New coronavirus single-chain antibody, quality control product and preparation method

文档序号：887890 发布日期：2021-03-23 浏览：15次中文

阅读说明：本技术 新冠病毒单链抗体及质控品和制备方法 (New coronavirus single-chain antibody, quality control product and preparation method ) 是由江章涛何雨禧魏道舜林晓涛邢智浩钱纯亘王刚林军李一荣潘运宝胡鹍于 2020-12-09 设计创作，主要内容包括：本发明涉及一种新冠病毒单链抗体及质控品和制备方法,从N段到C端依次包括轻链可变区和重链可变区,所述轻链可变区的氨基酸序列如SEQ ID NO:1所示,所述重链可变区的氨基酸序列如SEQ ID NO:2所示。本发明的新冠病毒单链抗体灵敏度高、稳定性好,非常适合质控品的制备。而且,在此可变区序列基础上进一步使用基因重组技术构建形成无CH1片段的人鼠嵌合IgM抗体,从而提高分子稳定性和特异性结合能力。本发明制备的新冠病毒单链抗体可应用于化学发光平台进行质控品检测,可以有效测定活性,能克服一般的2019-nCoV IgM抗体检测灵敏度差、特异性低的缺陷。(The invention relates to a new coronavirus single-chain antibody, a quality control product and a preparation method thereof, which sequentially comprise a light chain variable region and a heavy chain variable region from an N section to a C end, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2. The new coronavirus single-chain antibody disclosed by the invention is high in sensitivity and good in stability, and is very suitable for preparation of quality control products. Furthermore, a gene recombination technology is further used for constructing and forming a human-mouse chimeric IgM antibody without a CH1 fragment on the basis of the variable region sequence, thereby improving the molecular stability and the specific binding capacity. The new coronavirus single-chain antibody prepared by the invention can be applied to a chemiluminescence platform for quality control detection, can effectively determine the activity, and can overcome the defects of poor detection sensitivity and low specificity of a general 2019-nCoV IgM antibody.)

1. A new coronavirus single-chain antibody is characterized by sequentially comprising a light chain variable region and a heavy chain variable region from an N section to a C end, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

2. The isolated coronavirus single-chain antibody of claim 1, further comprising a human IgM antibody light chain constant region and a human IgM antibody heavy chain constant region linked to the C-terminus of the heavy chain variable region in this order.

3. The novel single-chain antibody of claim 2, wherein the constant region of the human IgM antibody light chain has the amino acid sequence shown in SEQ ID NO. 3, and the constant region of the human IgM antibody heavy chain has the amino acid sequence shown in SEQ ID NO. 4.

4. An isolated nucleic acid encoding the novel single chain antibody against coronavirus according to any one of claims 1 to 3.

5. The isolated nucleic acid of claim 4, comprising a nucleotide fragment having the sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6.

6. The isolated nucleic acid of claim 4, comprising a nucleotide fragment having the sequence shown in SEQ ID NO. 7 and SEQ ID NO. 8.

7. A preparation method of a new coronavirus antibody quality control product is characterized by comprising the following steps:

mixing the new coronavirus antibody with the freeze-drying protective solution to obtain an antibody solution;

sequentially carrying out pre-freezing treatment, primary drying treatment and secondary drying treatment on the antibody solution to obtain the new coronavirus antibody quality control product;

the condition parameters of the pre-freezing treatment are as follows in sequence: treating the separator at-5 ℃ to-15 ℃ for 35-45 min, treating the separator at-45 ℃ to-55 ℃ for 80-100 min, treating the separator at-20 ℃ to-30 ℃ for 80-100 min, and treating the separator at-50 ℃ to-60 ℃ for 80-100 min; the condition parameters of the primary drying treatment are as follows in sequence: treating for 6-10 s under the conditions that the temperature of a clapboard is-50 ℃ to-60 ℃, the vacuum degree is 0.2mbar to 0.4mbar, treating for 1-3 s under the conditions that the temperature of the clapboard is-50 ℃ to-60 ℃, the vacuum degree is 0.08mbar to 0.12mbar, treating for 20-21 hours under the conditions that the temperature of the clapboard is-25 ℃ to-35 ℃, the vacuum degree is 0.08mbar to 0.12mbar, treating for 25min to 35min under the conditions that the temperature of the clapboard is-5 ℃ to-15 ℃, the vacuum degree is 0.08mbar to 0.12mbar, and treating for 50-70 min under the conditions that the temperature of the clapboard is-5 ℃ to-15 ℃ and the vacuum degree is 0.06mbar to 0.10 mbar; the condition parameters of the secondary drying treatment are as follows: the temperature of the clapboard is 5-15 ℃, and the vacuum degree is 0-0.005 mbar for 5-6 hours.

8. The method of claim 7, wherein the lyoprotectant solution comprises the following concentrations of components: 0.02-0.06M Tris-HCl, 0.006-0.010M Tris, 0.6-1.2 wt% sodium chloride, 2-6 wt% bovine serum albumin, 6-10 wt% mannitol, 1-3 wt% trehalose.

9. The method of claim 8, wherein the lyoprotectant solution further comprises the following concentrations of components: 0.8 to 1.2 weight percent of gelatin and 0.8 to 1.2 weight percent of hydroxyethyl starch.

10. A novel coronavirus antibody quality control product prepared by the preparation method of any one of claims 7-9.

Technical Field

The invention relates to the technical field of immunodetection, in particular to a novel coronavirus single-chain antibody, a quality control product and a preparation method thereof.

Background

The 2019 novel coronavirus (2019-nCoV) is named as '2019-nCoV' by the world health organization at 12 th month in 2020, and is named as 'Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)' by the International Committee for Classification of viruses at 11 th month in 2020. The novel coronavirus is a new strain 113 of coronavirus that has not been previously found in humans. Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). The similarity between 2019-nCoV and SARS virus in the whole genome is about 80%. From the current cognition, 2019-nCoV is weaker than SARS in toxicity, and in infectivity, because 2019-nCoV has long latency period and asymptomatic infectivity exists in an infected person, and the whole population is susceptible, the difficulty of propagation control is higher. Based on the epidemiological survey before the day, the latent period of the disease is generally 3-7 days, the shortest latent period is 1 day, the longest latent period is 14 days, and the disease still has infectivity during the latent period. The disease can be spread from person to person, mainly through spray and contact, and may present aerosol transmission risks in closed, unventilated places. The disease condition of people with low immunity is serious after infection, and children and infants also have diseases. After a person is infected with 2019-nCoV, common signs comprise respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like, and in more serious cases, the infection can cause pneumonia, severe acute respiratory syndrome, renal failure and even death.

After 2019-nCoV infection, the human body will produce IgM antibodies against the virus in about 7 days and IgG antibodies against the virus in about 14 days. The produced antibody is a protective antibody and can promote the recovery and self-healing of human body from virus infection. The 2019-nCoV antibody detection is widely applied to platforms such as ELISA, colloidal gold, lateral chromatography, chemiluminescence and the like, but the general quality control antibody cannot be accurately and effectively measured due to insufficient stability, sensitivity and the like.

Disclosure of Invention

Based on this, there is a need for a novel single-chain antibody against coronavirus which has good stability and sensitivity.

A new coronavirus single-chain antibody sequentially comprises a light chain variable region and a heavy chain variable region from an N section to a C end, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

In one embodiment, the antibody further comprises a human IgM antibody light chain constant region and a human IgM antibody heavy chain constant region linked sequentially to the C-terminus of the heavy chain variable region.

In one embodiment, the amino acid sequence of the constant region of the human IgM antibody light chain is represented by SEQ ID NO. 3 and the amino acid sequence of the constant region of the human IgM antibody heavy chain is represented by SEQ ID NO. 4.

The invention also provides an isolated nucleic acid encoding the novel coronavirus single-chain antibody.

In one embodiment, the nucleotide fragment comprises nucleotide fragments with sequences shown as SEQ ID NO. 5 and SEQ ID NO. 6.

In one embodiment, the nucleotide fragments with the sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8 are included.

The invention also provides a preparation method of the new coronavirus antibody quality control product, which comprises the following steps:

mixing the new coronavirus antibody with the freeze-drying protective solution to obtain an antibody solution;

sequentially carrying out pre-freezing treatment, primary drying treatment and secondary drying treatment on the antibody solution to obtain the new coronavirus antibody quality control product;

In one embodiment, the lyoprotectant solution contains the following concentrations of components: 0.02-0.06M Tris-HCl, 0.006-0.010M Tris, 0.6-1.2 wt% sodium chloride, 2-6 wt% bovine serum albumin, 6-10 wt% mannitol, 1-3 wt% trehalose.

In one embodiment, the lyoprotectant solution further comprises the following components at the following concentrations: 0.8 to 1.2 weight percent of gelatin and 0.8 to 1.2 weight percent of hydroxyethyl starch.

The invention also provides a new coronavirus antibody quality control product, which is prepared according to the preparation method.

The new coronavirus single-chain antibody disclosed by the invention is high in sensitivity and good in stability, and is very suitable for preparation of quality control products. Furthermore, a gene recombination technology is further used for constructing and forming a human-mouse chimeric IgM antibody without a CH1 fragment on the basis of the variable region sequence, thereby improving the molecular stability and the specific binding capacity. The new coronavirus single-chain antibody prepared by the invention can be applied to a chemiluminescence platform for quality control detection, can effectively determine the activity, and can overcome the defects of poor detection sensitivity and low specificity of a general 2019-nCoV IgM antibody.

The invention adopts a novel freeze-drying procedure of independent research, improves the traditional quick freeze-drying procedure by applying the technology of combining step-by-step partition plate temperature lifting and step-by-step vacuum degree changing, combines quality control antibody processing to prepare a new coronavirus antibody quality control product in a clinical immune freeze-dried powder form, has good in-vitro storage stability, has the advantages of long storage period, low viscosity, easiness in redissolution and uniform mixing, small matrix effect and the like, and is very important for the precision of test results of a detection system and a kit. The quality control product is suitable for a chemiluminescence detection platform and can be matched with a corresponding detection system and a corresponding kit for use. The invention also adds gelatin and hydroxyethyl starch dual high molecular polymer as a freeze-drying protective agent, thereby improving the glass transition temperature and further improving the freeze-drying shape of the new coronavirus antibody quality control product.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The novel coronavirus single-chain antibody provided by the embodiment of the invention sequentially comprises a light chain variable region and a heavy chain variable region from N section to C end, wherein the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2.

In one specific example, the novel coronavirus single chain antibody further comprises a human IgM antibody light chain constant region and a human IgM antibody heavy chain constant region linked to the C-terminus of the above heavy chain variable region in this order. The 2019-nCoV IgM antibody is a specific antibody which appears earliest in humoral immunity, and IgM antibody detection has important significance for early diagnosis of virus infection when the virus infects a human body.

In a specific example, the amino acid sequence of the constant region of the human IgM antibody light chain is represented by SEQ ID NO. 3, and the amino acid sequence of the constant region of the human IgM antibody heavy chain is represented by SEQ ID NO. 4. Thus, the gene recombination technology is used for constructing and forming the human-mouse chimeric IgM antibody without the CH1 fragment, and the molecular stability and the specific binding capacity can be improved by utilizing the method.

An isolated nucleic acid of one embodiment of the present invention, which encodes the novel coronavirus single chain antibody described above.

In one specific example, the isolated nucleic acid includes a nucleotide fragment having a sequence as set forth in SEQ ID NO 5 and SEQ ID NO 6. It will be appreciated that due to the degeneracy of the codons, the nucleic acid sequences capable of expressing the same protein have a variety of forms, the above being codon optimized nucleic acid sequences, but are not limited thereto.

In one specific example, the isolated nucleic acid comprises nucleotide fragments having the sequences shown in SEQ ID NO 7 and SEQ ID NO 8, encoding the human IgM antibody light chain constant region and the human IgM antibody heavy chain constant region, respectively.

The recombinant expression vector of an embodiment of the present invention is used for expressing the novel coronavirus single-chain antibody.

It is understood that the recombinant expression vector may further comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences). In one specific example, the initial vector may be selected from the pCMVp-NEO-BAN vector.

The host cell of an embodiment of the present invention contains the above recombinant expression vector. In one particular example, the host cell is a HEK293 cell.

The preparation method of the new coronavirus antibody quality control product provided by the embodiment of the invention comprises the following steps:

and mixing the new coronavirus antibody with the freeze-drying protective solution to obtain an antibody solution.

And sequentially carrying out pre-freezing treatment, primary drying treatment and secondary drying treatment on the antibody solution to obtain the new coronavirus antibody quality control product.

The condition parameters of the pre-freezing treatment are as follows in sequence: the temperature of the clapboard is between 5 ℃ below zero and 15 ℃ below zero for 35-45 min, the temperature of the clapboard is between 45 ℃ below zero and 55 ℃ below zero for 80-100 min, the temperature of the clapboard is between 20 ℃ below zero and 30 ℃ below zero for 80-100 min, and the temperature of the clapboard is between 50 ℃ below zero and 60 ℃ below zero for 80-100 min. The condition parameters of the primary drying treatment are as follows in sequence: the temperature of the clapboard is between 50 ℃ below zero and 60 ℃ below zero, the vacuum degree is between 0.2mbar and 0.4mbar, the temperature of the clapboard is between 50 ℃ below zero and 60 ℃ below zero, the vacuum degree is between 0.08mbar and 0.12mbar, the temperature of the clapboard is between 25 ℃ below zero and 35 ℃ below zero, the vacuum degree is between 0.08mbar and 0.12mbar, the temperature of the clapboard is between 5 ℃ below zero and 15 ℃ below zero, and the vacuum degree is between 0.06mbar and 0.10mbar, the temperature of the clapboard is between 50 ℃ below zero and 70 min. The condition parameters of the secondary drying treatment are as follows: the temperature of the clapboard is 5-15 ℃, and the vacuum degree is 0-0.005 mbar for 5-6 hours.

In a specific example, the concentration of the new coronavirus antibody in the antibody solution is 5 AU/mL-24 AU/mL.

In one specific example, the lyoprotectant solution contains the following concentrations of components: 0.02-0.06M Tris-HCl, 0.006-0.010M Tris, 0.6-1.2 wt% sodium chloride, 2-6 wt% bovine serum albumin, 6-10 wt% mannitol, 1-3 wt% trehalose.

In one specific example, the lyoprotectant solution also contains the following concentrations of components: 0.8 to 1.2 weight percent of gelatin and 0.8 to 1.2 weight percent of hydroxyethyl starch. Therefore, the double high molecular polymer of the gelatin and the hydroxyethyl starch is added as a freeze-drying protective agent, so that the glass transition temperature is increased, and the freeze-drying form of the new coronavirus antibody quality control product can be further improved.

The following are specific examples.

Example 1

1. Preparation of whole-mouse IgM monoclonal antibody

Animal immunization: protein synthesis was performed according to the N gene sequence published in chinese CDC, and N protein was synthesized by anhui universal biosynthesis, and the N gene sequence is shown below. And (3) immunizing a mouse by using the prepared 2019-nCoV N protein as an immunogen to prepare a whole-mouse-derived IgM monoclonal antibody. The N protein and Freund's adjuvant are mixed and emulsified completely, and injected into 3 Balb/c mice with 25 ug each, and the immunization is carried out 3 times with 15 days interval.

N gene sequence:

GGGGAACTTCTCCTGCTAGAATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTGG

cell fusion: 3 days before fusion, mice are injected with 25 mug of coupling protein in the abdominal cavity, spleen cells of the immunized mice are taken out, and2.0×10⁷SP2/0 myeloma cells and 2.0X 10⁸Uniformly mixing splenocytes of immunized Balb/c mice, centrifuging, discarding supernatant, slightly shaking and uniformly mixing, placing in a 37 ℃ water bath kettle, dropwise adding 1mL of PEG-1500 aqueous solution with the volume concentration of 50% within 90 seconds, then dropwise adding 20mL of 1640 culture medium, centrifuging, discarding supernatant, washing once again, centrifuging, discarding supernatant, and obtaining hybridoma cells. Hybridoma cells were selected on 96-well cell culture plates using HAT selection medium, and total fusion rate > 95% was detected under a microscope. The coated N protein is used for detecting the supernatant of the monoclonal cell hole, the cells in the hole with OD450 more than 0.8 are selected for subcloning, and finally, the cell clone with the reaction positive rate more than 99 percent to the natural N protein is obtained and is used as a hybridoma cell strain secreting the IgM monoclonal antibody resisting human N protein.

Cell cloning: cloning the obtained positive cell strain for 3 times by using a limiting dilution method to finally obtain 5 strains of the IgM monoclonal antibody hybridoma cell line producing high-titer anti-human N protein, carrying out amplification culture and freezing.

Preparing ascites: treating 6-8 week-old female Balb/c mice with paraffin for 10 days, and treating hybridoma cells with 2 × 10⁶One cell/mouse was injected intraperitoneally. Ascites rich in IgM antibody was obtained from the abdominal cavity of the mouse 7 days later, and the ascites titer was determined to be > 10⁵。

Antibody purification: protein G affinity chromatography was used. Firstly, preparing a protein G affinity chromatographic column, balancing the column by PBS, taking ascites to pass through the column, then washing the column by PBS, eluting by 50nmol/L glycine hydrochloride solution, collecting eluent, measuring the OD value of each collecting pipe, reserving the eluent in a peak area, dialyzing the eluent and collecting the eluent. The purified murine IgM monoclonal antibody was identified by SDS-PAGE electrophoresis and the purity was 98% or more. The variable region sequences of the antibodies are shown in table 1 below.

TABLE 1

2. Amplifying variable region gene of synthetic murine IgM monoclonal antibody and constant region gene of human IgM antibody

Taking a mouse IgM monoclonal antibody gene as an amplification template, utilizing a pair of light chain variable region primers and a pair of heavy chain variable region primers, and taking four nucleotides dNTPs as raw materials, and amplifying and synthesizing the mouse variable region gene under the action of DNA polymerase.

Taking a human IgM antibody gene as an amplification template, amplifying and synthesizing the human constant region gene under the action of DNA polymerase by using a pair of light chain constant region primers and a pair of heavy chain constant region primers and taking four nucleotides dNTPs as raw materials. The constant region gene without CH1 fragment was amplified and synthesized using the human constant region gene as a template, as shown in Table 2 below.

TABLE 2

3. Construction of mouse-human IgM light and heavy chain gene expression vector without CH1 fragment

Through gene recombination, the constant region gene of the human-derived CH 1-free fragment and the variable region gene of the mouse-derived fragment are spliced, the sequence of the spliced antibody gene is shown in table 3, and the spliced antibody gene is cloned to an expression vector pCMVp-NEO-BAN to construct a human-mouse IgM chimeric antibody gene expression vector without a CH1 fragment.

TABLE 3

IgM chimeric antibody expression and purification

IgM chimeric antibody expression: transfecting the expression vector plasmid of the human-mouse IgM chimeric antibody gene without the CH1 fragment into 293 cells, and collecting cell supernatants to obtain the human-mouse chimeric 2019-nCoV IgM antibody without the CH1 fragment.

Purifying the IgM chimeric antibody: protein G affinity chromatography was used. Firstly, preparing a protein G affinity chromatographic column, balancing the column by PBS, taking ascites to pass through the column, then washing the column by PBS, eluting by 50nmol/L glycine hydrochloride solution, collecting eluent, measuring the OD value of each collecting pipe, reserving the eluent in a peak area, dialyzing the eluent and collecting the eluent. The purified IgM chimeric antibody was identified by SDS-PAGE electrophoresis and had a purity of 98% or more.

IgM chimeric antibody Activity detection

The purified IgM chimeric antibody was diluted 10-fold, 50-fold and 100-fold with Tris buffer, and the purified normal monoclonal antibody and polyclonal antibody were selected as controls and were diluted 10-fold, 50-fold and 100-fold with Tris buffer. The novel coronavirus IgM antibody measuring kit produced by the company is selected to detect the activity of the antibody on a chemiluminescence platform, the activity of the visible IgM chimeric antibody is higher, the calculated concentration is higher than that of the IgM monoclonal antibody and the polyclonal antibody, and the luminous value and the concentration value are shown in Table 4.

TABLE 4

The purified IgM antibodies containing the variable region and the constant region not containing CH1 were diluted 10-fold, 50-fold, 100-fold, 300-fold and 600-fold with Tris buffer, respectively, and the purified IgM antibodies containing only the variable region and the purified IgM antibodies containing the variable region and the intact constant region were selected as controls and were diluted 10-fold, 50-fold, 100-fold, 300-fold and 600-fold with Tris buffer, respectively. The novel coronavirus IgM antibody measuring kit produced by the company is selected, the affinity and the sensitivity of the antibody are detected on a chemiluminescence platform, the affinity and the sensitivity of the IgM antibody containing the variable region and the constant region without CH1 are higher than those of the IgM antibody containing the variable region only and the IgM antibody containing the variable region and the complete constant region, and the comparison results of the affinity and the sensitivity are shown in tables 5 and 6.

TABLE 5

TABLE 6

The method comprises the steps of taking purified IgM antibody stock solution containing a variable region and a constant region without CH1 as a control group, selecting purified IgM antibody only containing the variable region and purified IgM antibody containing the variable region and a complete constant region as a control group, selecting an anti-RA 33 IgG antibody, a novel coronavirus IgG antibody and a novel coronavirus IgM antibody determination kit produced by a company, and detecting the specificity of the antibodies on a chemiluminescence platform, wherein the specific comparison results are shown in Table 7.

TABLE 7

Example 2 preparation of novel coronavirus IgM antibody quality control Material

Raw material treatment and concentration confirmation: taking out the IgM chimeric antibody raw material from a refrigerator under the preservation condition for unfreezing, processing the raw material in a biological safety cabinet of a hundred thousand-level production workshop, detecting by using a hepatitis B virus surface antigen detection kit, a hepatitis C virus antibody detection kit, a human immunodeficiency virus antigen antibody detection kit and a treponema pallidum antibody detection kit, selecting the raw material with negative or non-reactive detection results, and then determining the concentration of the 2019-nCoV IgM chimeric antibody raw material. Then, centrifugation and filtration were carried out to obtain a clarified antibody raw material, and the antibody concentration was confirmed in a hundred thousand-stage production plant.

Solution preparation: the preparation of the freeze-drying solution is carried out in a hundred thousand grade production workshop. The preparation process is as follows: taking 1L solution as an example, 0.04M Tris-HCl +0.008M Tris + 0.9% sodium chloride + 4% bovine serum albumin + 1% gelatin + 1% hydroxyethyl starch + 0.05% PC300+ 0.02% PS-3+ 8% mannitol + 2% trehalose + 3% F012, the weighing amount is calculated according to the content of each component. Adding Tris-HCl, Tris, sodium chloride, PC300 and other components, adding 800mL of pure water to dissolve, adjusting the pH value of the solution to be within 7.4 +/-0.5, heating to dissolve gelatin in advance, adding bovine serum albumin and hydroxyethyl starch to fine adjust the pH value of the solution to be within 7.4 +/-0.5, and adding pure water to supplement to 1L.

Preparing quality control products, subpackaging and freeze-drying: and if the processed IgM chimeric antibody raw material meets the requirements of the company, continuously performing preparation, subpackaging and freeze-drying of quality control products in a hundred thousand-level production workshop. 2019-nCoV IgM chimeric antibody material is added into the solution to prepare a dual-concentration quality control product with the concentration of (5 +/-30%) AU/mL and (20 +/-20%) AU/mL, the novel freeze-drying program and the conventional freeze-drying program are used for freeze-drying, the program is shown in Table 8, and the cover is pressed under vacuum after the freeze-drying is finished. The stability performance of the freeze-dried IgM antibody quality control product under the two procedures is verified respectively, and the heat stability and the redissolution stability of the quality control product obtained by using the novel freeze-drying procedure are superior to those of the conventional freeze-drying procedure, and the results are shown in tables 9 and 10.

TABLE 8

TABLE 9

Watch 10

And (3) quality control material assignment: sampling and redissolving and assigning values in a common workshop. The assignment steps are as follows: 5 chemiluminescence measuring instruments are prepared, and the quality control substances are repeatedly measured on each instrument by using a new crown IgM antibody measuring kit (chemiluminescence method) calibrated by the product for 5 days and 4 times per day. After 5 days of testing, 20 test data are obtained for each quality control substance concentration of each instrument. The detection result is rejected abnormal values according to Grubbs (Grubbs) criterion, the number of rejected outliers is not more than 2.5%, and when the outliers exceed 2.5%, whether the method is unstable or the operator is unfamiliar should be suspected. At this point, the data from this test cannot be used to check for problems and to re-start a new evaluation experiment after solving the problems. And (4) eliminating abnormal values from the detection result, calculating the mean value X and standard deviation SD of the remaining data, and determining the target value range (X +/-3 SD) of the quality control product.

Inspecting and assembling a semi-finished product: and (4) carrying out semi-finished product inspection on the next step of assignment, inspecting the accuracy and repeatability of the quality control product, carrying out product assembly and finished product inspection if the quality control product passes the inspection, and carrying out assignment on the quality control product again and carrying out semi-finished product inspection or processing the semi-finished product as an unqualified product if the quality control product does not pass the inspection.

And (4) finished product inspection and warehousing: and (4) carrying out finished product inspection in the next step of assembly, inspecting the accuracy and the repeatability of the quality control product, warehousing the finished product after the inspection is passed, and otherwise, processing the unqualified product.

Example 3 verification of stability of New coronavirus IgM antibody quality control product

In order to verify the performance of the 2019-nCoV IgM quality control product in extreme environments such as high temperature and the like, the product is subjected to heat treatment at 37 ℃ for 10 days, and the product stored at 2-8 ℃ is used as a reference; meanwhile, in order to determine the stability of the 2019-nCoV IgM quality control product after redissolution under different storage conditions, the product is respectively stored for 3 days, 30 days and 90 days at 20-25 ℃, 2-8 ℃ and-20 ℃ after redissolution, and the product stored at 2-8 ℃ is used as a control. A novel coronavirus IgM antibody measuring kit produced by a company is selected, and the luminous value and the concentration value of the 2019-nCoV IgM quality control product are measured under various conditions by a chemiluminescence platform, so that the result stability is good, and the relative deviation is within +/-10%, as shown in tables 11 and 12.

TABLE 11

TABLE 12

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 4

<211> 349

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Val Ile Ala Glu Leu Pro Pro Lys Val Ser Val Phe Val Pro Pro Arg

1 5 10 15

Asp Gly Phe Phe Gly Asn Pro Arg Lys Ser Lys Leu Ile Cys Gln Ala

20 25 30

Thr Gly Phe Ser Pro Arg Gln Ile Gln Val Ser Trp Leu Arg Glu Gly

35 40 45

Lys Gln Val Gly Ser Gly Val Thr Thr Asp Gln Val Gln Ala Glu Ala

50 55 60

Lys Glu Ser Gly Pro Thr Thr Tyr Lys Val Thr Ser Thr Leu Thr Ile

65 70 75 80

Lys Glu Ser Asp Trp Leu Ser Gln Ser Met Phe Thr Cys Arg Val Asp

85 90 95

His Arg Gly Leu Thr Phe Gln Gln Asn Ala Ser Ser Met Cys Val Pro

100 105 110

Asp Gln Asp Thr Ala Ile Arg Val Phe Ala Ile Pro Pro Ser Phe Ala

115 120 125

Ser Ile Phe Leu Thr Lys Ser Thr Lys Leu Thr Cys Leu Val Thr Asp

130 135 140

Leu Thr Thr Tyr Asp Ser Val Thr Ile Ser Trp Thr Arg Gln Asn Gly

145 150 155 160

Glu Ala Val Lys Thr His Thr Asn Ile Ser Glu Ser His Pro Asn Ala

165 170 175

Thr Phe Ser Ala Val Gly Glu Ala Ser Ile Cys Glu Asp Asp Trp Asn

180 185 190

Ser Gly Glu Arg Phe Thr Cys Thr Val Thr His Thr Asp Leu Pro Ser

195 200 205

Pro Leu Lys Gln Thr Ile Ser Arg Pro Lys Gly Val Ala Leu His Arg

210 215 220

Pro Asp Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn Leu Arg

225 230 235 240

Glu Ser Ala Thr Ile Thr Cys Leu Val Thr Gly Phe Ser Pro Ala Asp

245 250 255

Val Phe Val Gln Trp Met Gln Arg Gly Gln Pro Leu Ser Pro Glu Lys

260 265 270

Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gln Ala Pro Gly Arg Tyr

275 280 285

Phe Ala His Ser Ile Leu Thr Val Ser Glu Glu Glu Trp Asn Thr Gly

290 295 300

Glu Thr Tyr Thr Cys Val Val Ala His Glu Ala Leu Pro Asn Arg Val

305 310 315 320

Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu Tyr Asn

325 330 335

Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr Cys Tyr

340 345

<210> 5

<211> 399

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggattcac aggcccaggt tcttatgtta ctgctgctat gggtatctgg tacctgtggg 60

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 120

atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 180

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 240

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 360

cctccgacgt tcggtggagg caccaagctg gaaatcaaa 399

<210> 6

<211> 411

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtat ccaagcacag 60

atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc 120

tgcaaggctt ctggttatac cttcacagac tattcaatgc actgggtgaa gcaggctcca 180

ggaaagggtt taaagtggat gggctggata aacactgaga ctggtgagcc aacatatgca 240

gatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300

cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgctag aggttccgat 360

tactatgcta tggactactg gggtcaagga acctcagtca ccgtctcctc a 411

<210> 7

<211> 321

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

<210> 8

<211> 1047

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtgattgctg agctgcctcc caaagtgagc gtcttcgtcc caccccgcga cggcttcttc 60

ggcaaccccc gcaagtccaa gctcatctgc caggccacgg gtttcagtcc ccggcagatt 120

caggtgtcct ggctgcgcga ggggaagcag gtggggtctg gcgtcaccac ggaccaggtg 180

caggctgagg ccaaagagtc tgggcccacg acctacaagg tgaccagcac actgaccatc 240

aaagagagcg actggctcag ccagagcatg ttcacctgcc gcgtggatca caggggcctg 300

accttccagc agaatgcgtc ctccatgtgt gtccccgatc aagacacagc catccgggtc 360

ttcgccatcc ccccatcctt tgccagcatc ttcctcacca agtccaccaa gttgacctgc 420

ctggtcacag acctgaccac ctatgacagc gtgaccatct cctggacccg ccagaatggc 480

gaagctgtga aaacccacac caacatctcc gagagccacc ccaatgccac tttcagcgcc 540

gtgggtgagg ccagcatctg cgaggatgac tggaattccg gggagaggtt cacgtgcacc 600

gtgacccaca cagacctgcc ctcgccactg aagcagacca tctcccggcc caagggggtg 660

gccctgcaca ggcccgatgt ctacttgctg ccaccagccc gggagcagct gaacctgcgg 720

gagtcggcca ccatcacgtg cctggtgacg ggcttctctc ccgcggacgt cttcgtgcag 780

tggatgcaga gggggcagcc cttgtccccg gagaagtatg tgaccagcgc cccaatgcct 840

gagccccagg ccccaggccg gtacttcgcc cacagcatcc tgaccgtgtc cgaagaggaa 900

tggaacacgg gggagaccta cacctgcgtg gtggcccatg aggccctgcc caacagggtc 960

accgagagga ccgtggacaa gtccaccggt aaacccaccc tgtacaacgt gtccctggtc 1020

atgtccgaca cagctggcac ctgctac 1047

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