in the dark for 15 to 35 minutes; S45. Add 30 μL to 150 μL of stop solution to each well to terminate the reaction, and measure the OD450 value with a microplate reader; S46. Use a four-parameter fitting equation to draw a calibration curve, and calculate the concentration of the sample to be tested based on the calibration curve and the OD450 value of the sample to be tested.

2. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 1, characterized in that: In the step S41, the host residual protein antibody purified in the step S22 is coated with 0.02-0.2 M carbonate buffer.

3. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 2, characterized in that: The selected concentration of the calibrator in step S43 is 0-400 ng / mL.

4. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 3, characterized in that: In the step S43, a diluent containing 3% to 6wt% sucrose, 3.5% to 7wt% trehalose and 1% to 2wt% fish collagen is added to dilute the E. coli host residual protein labeled antibody.

5. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 4, characterized in that: The blocking solution used in step S42 contains 3-8 wt% sucrose, 2-6 wt% trehalose and 0.5-5 wt% fish collagen.

6. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 5, characterized in that: The test can be completed in just 2 hours using a one-step method that involves mixed incubation of the antigen and the detection antibody.

7. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 6, characterized in that: The antibody coverage rate was shown to be over 80%.

8. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 7, characterized in that: This method has no cross-reaction with the closely related Pichia yeast.

9. The method for preparing a universal detection kit for residual protein in E. coli host according to claim 8, characterized in that: It has high detection accuracy, with a sensitivity limit of quantification of 1.56 ng / mL, a high antibody coverage rate, and no cross-reaction with the closely related Pichia yeast. In addition, this method uses a one-step method of mixed incubation of antigen and detection antibody, which has simple steps, takes a short time, and calculates the sample concentration through a fixed standard concentration, with a low error rate.

10. Use of the ELISA detection kit for residual protein in E. coli host prepared by the method for preparing the universal detection kit for residual protein in E. coli host according to any one of claims 1 to 9 in a rapid and simple method for detecting residual protein content in E. coli host.

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