CN112630428A - Method and kit for detecting new coronavirus IgG/IgM total antibody - Google Patents
Method and kit for detecting new coronavirus IgG/IgM total antibody Download PDFInfo
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Abstract
The invention relates to the technical field of biological medicines, and particularly discloses a method and a kit for detecting a new coronavirus IgG/IgM total antibody. The detection method realizes the detection of the IgG/IgM total antibody by using a double-antigen sandwich technology, and specifically comprises the following steps: coating the recombinant new coronavirus RBD protein on a solid phase carrier, adding a sample to be detected into the solid phase carrier coated with the recombinant new coronavirus RBD protein, incubating and washing; adding biotin-labeled new coronavirus RBD protein into the washed solid phase carrier, incubating, washing, then developing color and combining with a standard curve to obtain the content of the antibody. The invention utilizes the technical principle of double-antigen sandwich to detect that the original liquid of the healthy human serum has no false positive and can well distinguish the healthy human from the patient; the method can promote the large-scale early screening emergency serological detection of the new coronavirus and the related vaccine research promotion and vaccine effect evaluation work.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a method and a kit for detecting a new coronavirus IgG/IgM total antibody.
Background
Enzyme-linked immunosorbent assay (ELISA), also known as enzyme-linked immunosorbent assay, is a qualitative and quantitative detection method in which soluble antigen or antibody is bound to solid phase carriers such as polystyrene and immunoreaction is carried out by utilizing specific binding of antigen and antibody. Since the 70 s of the last century, ELISA has been widely used in many medical biology fields as an in vitro diagnostic technique for detecting antigens, antibodies, haptens, etc.; in the aspect of practical application, the kit can be used for clinical diagnosis, disease monitoring, disease census and the like of diseases. Therefore, it is closely related to immunology, epidemiology, infectious disease, and the like.
Currently, no enzyme linked immunosorbent assay kit for detecting total antibodies of the new coronavirus (COVID-19) is available on the market in terms of reagents for researching the new coronavirus. Meanwhile, the existing conventional ELISA experimental methods mostly use indirect methods, only new coronavirus IgG antibodies can be detected, IgM (immunoglobulin M) generated in the infection window period cannot be detected, and the indirect method products are used for detecting the healthy human serum stock solution, so that the indirect method products have higher false positive test effect, and the indirect method products are not beneficial to large-scale early-screening emergency serology detection of the new coronavirus antibodies and related vaccine research promotion and vaccine effect evaluation work.
Therefore, the method is necessary to detect and detect the new coronavirus IgG/IgM total antibody by combining a double-antigen sandwich ELISA method, reduce the occurrence of higher false positive in the detection of the healthy human serum stock solution, and promote the large-scale early-screening emergency serological detection of the new coronavirus antibody and the related vaccine research promotion and vaccine effect evaluation work.
Disclosure of Invention
The invention aims to provide a kit for detecting a new coronavirus IgG/IgM total antibody, so as to overcome the defects that the existing kit is low in detection efficiency, high false positive exists in the detection of a healthy human serum stock solution and the like.
In view of the above, in order to achieve the above object, the technical solution of the present invention is:
a kit for detecting new coronavirus IgG/IgM total antibody comprises a new coronavirus RBD recombinant protein and a biotin labeled protein thereof.
According to an embodiment of the present invention, the novel coronavirus RBD recombinant protein is ABclonal RP 01258.
In the kit, the Biotin label is Biotin-maleimide.
Further, the kit of the invention also comprises a novel coronavirus RBD monoclonal antibody; preferably, the novel coronavirus RBD monoclonal antibody is ABClonal RM01763 or ABClonal RM 01764.
The invention also aims to provide application of the kit in preparing a novel coronavirus detection product.
The invention also aims to provide a method for detecting IgG/IgM total antibodies of the new coronavirus, which detects the IgG/IgM total antibodies of the new coronavirus by using a recombinant new coronavirus RBD and a biotin-labeled protein thereof in combination with a double-antigen sandwich ELISA method.
Further, the method comprises the following steps: coating the recombinant new coronavirus RBD protein on a solid phase carrier, adding a sample to be detected into the solid phase carrier coated with the recombinant new coronavirus RBD protein, incubating and washing; adding biotin-labeled novel coronavirus RBD protein into the washed solid phase carrier, incubating, washing, then developing color and combining with a standard curve to obtain the total antibody content.
Compared with the prior art, the invention has the beneficial effects that:
1. the kit and the detection method solve the technical problems that the total antibody cannot be quantified and the antibody cannot be tested in the window infection period by using an indirect ELISA detection method in the market, greatly shorten the screening time, and can be used for quickly detecting the total IgG/IgM of the new coronavirus.
2. The double-antigen sandwich method detection kit provided by the invention has no false positive in detection of the serum stock of healthy people, can well distinguish healthy people from patients, and can realize rapid detection of the serum of the patients and high-throughput screening of suspected people.
[ description of the drawings ]
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic diagram showing the result of detecting the RBD protein activity of each batch of recombinant new coronavirus according to the present invention.
FIG. 2 is a schematic diagram showing the result of detecting the activity of the biotin-labeled recombinant coronavirus RBD protein of the present invention.
FIG. 3 is a graph of a standard protein test by the double antigen sandwich ELISA method of the present invention.
FIG. 4 is a diagram showing the results of the detection of human serum stock by the double-antigen sandwich ELISA method according to the present invention.
FIG. 5 is a graph showing the indirect method of testing standard protein by the novel coronavirus IgG antibody kit of the comparative example.
FIG. 6 shows the indirect detection results of the novel coronavirus IgG antibody kit of the comparative example.
FIG. 7 shows the results of indirect detection of the novel coronavirus IgG antibody kit of the present invention in random healthy subjects.
[ detailed description ] embodiments
The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.
The detection kit provided by the invention comprises:
novel crown RBD protein: cloning the novel coronavirus RBD protein gene to an escherichia coli expression system to obtain recombinant escherichia coli; and culturing the recombinant escherichia coli, and separating and purifying a culture solution to obtain the recombinant new coronavirus RBD protein.
RBD rabbit monoclonal antibody: the rabbit monoclonal antibody is prepared by using the recombinant new coronavirus RBD protein.
Example establishment and detection of double antigen sandwich method of new coronavirus IgG/IgM total antibody kit product
1.1 verification of the Activity of the envelope protein
The new crown RBD protein binds to its receptor ACE2 when active. When the activity of the envelope protein is tested, 4 different batches of proteins (with the product numbers of RP01258, RP01278 and RP01282 respectively and the product numbers of RP01258_9603040401 and RP01258_9601050101 (two different batches of RP01258), RP01278_9601050901 and RP01282_9601060904) developed by ABClonal are selected for activity test, so as to select the batch with the best activity for developing a double antigen sandwich method of a new coronavirus IgG/IgM total antibody kit product, and the specific method is as follows:
1) recombinant ACE2 protein was coated onto a microplate at a concentration of 2-10ug/ml overnight at 4 ℃.
2) The well was drained and blocked with 2% BSA at 37 ℃ for 2 hours.
3) The liquid in the wells was spun off, and the plates were washed with 0.1M PBS, 0.2% Twen-20, 350ul per well for 3 times, 1 minute each time; after completion, the microplate was vacuum packed and stored at 4 ℃ until use.
4) And (4) taking the microporous plate strips for standby from the plate frame, putting the rest plate strips back into the aluminum foil bag filled with the drying agent, and then sealing and storing again.
5) Add washing buffer 350. mu.L per well, leave for 40 seconds and discard the liquid, which is washed 3 times in total.
6) A blank control was prepared by adding 100. mu.L of the dilution to the blank wells.
7) Add 100. mu.L of each new coronal RBD protein to each of the other wells, seal the wells with sealing plate, and incubate at 37 ℃ for 2 hours.
8) Biotin-maleimide new crown RBD antibody working solution is prepared 15 minutes before use.
9) The liquid in the wells was discarded and the washing step in step 3) was repeated.
10) streptavidin-HRP working solution was prepared 15 minutes before use.
11) The liquid in the wells was discarded and the washing step in step 3) was repeated.
12) streptavidin-HRP working solution (100. mu.L/well) was added to each well and covered with a new plate-sealing membrane and incubated at 37 ℃ for 30 min.
13) And (4) preheating the microplate reader.
14) The liquid in the wells was discarded and the washing step in step 3) was repeated.
15) TMB substrate (100. mu.L/well) was added to the wells and incubated at 37 ℃ for 15-20 minutes in the absence of light.
16) Stop buffer (50. mu.L/well) was added and immediately placed in the microplate reader and OD 450nm was measured in each well over 5 min.
Test results as shown in figure 1, from the activity test data, the RP01258 activity of two batches was identical and superior to the other two batches of protein, so we selected the RP01258 with the best activity for coating.
1.2 Biotin-labeled protein Activity verification
Constructing a novel coronavirus RBD Biotin labeled protein, selecting Biotin-maleimide for labeling, and testing the activity of the Biotin labeled protein, selecting a novel coronavirus RBD protein (RP01258) developed by ABClonal for activity test to select a batch with the best activity for developing a double-antigen sandwich method of a novel coronavirus IgG/IgM total antibody kit product, wherein the specific method comprises the following steps:
1) recombinant ACE2 protein was coated onto a microplate at a concentration of 2-10ug/ml overnight at 4 ℃.
2) The well was drained and blocked with 2% BSA at 37 ℃ for 2 hours.
3) The liquid in the wells was spun off, and the plates were washed with 0.1M PBS, 0.2% Twen-20, 350ul per well for 3 times, 1 minute each time; after completion, the microplate was vacuum packed and stored at 4 ℃ until use.
4) And (4) taking the microporous plate strips for standby from the plate frame, putting the rest plate strips back into the aluminum foil bag filled with the drying agent, and then sealing and storing again.
5) Add washing buffer 350. mu.L per well, leave for 40 seconds and discard the liquid, which is washed 3 times in total.
6) A blank control was prepared by adding 100. mu.L of the dilution to the blank wells.
7) Add 100. mu.L of different concentrations of neo-coronary RBD protein or biotin-labeled neo-coronary RBD protein to other wells, seal the wells with sealing plate, and incubate at 37 ℃ for 2 hours.
8) New crown RBD antibody working solution is prepared 15 minutes before use.
9) The liquid in the wells was discarded and the washing step in step 3) was repeated.
10) New crown RBD antibody working solution (100. mu.L/well) was added to each well, and a new sealing membrane was applied thereto, followed by incubation at 37 ℃ for 60 minutes.
11) A goat anti-rabbit-HRP working solution is prepared 15 minutes before use.
12) The liquid in the wells was discarded and the washing step in step 3) was repeated.
13) Goat anti-rabbit-HRP working solution (100. mu.L/well) was added to each well and covered with a new sealing membrane and incubated at 37 ℃ for 30 min.
14) And (4) preheating the microplate reader.
15) The liquid in the wells was discarded and the washing step in step 3) was repeated.
16) TMB substrate (100. mu.L/well) was added to the wells and incubated at 37 ℃ for 15-20 minutes in the absence of light.
17) Stop buffer (50. mu.L/well) was added and immediately placed in the microplate reader and OD 450nm was measured in each well over 5 min.
The test result is shown in figure 2, from the activity test data, the biotin activity test data of the new crown RBD protein (RP01258) developed by ABClonal shows that the protein activity is not affected after the biotin is labeled, and the method can be used for developing a double-antigen sandwich method of a new crown virus IgG/IgM total antibody kit product.
1.3 establishment of double antigen sandwich method for new coronavirus IgG/IgM total antibody kit product
1) According to the concentration of 1-4ug/ml, the recombinant new coronavirus RBD protein is coated on a micropore plate and stays overnight at 4 ℃.
2) The well was drained and blocked with 2% BSA at 37 ℃ for 2 hours.
3) The liquid in the wells was spun off, and the plates were washed with 0.1M PBS, 0.2% Twen-20, 350ul per well for 3 times, 1 minute each time; after completion, the microplate was vacuum packed and stored at 4 ℃ until use.
4) And (4) taking the microporous plate strips for standby from the plate frame, putting the rest plate strips back into the aluminum foil bag filled with the drying agent, and then sealing and storing again.
5) Add washing buffer 350. mu.L per well, leave for 40 seconds and discard the liquid, which is washed 3 times in total.
6) A blank control was prepared by adding 100. mu.L of the dilution to the blank wells.
7) mu.L of RBD rabbit mAb (ABClonal RM01763 or RM01764) was added to each of the other wells at different concentrations, the wells were sealed with sealing plate membranes, and incubated at 37 ℃ for 2 hours.
8) The biotin recombinant RBD protein working solution is prepared 15 minutes before use.
9) The liquid in the wells was discarded and the washing step in step 3) was repeated.
10) Biotin recombinant RBD protein working solution (100. mu.L/well) was added to each well, and a new sealing membrane was applied thereto, followed by incubation at 37 ℃ for 1 hour.
11) streptavidin-HRP working solution was prepared 15 minutes before use.
12) The liquid in the wells was discarded and the washing step in step 3) was repeated.
13) streptavidin-HRP working solution (100. mu.L/well) was added to each well and covered with a new plate-sealing membrane and incubated at 37 ℃ for 30 min.
14) And (4) preheating the microplate reader.
15) The well liquid was discarded and the washing step in step 3 was repeated.
16) TMB substrate (100. mu.L/well) was added to the wells. Incubate at 37 ℃ for 15-20 minutes in the absence of light.
17) Stop buffer (50. mu.L/well) was added and immediately placed in the microplate reader and OD 450nm was measured in each well over 5 min. If the correction wavelength can be selected, 570nm or 630nm is set. And subtracting the 570nm or 630nm reading from the 450nm reading in such a way as to correct and remove the OD of the non-chromogenic material, thereby obtaining a more accurate detection result. If the correction wavelength cannot be selected, the reading obtained will be too high, resulting in a reduction in the accuracy of the reading.
1.4 detection
Table 1 shows the results of testing standard proteins using the double antigen sandwich ELISA method.
Table 1:
| test for Standard protein concentration (ng/ml) | Standard protein (OD) |
| B | 0.106 |
| 1.56 | 0.149 |
| 3.12 | 0.189 |
| 6.25 | 0.28 |
| 12.5 | 0.509 |
| 25 | 0.989 |
| 50 | 2.221 |
| 100 | 3.225 |
The standard protein curve obtained according to Table 1 is shown in FIG. 3.
The kit is used for detecting antigen RBD protein by a double-antibody sandwich method, the ELISA plate adopts a high-affinity anti-RBD protein antibody as a coating, a sample to be detected or a standard substance is added into the hole of the ELISA plate during detection, a biotinylated anti-RBD protein antibody is added, streptavidin HRP is added after reaction, and finally single-component TMB substrate liquid is added. If the sample to be detected contains RBD protein, TMB substrate liquid is developed under the catalysis of HRP, after the termination liquid is added to stop the development, the OD value is read on an enzyme-linked immunosorbent assay, the light absorption value is in positive correlation with the concentration of the RBD protein in the sample to be detected, and the content of the RBD protein in the sample to be detected can be calculated according to a standard curve.
FIG. 4 is a schematic diagram showing the comparison of the results of the human serum stock solution detection by the double-antigen sandwich ELISA method according to the present invention, wherein 8 healthy persons and 4 convalescent patients are used as each case of the blank control group and the positive control group, and the serum test results of the healthy persons and the convalescent patients show that the human serum stock solution detection of the healthy persons is not positive (OD <0.18), and the convalescent patients should produce antibodies in vivo and show positive with different titers.
Comparative example 1 establishment and detection of new coronavirus IgG antibody kit product indirect method
1. Establishment of new coronavirus IgG antibody kit product indirect method
1) According to the concentration of 1-4ug/ml, the recombinant new coronavirus RBD protein is coated on a micropore plate and stays overnight at 4 ℃.
2) The well was drained and blocked with 2% BSA at 37 ℃ for 2 hours.
3) The liquid in the wells was spun off, and the plates were washed with 0.1M PBS, 0.2% Twen-20, 350ul per well for 3 times, 1 minute each time; after completion, the microplate was vacuum packed and stored at 4 ℃ until use.
4) And (4) taking the microporous plate strips for standby from the plate frame, putting the rest plate strips back into the aluminum foil bag filled with the drying agent, and then sealing and storing again.
5) Add washing buffer 350. mu.L per well, leave for 40 seconds and discard the liquid, which is washed 3 times in total.
6) A blank control was prepared by adding 100. mu.L of the dilution to the blank wells.
7) And adding 100 mu L of RBD rabbit monoclonal antibody with different concentrations and detection samples into other wells, sealing the wells with a sealing plate membrane, and incubating for 2 hours at 37 ℃.
8) Working solution of HRP-labeled goat anti-rabbit secondary antibody is prepared 15 minutes before use.
9) The well liquid was discarded and the washing step in step 3 was repeated.
10) Working solution of HRP-labeled goat anti-rabbit secondary antibody (100. mu.L/well) was added to each well, and a new plate sealing membrane was covered and incubated at 37 ℃ for 1 hour.
11) And (4) preheating the microplate reader.
12) The well liquid was discarded and the washing step in step 3 was repeated.
13) TMB substrate (100. mu.L/well) was added to the wells. Incubate at 37 ℃ for 15-20 minutes in the absence of light.
14) Stop buffer (50. mu.L/well) was added and immediately placed in the microplate reader and OD 450nm was measured in each well over 5 min. If the correction wavelength can be selected, 570nm or 630nm is set. And subtracting the 570nm or 630nm reading from the 450nm reading in such a way as to correct and remove the OD of the non-chromogenic material, thereby obtaining a more accurate detection result. If the correction wavelength cannot be selected, the reading obtained will be too high, resulting in a reduction in the accuracy of the reading.
2. Data processing and detection results
Table 2 is data for testing standard proteins using the indirect ELISA method.
Table 2:
| test for Standard protein concentration (pg/ml) | Standard protein (OD) |
| 0 | 0.056 |
| 39 | 0.113 |
| 78 | 0.256 |
| 156 | 0.322 |
| 312 | 0.641 |
| 625 | 0.944 |
| 1250 | 1.552 |
| 2500 | 3.478 |
FIG. 5 is a standard curve obtained from the data of Table 2.
FIG. 6 shows the results of the indirect method of the present invention for detecting the serum of 16 randomly sampled healthy people, and the present invention adopts 1 example of each of the blank control group and the positive control group, and shows that the 16 randomly sampled healthy people have positive OD values (OD ≥ 0.18) of different degrees, indicating higher false positives.
FIG. 7 shows the results of the indirect method of the present invention for the serum detection of 8 healthy patients and 4 convalescent patients in example 1, wherein 1 of each of the blank control group and the positive control group shows positive OD values (OD ≥ 0.18) of different degrees among the 8 randomly selected healthy people, and also shows higher false positives.
Claims (8)
1. A kit for detecting new coronavirus IgG/IgM total antibody is characterized by comprising a new coronavirus RBD recombinant protein and a biotin labeled protein thereof.
2. The kit according to claim 1, characterized in that said novel coronavirus RBD recombinant protein is ABClonal RP 01258.
3. The kit of claim 1, wherein the Biotin label is Biotin-maleimide.
4. The kit of claim 1, further comprising a novel coronavirus RBD mab.
5. The kit of claim 4, wherein the novel coronavirus RBD mab is ABClonal RM01763 or ABClonal RM 01764.
6. Use of the kit of claim 1 for the preparation of a novel coronavirus detection product.
7. The method for detecting the new coronavirus IgG/IgM total antibody is characterized in that the new coronavirus IgG/IgM total antibody is detected by utilizing a recombinant new coronavirus RBD and a biotin-labeled protein thereof in combination with a double-antigen sandwich ELISA method.
8. The method of claim 7, wherein the recombinant new coronavirus RBD protein is coated on a solid phase carrier, a sample to be detected is added to the solid phase carrier coated with the recombinant new coronavirus RBD protein, and the sample is incubated and washed; adding biotin-labeled novel coronavirus RBD protein into the washed solid phase carrier, incubating, washing, then developing color and combining with a standard curve to obtain the total antibody content.
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| CN113238046A (en) * | 2021-04-30 | 2021-08-10 | 深圳迈瑞生物医疗电子股份有限公司 | Kit for detecting coronavirus antibody and detection method of coronavirus antibody |
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| CN113624965A (en) * | 2021-08-05 | 2021-11-09 | 中国人民解放军军事科学院军事医学研究院 | Application of N protein specific IgG4 in screening novel coronavirus infectors and vaccinators |
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