阅读说明：本技术 一种新冠病毒检测用单抗 (Monoclonal antibody for detecting new coronavirus ) 是由 刘立成 吴刚 闫广为 张治业 于 2021-09-08 设计创作，主要内容包括：本发明公开了一种新冠病毒检测用单抗,所述新冠病毒检测用单抗的配方如下：以重量计：制备腹水,proteinG纯化的抗体1-3mg；制备腹水,抗原亲和纯化的抗体0.5-1.5mg；制备腹水,硫酸铵沉淀纯化抗体1-3mg；细胞上清扩大培养,proteinG纯化的抗体1-3mg；细胞上清扩大培养,抗原亲和纯化的抗体0.5-1.5mg,新冠病毒检测用单抗的制备方法,包括如下步骤：S1、订购小鼠,S2、小鼠免疫程序,S3、融合,S4、融合检测,S5、单克隆；本发明提供的一种新冠病毒检测用单抗,可以有效对新冠病毒进行检测,解决了核酸检测容易出现漏诊的问题,当核酸检测阴性时,将IgM和IgG抗体检测增加进去,可作为核酸诊断的补充手段,极大提高了新冠病毒检测的准确性。(The invention discloses a monoclonal antibody for detecting new coronavirus, which has the following formula: by weight: preparing 1-3mg of ascites, proteinG purified antibody; preparing ascites, and 0.5-1.5mg of antibody subjected to antigen affinity purification; preparing ascites, precipitating and purifying antibody 1-3mg by ammonium sulfate; amplifying and culturing cell supernatant, and purifying antibody 1-3mg of proteinG; the cell supernatant is enlarged and cultured, the antibody of antigen affinity purification is 0.5-1.5mg, the preparation method of the monoclonal antibody for detecting the new coronavirus comprises the following steps: s1, ordering mice, S2, mouse immunization program, S3, fusion, S4, fusion detection, S5 and monoclonal; the monoclonal antibody for detecting the new coronavirus provided by the invention can effectively detect the new coronavirus, solves the problem that missed diagnosis is easy to occur in nucleic acid detection, and can be used as a supplementary means for nucleic acid diagnosis by adding IgM and IgG antibody detection when the nucleic acid detection is negative, thereby greatly improving the accuracy of new coronavirus detection.)

1. A monoclonal antibody for detecting a novel coronavirus is characterized in that: the monoclonal antibody for detecting the novel coronavirus has the following formula: by weight: preparing 1-3mg of ascites, proteinG purified antibody; preparing ascites, and 0.5-1.5mg of antibody subjected to antigen affinity purification; preparing ascites, precipitating and purifying antibody 1-3mg by ammonium sulfate; amplifying and culturing cell supernatant, and purifying antibody 1-3mg of proteinG; the cell supernatant is cultured in an enlarged way, and the antibody is purified by antigen affinity and is 0.5-1.5 mg.

2. The monoclonal antibody for detecting a novel coronavirus according to claim 1, which is characterized in that: the monoclonal antibody for detecting the novel coronavirus has the following formula: by weight: preparing 3mg of ascites, proteinG purified antibody; preparing ascites, and 1.5mg of antibody subjected to antigen affinity purification; preparing ascites, precipitating and purifying antibody 3mg by ammonium sulfate; the cell supernatant was expanded and cultured, and 3mg of antibody purified by proteinG; the cell supernatant was cultured in a large scale, and 1.5mg of the antibody was affinity-purified.

3. The monoclonal antibody for detecting a novel coronavirus according to claim 1, which is characterized in that: the monoclonal antibody for detecting the novel coronavirus has the following formula: by weight: preparing 2mg of ascites, proteinG purified antibody; preparing ascites, and 1mg of antibody subjected to antigen affinity purification; preparing ascites, and precipitating and purifying the antibody 2mg by ammonium sulfate; the cell supernatant is expanded and cultured, and the antibody purified by proteinG is 2 mg; the cell supernatant was cultured in a large scale, and 1mg of the antibody was affinity-purified.

4. The monoclonal antibody for detecting a novel coronavirus according to claim 1, which is characterized in that: the monoclonal antibody for detecting the novel coronavirus has the following formula: by weight: preparing 1mg of ascites, proteinG purified antibody; preparing ascites, and 0.5mg of antibody subjected to antigen affinity purification; preparing ascites, and precipitating and purifying the antibody 1mg by ammonium sulfate; the cell supernatant was expanded and cultured, and the amount of antibody purified by proteinG was 1 mg; the cell supernatant was cultured in a large scale, and 0.5mg of the antibody was affinity-purified.

5. The method for producing the monoclonal antibody for detecting a novel coronavirus according to any one of claims 1 to 4, wherein the method comprises: the method comprises the following steps:

s1, ordering mice;

s2, mouse immunization program: injecting antigen twice, and detecting the serum titer of the mouse by adopting an ELISA method;

s3, fusion: culturing cells, changing the HT medium by half on day 8, and drawing monoclonals under a microscope after day 10;

s4, fusion detection: making positive control on the serum of the immune mouse, and determining the OD value at 450nm/620nm by using an enzyme-labeling instrument;

s5, monoclonal: and (3) selecting 20 positive parent cells, culturing and subculturing for three times, and selecting cell strains with larger OD values to prepare the antibody.

6. The method for preparing the monoclonal antibody for detecting the novel coronavirus according to claim 5, wherein the method comprises the following steps: the immunization program protocol in step S2 includes the following steps:

s201, an antigen preparation method: the antigen was mixed in equal volumes with Quickantibody-Mouse5W following approximately 20ug of immunization per Mouse;

s202, collecting blank serum before immunization, marking the mouse by cutting the fingers, holding the neck skin of the mouse to enable the abdomen of the mouse to face upwards, cutting the right front toe to be No. 1, the left front toe to be No. 2, the right rear toe to be No. 3, the left rear toe to be No. 4 and the No. 5 toe to be uncut, fixing the mouse in a fixer, cutting the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood for separating the serum by using an EP (ethylene propylene glycol) tube, collecting about 3-5 ul of blood, and marking by handwriting, and storing at-20 ℃;

s203, day 0 of primary immunization: mixing 10-20ug protein antigen with QuickAntibody-Mouse5W in equal volume, mixing, injecting into the hind leg muscle of Mouse, collecting blank serum before primary immunization;

s204, day 21 of second immunization: an equivalent primary immunization;

s205, titer detection day 35: the ELISA method is adopted to detect the serum titer of the mouse, the toe-off labeling method is adopted to mark the mouse, the mouse is fixed in a fixer, the tail tip of the mouse is cut off by an ophthalmic scissors, about 20ul of blood is collected by an EP tube, and the serum is separated for detecting the ELISA titer.

7. The method for preparing the monoclonal antibody for detecting the novel coronavirus according to claim 6, wherein the method comprises the following steps: the method for detecting titer in step S205 includes the following steps:

s2051, antigen coating: determining the concentration of antigen coating to be 1ug/ml, adding 96-well plates into each well with the concentration of 100ul, coating 6 strips for 72 wells, and keeping the temperature at 4 ℃ overnight or in a 37 ℃ incubator for 1 h;

s2052, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s2053, washing: washing with PBST for three times, and patting to dry;

s2054, adding a primary antibody: starting from the second well, 100ul pbs was added to each well, and the sera from immunized mice were taken as 1: 65 dilution, taking about 200ul, adding into a first hole coated with a plate, taking 100ul from the first hole, adding into a second hole, sucking 100ul after blowing and sucking for 7 times, adding into a third hole, sequentially diluting to a 11 th hole, reserving 100ul PBS in a 12 th hole, adding 100ul blank serum in a 6 th row as negative control, and performing incubator action at 37 ℃ for 1 h;

s2055, washing: washing with PBST for three times, and patting to dry;

s2056, adding an enzyme-labeled secondary antibody: diluting HRP goat anti-mouse secondary antibody at a ratio of 1:5000, allowing reaction in 100ul per well at 37 ℃ in a incubator for 1 h;

s2057, washing: washing with PBST for 3-5 times, and patting to dry;

s2058, color development: adding soluble TMB single-component color developing solution into the mixture, performing the action of 100ul per hole in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s2059, termination: the addition of 30ul of 2M H2SO4 was terminated, the solution turned yellow, and OD450/620 was read using a microplate reader, positive for OD values greater than 0.2, and for blank sera no greater than 0.1.

8. The method for preparing the monoclonal antibody for detecting the novel coronavirus according to claim 5, wherein the method comprises the following steps: the fusion method in step S3 includes the following steps:

s301, selecting a mouse, killing the mouse painlessly, performing aseptic separation on the spleen, grinding the mouse by using a filter screen, centrifuging to obtain B cells and erythrocytes, cracking the erythrocytes for 5 minutes by using 10mlB cell separation liquid, centrifuging at 1000rpm for 10 minutes to obtain the B cells, and respectively suspending the 1 × 108 mouse spleen cells and the 2 × 107 myeloma cells in 25ml of serum-free culture medium;

s302, centrifuging for 5-10min at 400g of 200-one, discarding supernatant, lightly tapping the bottom of the tube, fully scattering precipitate, placing the centrifugal tube in a water bath at 37 ℃ for heat preservation until fusion begins, adding 1ml of 50% PEG1450 preheated at 37 ℃ into the centrifugal tube at uniform speed, using 1ml of a pipette for 1min, continuously stirring by using a pipette tip during the time, and continuously stirring for 1-2 min by using the pipette tip;

s303, adding 1ml of culture medium preheated at 37 ℃ into the fused mixture at a constant speed, stirring for 1min by using a pipette tip continuously, adding 3ml of culture medium preheated at 37 ℃ at a constant speed, stirring for 3min by using a pipette tip continuously, adding 10ml of culture medium preheated at 37 ℃, and incubating for 5min at 37 ℃;

s304 and 200-.

9. The method for preparing the monoclonal antibody for detecting the novel coronavirus according to claim 5, wherein the method comprises the following steps: the fusion detection in step S4 includes the following steps:

s401, antigen coating: coating the plate according to the number of fusion plates or fusion positive cell holes, determining the concentration of the antigen coating to be 1ug/ml, adding 100ul of the antigen coating into a 96-well plate per hole, and standing overnight at 4 ℃;

s402, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s403, washing: washing with PBST for three times, and patting to dry;

s404, adding a primary antibody: aseptically taking about 100ul of cell supernatant containing the monoclonal antibody, adding the cell supernatant into the holes of the coated plate, using a blank culture medium as a negative control, using serum of an immune mouse as a positive control, and performing incubator action at 37 ℃ for 1 h;

s405, washing: washing with PBST for three times, and patting to dry;

s406, adding an enzyme-labeled secondary antibody: taking a secondary antibody of a mouse, a goat anti-mouse or rabbit anti-mouse IgG and the like, wherein the Ig type of the secondary antibody can basically determine the Ig type of the monoclonal antibody to be prepared, diluting the secondary antibody according to the ratio of 1:5000 to 1:10000, performing 100ul per hole, and performing incubator at 37 ℃ for 1 h;

s407, washing: washing with PBST for 3-5 times, and patting to dry;

s408, color development: adding a TMB single-component substrate solution into the reaction kettle, allowing 100ul of the substrate solution to act in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s409, terminating: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and was measured: and (3) measuring the OD value at 450nm/620nm by using a microplate reader, and judging the test well to be positive if the test well is more than 2.1 and the numerical value is not less than 0.2.

10. The method for preparing the monoclonal antibody for detecting the novel coronavirus according to claim 5, wherein the method comprises the following steps: the single cloning process in the step S5 includes the following steps:

s501, transferring 20 parent clones to a 24-well plate for culture, and carrying out passage for 3 times;

s502, culturing 3 rd generation cells to the bottom of the hole by 40% -70%, and detecting the titer and subtype identification of the supernatant ELISA; and (3) independently selecting 5 positive cells, freezing and storing 4 positive cells in each cell, collecting supernatant, labeling by a labeling machine, and independently selecting 1 positive cell from 106 positive cells/cell to prepare an antibody.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a monoclonal antibody for detecting a novel coronavirus.

Background

The detection method of the novel coronavirus mainly comprises a nucleic acid detection method, an antibody detection method and an antigen detection method, and the detection rate of the antigen detection is low, so that the detection of the novel coronavirus mainly focuses on the detection of the antibody and the nucleic acid at present.

The requirement of nucleic acid detection on detection equipment or a platform is high, the high-sensitivity RT-PCR instrument is expensive, the requirements on the cleanliness and operators of laboratories are high, in addition, the nucleic acid detection takes longer, the results can be reported within 24 hours at the fastest time in consideration of sample transportation and sample overstock, compared with nucleic acid detection, the blood sample detected by the antibody is easier to obtain, the quality of the sample is ensured, the operation is simple and quick, the risk of infection of medical staff in the sample collection and detection process is reduced to a great extent, the screening work in a basic laboratory is easier to be carried out, and the like, if nucleic acid detection of viral ribonucleic acid (RNA) is said to be direct evidence of the presence of the virus, then antibody detection is the production of stimulated antibodies in the patient's blood, which is indirect evidence, the kit has clinical suggestion that IgM and IgG antibody detection is increased when nucleic acid detection is negative.

The antibody detection can make up for the defect that the nucleic acid detection is easy to cause missed diagnosis, and becomes an important new coronavirus detection means.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a novel monoclonal antibody for detecting coronavirus.

In order to achieve the purpose, the invention provides the following technical scheme: the monoclonal antibody for detecting the new coronavirus comprises the following formula: by weight: preparing 1-3mg of ascites, proteinG purified antibody; preparing ascites, and 0.5-1.5mg of antibody subjected to antigen affinity purification; preparing ascites, precipitating and purifying antibody 1-3mg by ammonium sulfate; amplifying and culturing cell supernatant, and purifying antibody 1-3mg of proteinG; cell supernatant amplification culture, antigen affinity purification of antibody 0.5-1.5mg

Preferably, the formula of the monoclonal antibody for detecting the new coronavirus is as follows: by weight: preparing 3mg of ascites, proteinG purified antibody; preparing ascites, and 1.5mg of antibody subjected to antigen affinity purification; preparing ascites, precipitating and purifying antibody 3mg by ammonium sulfate; the cell supernatant was expanded and cultured, and 3mg of antibody purified by proteinG; the cell supernatant was cultured in a large scale, and 1.5mg of the antibody was affinity-purified.

Preferably, the formula of the monoclonal antibody for detecting the new coronavirus is as follows: by weight: preparing 2mg of ascites, proteinG purified antibody; preparing ascites, and 1mg of antibody subjected to antigen affinity purification; preparing ascites, and precipitating and purifying the antibody 2mg by ammonium sulfate; the cell supernatant is expanded and cultured, and the antibody purified by proteinG is 2 mg; the cell supernatant was cultured in a large scale, and 1mg of the antibody was affinity-purified.

Preferably, the formula of the monoclonal antibody for detecting the new coronavirus is as follows: by weight: preparing 1mg of ascites, proteinG purified antibody; preparing ascites, and 0.5mg of antibody subjected to antigen affinity purification; preparing ascites, and precipitating and purifying the antibody 1mg by ammonium sulfate; the cell supernatant was expanded and cultured, and the amount of antibody purified by proteinG was 1 mg; the cell supernatant was cultured in a large scale, and 0.5mg of the antibody was affinity-purified.

The invention also provides a preparation method of the monoclonal antibody for detecting the novel coronavirus, which comprises the following steps:

s1, ordering mice;

s2, mouse immunization program: injecting antigen twice, and detecting the serum titer of the mouse by adopting an ELISA method;

s3, fusion: culturing cells, changing the HT medium by half on day 8, and drawing monoclonals under a microscope after day 10;

s4, fusion detection: making positive control on the serum of the immune mouse, and determining the OD value at 450nm/620nm by using an enzyme-labeling instrument;

s5, monoclonal: and (3) selecting 20 positive parent cells, culturing and subculturing for three times, and selecting cell strains with larger OD values to prepare the antibody.

Preferably, the immunization program protocol in step S2 includes the following steps:

s201, an antigen preparation method: the antigen was mixed in equal volumes with Quickantibody-Mouse5W following approximately 20ug of immunization per Mouse;

s202, collecting blank serum before immunization, marking the mouse by cutting the fingers, holding the neck skin of the mouse to enable the abdomen of the mouse to face upwards, cutting the right front toe to be No. 1, the left front toe to be No. 2, the right rear toe to be No. 3, the left rear toe to be No. 4 and the No. 5 toe to be uncut, fixing the mouse in a fixer, cutting the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood for separating the serum by using an EP (ethylene propylene glycol) tube, collecting about 3-5 ul of blood, and marking by handwriting, and storing at-20 ℃;

s203, day 0 of primary immunization: mixing 10-20ug protein antigen with QuickAntibody-Mouse5W in equal volume, mixing, injecting into the hind leg muscle of Mouse, collecting blank serum before primary immunization;

s204, day 21 of second immunization: an equivalent primary immunization;

s205, titer detection day 35: the ELISA method is adopted to detect the serum titer of the mouse, the toe-off labeling method is adopted to mark the mouse, the mouse is fixed in a fixer, the tail tip of the mouse is cut off by an ophthalmic scissors, about 20ul of blood is collected by an EP tube, and the serum is separated for detecting the ELISA titer.

Preferably, the method for detecting titer in step S205 comprises the following steps:

s2051, antigen coating: determining the concentration of antigen coating to be 1ug/ml, adding 96-well plates into each well with the concentration of 100ul, coating 6 strips for 72 wells, and keeping the temperature at 4 ℃ overnight or in a 37 ℃ incubator for 1 h;

s2052, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s2053, washing: washing with PBST for three times, and patting to dry;

s2054, adding a primary antibody: starting from the second well, 100ul pbs was added to each well, and the sera from immunized mice were taken as 1: 65 dilution, taking about 200ul, adding into a first hole coated with a plate, taking 100ul from the first hole, adding into a second hole, sucking 100ul after blowing and sucking for 7 times, adding into a third hole, sequentially diluting to a 11 th hole, reserving 100ul PBS in a 12 th hole, adding 100ul blank serum in a 6 th row as negative control, and performing incubator action at 37 ℃ for 1 h;

s2055, washing: washing with PBST for three times, and patting to dry;

s2056, adding an enzyme-labeled secondary antibody: diluting HRP goat anti-mouse secondary antibody at a ratio of 1:5000, allowing reaction in 100ul per well at 37 ℃ in a incubator for 1 h;

s2057, washing: washing with PBST for 3-5 times, and patting to dry;

s2058, color development: adding soluble TMB single-component color developing solution into the mixture, performing the action of 100ul per hole in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s2059, termination: the addition of 30ul of 2M H2SO4 was terminated, the solution turned yellow, and OD450/620 was read using a microplate reader, positive for OD values greater than 0.2, and for blank sera no greater than 0.1.

Preferably, the fusion method in step S3 includes the following steps:

s301, selecting a mouse, killing the mouse painlessly, performing aseptic separation on the spleen, grinding the mouse by using a filter screen, centrifuging to obtain B cells and erythrocytes, cracking the erythrocytes for 5 minutes by using 10mlB cell separation liquid, centrifuging at 1000rpm for 10 minutes to obtain the B cells, and respectively suspending the 1 × 108 mouse spleen cells and the 2 × 107 myeloma cells in 25ml of serum-free culture medium;

s302, centrifuging for 5-10min at 400g of 200-one, discarding supernatant, lightly tapping the bottom of the tube, fully scattering precipitate, placing the centrifugal tube in a water bath at 37 ℃ for heat preservation until fusion begins, adding 1ml of 50% PEG1450 preheated at 37 ℃ into the centrifugal tube at uniform speed, using 1ml of a pipette for 1min, continuously stirring by using a pipette tip during the time, and continuously stirring for 1-2 min by using the pipette tip;

s303, adding 1ml of culture medium preheated at 37 ℃ into the fused mixture at a constant speed, stirring for 1min by using a pipette tip continuously, adding 3ml of culture medium preheated at 37 ℃ at a constant speed, stirring for 3min by using a pipette tip continuously, adding 10ml of culture medium preheated at 37 ℃, and incubating for 5min at 37 ℃;

s304 and 200-.

Preferably, the fusion detection in step S4 includes the following steps:

s401, antigen coating: coating the plate according to the number of fusion plates or fusion positive cell holes, determining the concentration of the antigen coating to be 1ug/ml, adding 100ul of the antigen coating into a 96-well plate per hole, and standing overnight at 4 ℃;

s402, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s403, washing: washing with PBST for three times, and patting to dry;

s404, adding a primary antibody: aseptically taking about 100ul of cell supernatant containing the monoclonal antibody, adding the cell supernatant into the holes of the coated plate, using a blank culture medium as a negative control, using serum of an immune mouse as a positive control, and performing incubator action at 37 ℃ for 1 h;

s405, washing: washing with PBST for three times, and patting to dry;

s406, adding an enzyme-labeled secondary antibody: taking a secondary antibody of a mouse, a goat anti-mouse or rabbit anti-mouse IgG and the like, wherein the Ig type of the secondary antibody can basically determine the Ig type of the monoclonal antibody to be prepared, diluting the secondary antibody according to the ratio of 1:5000 to 1:10000, performing 100ul per hole, and performing incubator at 37 ℃ for 1 h;

s407, washing: washing with PBST for 3-5 times, and patting to dry;

s408, color development: adding a TMB single-component substrate solution into the reaction kettle, allowing 100ul of the substrate solution to act in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s409, terminating: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and was measured: and (3) measuring the OD value at 450nm/620nm by using a microplate reader, and judging the test well to be positive if the test well is more than 2.1 and the numerical value is not less than 0.2.

Preferably, the single cloning process in step S5 includes the following steps:

s501, transferring 20 parent clones to a 24-well plate for culture, and carrying out passage for 3 times;

s502, culturing 3 rd generation cells to the bottom of the hole by 40% -70%, and detecting the titer and subtype identification of the supernatant ELISA; and (3) independently selecting 5 positive cells, freezing and storing 4 positive cells in each cell, collecting supernatant, labeling by a labeling machine, and independently selecting 1 positive cell from 106 positive cells/cell to prepare an antibody.

The invention has the technical effects and advantages that:

the monoclonal antibody for detecting the new coronavirus provided by the invention can effectively detect the new coronavirus, solves the problem that missed diagnosis is easy to occur in nucleic acid detection, and can be used as a supplementary means for nucleic acid diagnosis by adding IgM and IgG antibody detection when the nucleic acid detection is negative, thereby greatly improving the accuracy of new coronavirus detection.

Drawings

Fig. 1 is a schematic structural view of a pipette body of the present invention;

FIG. 2 is a schematic view of a threaded rod and threaded bore of the present invention;

FIG. 3 is a flow chart of the preparation method of the monoclonal antibody for detecting the novel coronavirus of the present invention;

FIG. 4 is a flow chart of the titer detection method of the present invention;

FIG. 5 is a flow chart of fusion detection according to the present invention.

In the figure: 001. a support plate; 002. a fixing plate; 003. a placement groove; 004. culturing the plate; 005. a fixing hole; 006. a channel; 007. pulling a plate; 008. groove drawing; 009. a pipettor body; 010. a threaded rod; 011. a threaded bore.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example 1

The monoclonal antibody for detecting the new coronavirus comprises the following formula: by weight: preparing 3mg of ascites, proteinG purified antibody; preparing ascites, and 1.5mg of antibody subjected to antigen affinity purification; preparing ascites, precipitating and purifying antibody 3mg by ammonium sulfate; the cell supernatant was expanded and cultured, and 3mg of antibody purified by proteinG; the cell supernatant was cultured in a large scale, and 1.5mg of the antibody was affinity-purified.

A preparation method of a monoclonal antibody for detecting a novel coronavirus comprises the following steps:

s1, ordering mice;

s2, mouse immunization program: injecting antigen twice, and detecting the serum titer of the mouse by adopting an ELISA method, wherein the immunization program scheme comprises the following steps:

s201, an antigen preparation method: the antigen was mixed in equal volumes with Quickantibody-Mouse5W following approximately 20ug of immunization per Mouse;

s202, collecting blank serum before immunization, marking the mouse by cutting the fingers, holding the neck skin of the mouse to enable the abdomen of the mouse to face upwards, cutting the right front toe to be No. 1, the left front toe to be No. 2, the right rear toe to be No. 3, the left rear toe to be No. 4 and the No. 5 toe to be uncut, fixing the mouse in a fixer, cutting the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood for separating the serum by using an EP (ethylene propylene glycol) tube, collecting about 3-5 ul of blood, and marking by handwriting, and storing at-20 ℃;

s203, day 0 of primary immunization: mixing 10-20ug protein antigen with QuickAntibody-Mouse5W in equal volume, mixing, injecting into the hind leg muscle of Mouse, collecting blank serum before primary immunization;

s204, day 21 of second immunization: an equivalent primary immunization;

s205, titer detection day 35: detecting the serum titer of a mouse by adopting an ELISA method, marking the mouse by adopting a toe-off marking method, fixing the mouse in a fixer, shearing the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood by using an EP tube, and separating the serum for detecting the ELISA titer;

the titer detection method comprises the following steps:

s2051, antigen coating: determining the concentration of antigen coating to be 1ug/ml, adding 96-well plates into each well with the concentration of 100ul, coating 6 strips for 72 wells, and keeping the temperature at 4 ℃ overnight or in a 37 ℃ incubator for 1 h;

s2052, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s2053, washing: washing with PBST for three times, and patting to dry;

s2054, adding a primary antibody: starting from the second well, 100ul pbs was added to each well, and the sera from immunized mice were taken as 1: 65 dilution, taking about 200ul, adding into a first hole coated with a plate, taking 100ul from the first hole, adding into a second hole, sucking 100ul after blowing and sucking for 7 times, adding into a third hole, sequentially diluting to a 11 th hole, reserving 100ul PBS in a 12 th hole, adding 100ul blank serum in a 6 th row as negative control, and performing incubator action at 37 ℃ for 1 h;

s2055, washing: washing with PBST for three times, and patting to dry;

s2056, adding an enzyme-labeled secondary antibody: diluting HRP goat anti-mouse secondary antibody at a ratio of 1:5000, allowing reaction in 100ul per well at 37 ℃ in a incubator for 1 h;

s2057, washing: washing with PBST for 3-5 times, and patting to dry;

s2058, color development: adding soluble TMB single-component color developing solution into the mixture, performing the action of 100ul per hole in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s2059, termination: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and the OD read using a microplate reader_450/620Positive by OD value greater than 0.2, blank serum OD value not greater than 0.1;

s3, fusion: culturing cells, half replacing the HT medium on day 8, and drawing monoclonals under a microscope after day 10, wherein the fusion method comprises the following steps:

s301, selecting a mouse, killing the mouse painlessly, performing aseptic separation on the spleen, grinding the mouse by using a filter screen, centrifuging to obtain B cells and erythrocytes, cracking the erythrocytes by using 10mlB cell separation liquid for 5 minutes, centrifuging at 1000rpm for 10 minutes to obtain the B cells, and respectively performing 1 × 10 cell separation on the B cells and the erythrocytes by using the centrifugation liquid⁸Mouse splenocytes and 2X 10⁷Myeloma cells were resuspended in 25ml serum-free medium;

s302, centrifuging for 5-10min at 400g of 200-one, discarding supernatant, lightly tapping the bottom of the tube, fully scattering precipitate, placing the centrifugal tube in a water bath at 37 ℃ for heat preservation until fusion begins, adding 1ml of 50% PEG1450 preheated at 37 ℃ into the centrifugal tube at uniform speed, using 1ml of a pipette for 1min, continuously stirring by using a pipette tip during the time, and continuously stirring for 1-2 min by using the pipette tip;

s303, adding 1ml of culture medium preheated at 37 ℃ into the fused mixture at a constant speed, stirring for 1min by using a pipette tip continuously, adding 3ml of culture medium preheated at 37 ℃ at a constant speed, stirring for 3min by using a pipette tip continuously, adding 10ml of culture medium preheated at 37 ℃, and incubating for 5min at 37 ℃;

s304, 200-400g centrifugation for 5-10min, cell taking, supernatant discarding, cell resuspension by 230ml of selective medium, laying 10 blocks of 96 cell culture plates, cell culture by the conventional method, half-changing the HT medium on day 8, drawing a single clone under a microscope after day 10, and fusion detection on days 12-15;

s4, fusion detection: the serum of the immune mouse is used as positive control, an enzyme-labeling instrument is used for measuring OD values at 450nm/620nm, and fusion detection comprises the following steps:

s401, antigen coating: coating the plate according to the number of fusion plates or fusion positive cell holes, determining the concentration of the antigen coating to be 1ug/ml, adding 100ul of the antigen coating into a 96-well plate per hole, and standing overnight at 4 ℃;

s402, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s403, washing: washing with PBST for three times, and patting to dry;

s404, adding a primary antibody: aseptically taking about 100ul of cell supernatant containing the monoclonal antibody, adding the cell supernatant into the holes of the coated plate, using a blank culture medium as a negative control, using serum of an immune mouse as a positive control, and performing incubator action at 37 ℃ for 1 h;

s405, washing: washing with PBST for three times, and patting to dry;

s406, adding an enzyme-labeled secondary antibody: taking a secondary antibody of a mouse, a goat anti-mouse or rabbit anti-mouse IgG and the like, wherein the Ig type of the secondary antibody can basically determine the Ig type of the monoclonal antibody to be prepared, diluting the secondary antibody according to the ratio of 1:5000 to 1:10000, performing 100ul per hole, and performing incubator at 37 ℃ for 1 h;

s407, washing: washing with PBST for 3-5 times, and patting to dry;

s408, color development: adding a TMB single-component substrate solution into the reaction kettle, allowing 100ul of the substrate solution to act in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s409, terminating: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and was measured: determining OD value at 450nm/620nm by using an enzyme-labeling instrument, and determining that the experimental hole is more than 2.1 and the numerical value is not less than 0.2 as positive;

s5, monoclonal: selecting 20 positive parent cells, culturing and subculturing for three times, selecting a cell strain with a larger OD value to prepare an antibody, wherein the monoclonal process comprises the following steps:

s501, transferring 20 parent clones to a 24-well plate for culture, and carrying out passage for 3 times;

s502, culturing 3 rd generation cells to the bottom of the hole by 40% -70%, and detecting the titer and subtype identification of the supernatant ELISA; and (3) independently selecting 5 positive cells, freezing and storing 4 positive cells in each cell, collecting supernatant, labeling by a labeling machine, and independently selecting 1 positive cell from 106 positive cells/cell to prepare an antibody.

The pipettor in this embodiment has the advantage of fixing the use position, the shortcoming that the position of stirring cannot be fixed in the use process of a traditional handheld pipettor is solved, specifically including support plate 001, both sides are fixed with fixed plate 002 around the top of support plate 001, place groove 003 is opened at the top of support plate 001 and the inboard that is located two fixed plate 002, culture plate 004 is placed in the inner cavity of place groove 003, fixed hole 005 is opened at the top of culture plate 004, channel 006 corresponding to fixed hole 005 is opened at the top of the inboard of fixed plate 002, arm-tie 007 is fastened to the inner cavity of channel 006, arm-tie 008 is opened on the surface of arm-tie 007, pipettor body 009 is arranged below arm-tie 007, threaded rod 010 is fixedly installed at the top of pipettor body 009, threaded hole 011 matched with threaded rod 010 is opened at the bottom of arm-tie 007, and the inner wall threaded connection of threaded hole 011 is opened at the top of threaded rod 010;

the user is when using pipettor body 009, can cultivate the pipettor body 009 of the corresponding quantity of specification installation of board 004 according to the below, use through the cooperation of screw hole 011 and threaded rod 010, can conveniently carry out the dismouting to pipettor body 009, setting through standing groove 003, can fix the position of cultivateing board 004, setting through arm-tie 007, can adjust the position of pipettor body 009, setting through channel 006, can fix the position of arm-tie 007, setting through fixed orifices 005, can insert the inner chamber of fixed orifices 005 with the centrifuge tube, fix its position, setting through arm-tie 008, can convenience of customers pulling arm-tie 007 remove the position of pipettor body 009.

Example 2

The monoclonal antibody for detecting the new coronavirus comprises the following formula: by weight: preparing 2mg of ascites, proteinG purified antibody; preparing ascites, and 1mg of antibody subjected to antigen affinity purification; preparing ascites, and precipitating and purifying the antibody 2mg by ammonium sulfate; the cell supernatant is expanded and cultured, and the antibody purified by proteinG is 2 mg; the cell supernatant was cultured in a large scale, and 1mg of the antibody was affinity-purified.

A preparation method of a monoclonal antibody for detecting a novel coronavirus comprises the following steps:

s1, ordering mice;

s2, mouse immunization program: injecting antigen twice, and detecting the serum titer of the mouse by adopting an ELISA method, wherein the immunization program scheme comprises the following steps:

s201, an antigen preparation method: the antigen was mixed in equal volumes with Quickantibody-Mouse5W following approximately 20ug of immunization per Mouse;

s202, collecting blank serum before immunization, marking the mouse by cutting the fingers, holding the neck skin of the mouse to enable the abdomen of the mouse to face upwards, cutting the right front toe to be No. 1, the left front toe to be No. 2, the right rear toe to be No. 3, the left rear toe to be No. 4 and the No. 5 toe to be uncut, fixing the mouse in a fixer, cutting the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood for separating the serum by using an EP (ethylene propylene glycol) tube, collecting about 3-5 ul of blood, and marking by handwriting, and storing at-20 ℃;

s203, day 0 of primary immunization: mixing 10-20ug protein antigen with QuickAntibody-Mouse5W in equal volume, mixing, injecting into the hind leg muscle of Mouse, collecting blank serum before primary immunization;

s204, day 21 of second immunization: an equivalent primary immunization;

s205, titer detection day 35: detecting the serum titer of a mouse by adopting an ELISA method, marking the mouse by adopting a toe-off marking method, fixing the mouse in a fixer, shearing the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood by using an EP tube, and separating the serum for detecting the ELISA titer;

the titer detection method comprises the following steps:

s2051, antigen coating: determining the concentration of antigen coating to be 1ug/ml, adding 96-well plates into each well with the concentration of 100ul, coating 6 strips for 72 wells, and keeping the temperature at 4 ℃ overnight or in a 37 ℃ incubator for 1 h;

s2052, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s2053, washing: washing with PBST for three times, and patting to dry;

s2054, adding a primary antibody: starting from the second well, 100ul pbs was added to each well, and the sera from immunized mice were taken as 1: 65 dilution, taking about 200ul, adding into a first hole coated with a plate, taking 100ul from the first hole, adding into a second hole, sucking 100ul after blowing and sucking for 7 times, adding into a third hole, sequentially diluting to a 11 th hole, reserving 100ul PBS in a 12 th hole, adding 100ul blank serum in a 6 th row as negative control, and performing incubator action at 37 ℃ for 1 h;

s2055, washing: washing with PBST for three times, and patting to dry;

s2056, adding an enzyme-labeled secondary antibody: diluting HRP goat anti-mouse secondary antibody at a ratio of 1:5000, allowing reaction in 100ul per well at 37 ℃ in a incubator for 1 h;

s2057, washing: washing with PBST for 3-5 times, and patting to dry;

s2058, color development: adding soluble TMB single-component color developing solution into the mixture, performing the action of 100ul per hole in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s2059, termination: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and the OD read using a microplate reader_450/620Positive by OD value greater than 0.2, blank serum OD value not greater than 0.1;

s3, fusion: culturing cells, half replacing the HT medium on day 8, and drawing monoclonals under a microscope after day 10, wherein the fusion method comprises the following steps:

s301, selecting a mouse, killing the mouse without pain,aseptically separating spleen, grinding with filter screen, centrifuging to obtain B cells and erythrocytes, lysing erythrocytes with 10mlB cell separating medium for 5min, centrifuging at 1000rpm for 10min to obtain B cells, and mixing with the supernatant of 1 × 10⁸Mouse splenocytes and 2X 10⁷Myeloma cells were resuspended in 25ml serum-free medium;

s302, centrifuging for 5-10min at 400g of 200-one, discarding supernatant, lightly tapping the bottom of the tube, fully scattering precipitate, placing the centrifugal tube in a water bath at 37 ℃ for heat preservation until fusion begins, adding 1ml of 50% PEG1450 preheated at 37 ℃ into the centrifugal tube at uniform speed, using 1ml of a pipette for 1min, continuously stirring by using a pipette tip during the time, and continuously stirring for 1-2 min by using the pipette tip;

s303, adding 1ml of culture medium preheated at 37 ℃ into the fused mixture at a constant speed, stirring for 1min by using a pipette tip continuously, adding 3ml of culture medium preheated at 37 ℃ at a constant speed, stirring for 3min by using a pipette tip continuously, adding 10ml of culture medium preheated at 37 ℃, and incubating for 5min at 37 ℃;

s304, 200-400g centrifugation for 5-10min, cell taking, supernatant discarding, cell resuspension by 230ml of selective medium, laying 10 blocks of 96 cell culture plates, cell culture by the conventional method, half-changing the HT medium on day 8, drawing a single clone under a microscope after day 10, and fusion detection on days 12-15;

s4, fusion detection: the serum of the immune mouse is used as positive control, an enzyme-labeling instrument is used for measuring OD values at 450nm/620nm, and fusion detection comprises the following steps:

s401, antigen coating: coating the plate according to the number of fusion plates or fusion positive cell holes, determining the concentration of the antigen coating to be 1ug/ml, adding 100ul of the antigen coating into a 96-well plate per hole, and standing overnight at 4 ℃;

s402, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s403, washing: washing with PBST for three times, and patting to dry;

s404, adding a primary antibody: aseptically taking about 100ul of cell supernatant containing the monoclonal antibody, adding the cell supernatant into the holes of the coated plate, using a blank culture medium as a negative control, using serum of an immune mouse as a positive control, and performing incubator action at 37 ℃ for 1 h;

s405, washing: washing with PBST for three times, and patting to dry;

s406, adding an enzyme-labeled secondary antibody: taking a secondary antibody of a mouse, a goat anti-mouse or rabbit anti-mouse IgG and the like, wherein the Ig type of the secondary antibody can basically determine the Ig type of the monoclonal antibody to be prepared, diluting the secondary antibody according to the ratio of 1:5000 to 1:10000, performing 100ul per hole, and performing incubator at 37 ℃ for 1 h;

s407, washing: washing with PBST for 3-5 times, and patting to dry;

s408, color development: adding a TMB single-component substrate solution into the reaction kettle, allowing 100ul of the substrate solution to act in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s409, terminating: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and was measured: determining OD value at 450nm/620nm by using an enzyme-labeling instrument, and determining that the experimental hole is more than 2.1 and the numerical value is not less than 0.2 as positive;

s5, monoclonal: selecting 20 positive parent cells, culturing and subculturing for three times, selecting a cell strain with a larger OD value to prepare an antibody, wherein the monoclonal process comprises the following steps:

s501, transferring 20 parent clones to a 24-well plate for culture, and carrying out passage for 3 times;

s502, culturing 3 rd generation cells to the bottom of the hole by 40% -70%, and detecting the titer and subtype identification of the supernatant ELISA; and (3) independently selecting 5 positive cells, freezing and storing 4 positive cells in each cell, collecting supernatant, labeling by a labeling machine, and independently selecting 1 positive cell from 106 positive cells/cell to prepare an antibody.

The pipettor in this embodiment has the advantage of fixing the use position, the shortcoming that the position of stirring cannot be fixed in the use process of a traditional handheld pipettor is solved, specifically including support plate 001, both sides are fixed with fixed plate 002 around the top of support plate 001, place groove 003 is opened at the top of support plate 001 and the inboard that is located two fixed plate 002, culture plate 004 is placed in the inner cavity of place groove 003, fixed hole 005 is opened at the top of culture plate 004, channel 006 corresponding to fixed hole 005 is opened at the top of the inboard of fixed plate 002, arm-tie 007 is fastened to the inner cavity of channel 006, arm-tie 008 is opened on the surface of arm-tie 007, pipettor body 009 is arranged below arm-tie 007, threaded rod 010 is fixedly installed at the top of pipettor body 009, threaded hole 011 matched with threaded rod 010 is opened at the bottom of arm-tie 007, and the inner wall threaded connection of threaded hole 011 is opened at the top of threaded rod 010;

the user is when using pipettor body 009, can cultivate the pipettor body 009 of the corresponding quantity of specification installation of board 004 according to the below, use through the cooperation of screw hole 011 and threaded rod 010, can conveniently carry out the dismouting to pipettor body 009, setting through standing groove 003, can fix the position of cultivateing board 004, setting through arm-tie 007, can adjust the position of pipettor body 009, setting through channel 006, can fix the position of arm-tie 007, setting through fixed orifices 005, can insert the inner chamber of fixed orifices 005 with the centrifuge tube, fix its position, setting through arm-tie 008, can convenience of customers pulling arm-tie 007 remove the position of pipettor body 009.

Example 3

The monoclonal antibody for detecting the new coronavirus comprises the following formula: by weight: preparing 1mg of ascites, proteinG purified antibody; preparing ascites, and 0.5mg of antibody subjected to antigen affinity purification; preparing ascites, and precipitating and purifying the antibody 1mg by ammonium sulfate; the cell supernatant was expanded and cultured, and the amount of antibody purified by proteinG was 1 mg; the cell supernatant was cultured in a large scale, and 0.5mg of the antibody was affinity-purified.

A preparation method of a monoclonal antibody for detecting a novel coronavirus comprises the following steps:

s1, ordering mice;

s2, mouse immunization program: injecting antigen twice, and detecting the serum titer of the mouse by adopting an ELISA method, wherein the immunization program scheme comprises the following steps:

s201, an antigen preparation method: the antigen was mixed in equal volumes with Quickantibody-Mouse5W following approximately 20ug of immunization per Mouse;

s202, collecting blank serum before immunization, marking the mouse by cutting the fingers, holding the neck skin of the mouse to enable the abdomen of the mouse to face upwards, cutting the right front toe to be No. 1, the left front toe to be No. 2, the right rear toe to be No. 3, the left rear toe to be No. 4 and the No. 5 toe to be uncut, fixing the mouse in a fixer, cutting the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood for separating the serum by using an EP (ethylene propylene glycol) tube, collecting about 3-5 ul of blood, and marking by handwriting, and storing at-20 ℃;

s203, day 0 of primary immunization: mixing 10-20ug protein antigen with QuickAntibody-Mouse5W in equal volume, mixing, injecting into the hind leg muscle of Mouse, collecting blank serum before primary immunization;

s204, day 21 of second immunization: an equivalent primary immunization;

s205, titer detection day 35: detecting the serum titer of a mouse by adopting an ELISA method, marking the mouse by adopting a toe-off marking method, fixing the mouse in a fixer, shearing the tail tip of the mouse by an ophthalmic scissors, collecting about 20ul of blood by using an EP tube, and separating the serum for detecting the ELISA titer;

the titer detection method comprises the following steps:

s2051, antigen coating: determining the concentration of antigen coating to be 1ug/ml, adding 96-well plates into each well with the concentration of 100ul, coating 6 strips for 72 wells, and keeping the temperature at 4 ℃ overnight or in a 37 ℃ incubator for 1 h;

s2052, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s2053, washing: washing with PBST for three times, and patting to dry;

s2054, adding a primary antibody: starting from the second well, 100ul pbs was added to each well, and the sera from immunized mice were taken as 1: 65 dilution, taking about 200ul, adding into a first hole coated with a plate, taking 100ul from the first hole, adding into a second hole, sucking 100ul after blowing and sucking for 7 times, adding into a third hole, sequentially diluting to a 11 th hole, reserving 100ul PBS in a 12 th hole, adding 100ul blank serum in a 6 th row as negative control, and performing incubator action at 37 ℃ for 1 h;

s2055, washing: washing with PBST for three times, and patting to dry;

s2056, adding an enzyme-labeled secondary antibody: diluting HRP goat anti-mouse secondary antibody at a ratio of 1:5000, allowing reaction in 100ul per well at 37 ℃ in a incubator for 1 h;

s2057, washing: washing with PBST for 3-5 times, and patting to dry;

s2058, color development: adding soluble TMB single-component color developing solution into the mixture, performing the action of 100ul per hole in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s2059, termination: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and the OD read using a microplate reader_450/620Positive by OD value greater than 0.2, blank serum OD value not greater than 0.1;

s3, fusion: culturing cells, half replacing the HT medium on day 8, and drawing monoclonals under a microscope after day 10, wherein the fusion method comprises the following steps:

s301, selecting a mouse, killing the mouse painlessly, performing aseptic separation on the spleen, grinding the mouse by using a filter screen, centrifuging to obtain B cells and erythrocytes, cracking the erythrocytes by using 10mlB cell separation liquid for 5 minutes, centrifuging at 1000rpm for 10 minutes to obtain the B cells, and respectively performing 1 × 10 cell separation on the B cells and the erythrocytes by using the centrifugation liquid⁸Mouse splenocytes and 2X 10⁷Myeloma cells were resuspended in 25ml serum-free medium;

s302, centrifuging for 5-10min at 400g of 200-one, discarding supernatant, lightly tapping the bottom of the tube, fully scattering precipitate, placing the centrifugal tube in a water bath at 37 ℃ for heat preservation until fusion begins, adding 1ml of 50% PEG1450 preheated at 37 ℃ into the centrifugal tube at uniform speed, using 1ml of a pipette for 1min, continuously stirring by using a pipette tip during the time, and continuously stirring for 1-2 min by using the pipette tip;

s303, adding 1ml of culture medium preheated at 37 ℃ into the fused mixture at a constant speed, stirring for 1min by using a pipette tip continuously, adding 3ml of culture medium preheated at 37 ℃ at a constant speed, stirring for 3min by using a pipette tip continuously, adding 10ml of culture medium preheated at 37 ℃, and incubating for 5min at 37 ℃;

s304, 200-400g centrifugation for 5-10min, cell taking, supernatant discarding, cell resuspension by 230ml of selective medium, laying 10 blocks of 96 cell culture plates, cell culture by the conventional method, half-changing the HT medium on day 8, drawing a single clone under a microscope after day 10, and fusion detection on days 12-15;

s4, fusion detection: the serum of the immune mouse is used as positive control, an enzyme-labeling instrument is used for measuring OD values at 450nm/620nm, and fusion detection comprises the following steps:

s401, antigen coating: coating the plate according to the number of fusion plates or fusion positive cell holes, determining the concentration of the antigen coating to be 1ug/ml, adding 100ul of the antigen coating into a 96-well plate per hole, and standing overnight at 4 ℃;

s402, sealing: discarding the supernatant, washing with PBST for three times, adding 300ul of blocking solution into each well, and standing at 37 ℃ for 1 h;

s403, washing: washing with PBST for three times, and patting to dry;

s404, adding a primary antibody: aseptically taking about 100ul of cell supernatant containing the monoclonal antibody, adding the cell supernatant into the holes of the coated plate, using a blank culture medium as a negative control, using serum of an immune mouse as a positive control, and performing incubator action at 37 ℃ for 1 h;

s405, washing: washing with PBST for three times, and patting to dry;

s406, adding an enzyme-labeled secondary antibody: taking a secondary antibody of a mouse, a goat anti-mouse or rabbit anti-mouse IgG and the like, wherein the Ig type of the secondary antibody can basically determine the Ig type of the monoclonal antibody to be prepared, diluting the secondary antibody according to the ratio of 1:5000 to 1:10000, performing 100ul per hole, and performing incubator at 37 ℃ for 1 h;

s407, washing: washing with PBST for 3-5 times, and patting to dry;

s408, color development: adding a TMB single-component substrate solution into the reaction kettle, allowing 100ul of the substrate solution to act in a 37 ℃ incubator for 7-8 min, and displaying a positive result as blue;

s409, terminating: the addition of 30ul of 2M H2SO4 was stopped, the solution turned yellow and was measured: determining OD value at 450nm/620nm by using an enzyme-labeling instrument, and determining that the experimental hole is more than 2.1 and the numerical value is not less than 0.2 as positive;

s5, monoclonal: selecting 20 positive parent cells, culturing and subculturing for three times, selecting a cell strain with a larger OD value to prepare an antibody, wherein the monoclonal process comprises the following steps:

s501, transferring 20 parent clones to a 24-well plate for culture, and carrying out passage for 3 times;

s502, culturing 3 rd generation cells to the bottom of the hole by 40% -70%, and detecting the titer and subtype identification of the supernatant ELISA; and (3) independently selecting 5 positive cells, freezing and storing 4 positive cells in each cell, collecting supernatant, labeling by a labeling machine, and independently selecting 1 positive cell from 106 positive cells/cell to prepare an antibody.

The pipettor in this embodiment has the advantage of fixing the use position, the shortcoming that the position of stirring cannot be fixed in the use process of a traditional handheld pipettor is solved, specifically including support plate 001, both sides are fixed with fixed plate 002 around the top of support plate 001, place groove 003 is opened at the top of support plate 001 and the inboard that is located two fixed plate 002, culture plate 004 is placed in the inner cavity of place groove 003, fixed hole 005 is opened at the top of culture plate 004, channel 006 corresponding to fixed hole 005 is opened at the top of the inboard of fixed plate 002, arm-tie 007 is fastened to the inner cavity of channel 006, arm-tie 008 is opened on the surface of arm-tie 007, pipettor body 009 is arranged below arm-tie 007, threaded rod 010 is fixedly installed at the top of pipettor body 009, threaded hole 011 matched with threaded rod 010 is opened at the bottom of arm-tie 007, and the inner wall threaded connection of threaded hole 011 is opened at the top of threaded rod 010;

the user is when using pipettor body 009, can cultivate the pipettor body 009 of the corresponding quantity of specification installation of board 004 according to the below, use through the cooperation of screw hole 011 and threaded rod 010, can conveniently carry out the dismouting to pipettor body 009, setting through standing groove 003, can fix the position of cultivateing board 004, setting through arm-tie 007, can adjust the position of pipettor body 009, setting through channel 006, can fix the position of arm-tie 007, setting through fixed orifices 005, can insert the inner chamber of fixed orifices 005 with the centrifuge tube, fix its position, setting through arm-tie 008, can convenience of customers pulling arm-tie 007 remove the position of pipettor body 009.

Table 1, the formulation composition when implemented according to examples 1-3 gives the following table:

in summary, the following steps: the monoclonal antibody for detecting the new coronavirus provided by the invention can effectively detect the new coronavirus, solves the problem that missed diagnosis is easy to occur in nucleic acid detection, and can be used as a supplementary means for nucleic acid diagnosis by adding IgM and IgG antibody detection when the nucleic acid detection is negative, thereby greatly improving the accuracy of new coronavirus detection.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.