CN113252908B

CN113252908B - Flow type kit for detecting novel coronavirus neutralizing antibody and detection method

Info

Publication number: CN113252908B
Application number: CN202110639635.6A
Authority: CN
Inventors: 严崇虎; 李国平
Original assignee: Pan Peptide Biotechnology Zhejiang Co ltd
Current assignee: Pan Peptide Biotechnology Zhejiang Co ltd
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2021-09-28
Anticipated expiration: 2041-06-09
Also published as: CN113252908A

Abstract

The invention provides a flow type kit for detecting a novel coronavirus neutralizing antibody and a detection method; the kit comprises a magnetic bead-S-RBD compound and a mouse anti-human IgG-biotin compound. The detection method comprises the following steps: capturing neutralizing antibodies in plasma by the magnetic bead-S-RBD complex and crosslinking the mouse anti-human IgG-biotin complex and SA-PE; and (6) performing computer detection to obtain data. The kit can be used for determining the content of the new coronavirus neutralizing antibody in a sample by combining a flow cytometry method, the result can be obtained within 30min, the neutralizing antibody can be quickly diagnosed by using the kit only through simple operation, the using amount of a blood sample is very small, only 1ul of blood plasma or serum is needed, the sensitivity of the detection result is very high, and the trace neutralizing antibody can be detected.

Description

Flow type kit for detecting novel coronavirus neutralizing antibody and detection method

Technical Field

The invention relates to the technical field of in-vitro detection, in particular to a flow type kit for detecting a novel coronavirus neutralizing antibody and a detection method.

Background

The world health organization formally named the new coronavirus causing the new pneumonia epidemic of the current time as '2019-nCoV' in 12 months in 2020. On day 11/2/2020, the world health organization named "COVID-19" for pneumonia infected with the novel coronavirus. Meanwhile, the International Committee for Classification of viruses has named the novel coronavirus "SARS-CoV-2".

Pneumonia caused by the novel coronavirus is rapidly spread to various countries around the world, such as Asia, Australia, Europe, America, Africa, etc. The symptoms of the attack are respiratory tract symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.

Due to the dramatic increase of the number of infected people and dead people in the world, the vaccine development is not slow enough, and the successful control of the number of infected people with the new coronary pneumonia mainly depends on the vaccine immunity. Therefore, the quality of the immune effect of the vaccine plays a decisive role in the prevention and control of the new coronary pneumonia. The detection of the level of the new crown neutralizing antibody is a main method for evaluating the immune effect of the vaccine, and in addition, the detection of the level of the new crown antibody has important significance for the formulation of the immune program of the new crown vaccine. Therefore, a rapid, accurate, highly sensitive and easy-to-use method for detecting neutralizing antibodies is important for the treatment of new coronary pneumonia and the evaluation of the effectiveness of vaccines.

At present, common related detection technologies include virus separation and identification, immunofluorescence antibody experiments, immune colloidal gold technology, forward indirect hemagglutination tests, neutralization tests, enzyme-linked immunosorbent assays, reverse transcription-polymerase chain reactions, fluorescence quantification technologies and gene chip technologies. The conventional detection technology of the neutralizing antibody is a neutralization test, needs to use viruses or pseudoviruses, is carried out in a high-grade microorganism laboratory, has very high requirements on personnel, equipment and operation, and cannot meet the current clinical requirements.

Furthermore, the prior art has the following disadvantages: 1. during detection, the dosage of blood plasma or blood serum is large; 2. the sensitivity is low, and when the content of the new crown neutralizing antibody in the plasma or serum sample is extremely low, the new crown neutralizing antibody cannot be detected; 3, the weak change of the concentration of the new crown neutralizing antibody in the blood can not be accurately monitored, and the resolution is low; 4. in the detection, when the amount of plasma or serum is large, the detection result is very likely to be disturbed, and false positive or false negative may occur, and therefore, it is necessary to add another chemical reagent (for example, HAMA) for eliminating the disturbance to the detection kit, which increases the reagent cost and complicates the production process.

Disclosure of Invention

In order to solve the problems, the invention provides a flow type kit and a detection method for detecting a novel coronavirus neutralizing antibody, the kit can be used for determining the content of the novel coronavirus neutralizing antibody in a sample by combining a flow cytometry method, the result can be obtained within 30min, the kit can be used for quickly diagnosing the neutralizing antibody only by simple operation, the blood sample consumption is very small, only 1ul of blood plasma or serum is needed, the detection result sensitivity is extremely high, and the trace neutralizing antibody can be detected.

In one aspect, the invention provides a flow kit for detecting neutralizing antibodies against a novel coronavirus, which comprises a magnetic bead-S-RBD complex and a mouse anti-human IgG-biotin complex.

In another aspect, the present invention provides a method for preparing a flow kit for detecting neutralizing antibodies against a novel coronavirus, comprising the steps of:

s10: obtaining magnetic beads and S-RBD, and chemically crosslinking to obtain magnetic bead-S-RBD compounds;

s20: acquiring biotin and mouse anti-human IgG, and chemically crosslinking to obtain a mouse anti-human IgG-biotin compound.

Further, the S10 includes:

s11: using NHS and EDC to activate the magnetic beads, and separating and cleaning to obtain activated magnetic beads;

s12: and adding S-RBD into the activated magnetic beads, blowing, beating and uniformly mixing, and shaking and coating in a dark place to obtain the magnetic bead-S-RBD compound.

Further, the mass ratio of the NHS to the EDC is 2: 1.

further, the S20 includes the following steps:

s21: desalting the mouse anti-human IgG to obtain desalted mouse anti-human IgG;

s22: and adding an NHS-biotin solution into the desalted mouse anti-human IgG, and oscillating at room temperature in a dark place to obtain the mouse anti-human IgG-biotin compound.

Further, in the NHS-biotin solution, the solvent is DMSO.

In another aspect, the present invention also provides a method for using a flow kit for detecting neutralizing antibodies against a novel coronavirus, comprising the following steps:

s30: adding the magnetic bead-S-RBD compound, the plasma and the diluent into a 96-well plate for immunoreaction, and capturing a neutralizing antibody in the plasma by the magnetic bead-S-RBD compound to form a magnetic bead-S-RBD-neutralizing antibody compound;

s40: the magnetic bead-S-RBD-neutralizing antibody compound and mouse anti-human IgG-biotin carry out immunoreaction to form a magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin compound;

s50: the magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin compound reacts with SA-PE to form a magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin-SA-PE compound; and (5) performing computer analysis to obtain detection data.

The PE is used as a fluorescent marker and plays a role in amplifying a fluorescent signal, when the number of the neutralizing antibodies in a plasma or serum sample is more, the formed final complex is more, the fluorescent signal is stronger, otherwise, the final complex is less, the fluorescent signal is weaker, and the intensity of the fluorescent signal is in direct proportion to the number of the neutralizing antibodies in the sample.

Further, the detection data is the fluorescence intensity value of the magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin-SA-PE compound.

Further, the cutoff value was set to 1ul of the fluorescence value of the mixture of the magnetic bead-S-RBD complex and 150ul of the wash solution × 2.5; and when the fluorescence intensity value of the magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin-SA-PE compound is greater than or equal to the cutoff value, judging the compound to be positive, otherwise, judging the compound to be negative.

In summary, the present application may have one or more of the following advantages or benefits:

(1) the flow type kit for detecting the novel coronavirus neutralizing antibody can quickly detect the novel coronavirus neutralizing antibody, is simple to operate, intuitive in result, safe and cheap, and is suitable for batch quick detection.

(2) The dosage of the plasma or serum sample is very small, and only 1ul is needed.

(3) The detection sensitivity is extremely high, and the neutralization antibody with a trace amount of 10-30pg/ml can be detected.

(4) The detection precision is high, the weak change of the concentration of the new crown neutralizing antibody in blood can be accurately monitored, and the resolution is high.

(5) The dosage of the main components of the reagent is very small, and the dosage of the magnetic bead-S-RBD is only 1ul (the concentration is 500 ug/ml), thereby greatly reducing the cost of the reagent.

(6) During detection, the dosage of the required blood plasma or blood serum is very small, so that the anti-interference capability of the kit is very strong, the detection result is not interfered by other substances in the blood plasma or blood serum, and the detection result is very accurate. And an anti-interference chemical reagent is not required to be additionally added, so that the cost is further saved.

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 description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a schematic diagram of the principle of the present invention.

FIG. 2 is a graph showing fluorescence intensity values of a negative sample 1 according to a second embodiment of the present invention.

FIG. 3 is a graph showing fluorescence intensity values of a second example positive sample 1 according to the present invention.

FIG. 4 is a diagram showing the detection effect of colloidal gold test paper of a certain company.

Fig. 5 is a cutoff value of the second embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

[ first embodiment ] A method for manufacturing a semiconductor device

The embodiment provides a flow-type kit for detecting a novel coronavirus neutralizing antibody and a preparation method thereof, wherein the kit comprises a magnetic bead-S-RBD (restricted bead-based binding domain) -mouse anti-human IgG-biotin compound, and the detection principle of the kit is shown in figure 1; the preparation method comprises the following steps.

Magnetic bead crosslinked S-RBD

Activation of magnetic beads

1. Fully and uniformly mixing suspended magnetic beads by using a vortex mixer, taking 2 mg of magnetic bead suspension into a trace thick-wall reaction bottle, placing the trace thick-wall reaction bottle on a magnetic separation rack for about 1min, and carefully removing supernatant;

2. adding 200ul MES buffer solution, mixing the suspended magnetic microspheres with a vortex mixer, placing the micro thick-wall reaction bottle on a magnetic separation frame for about 1min, carefully removing the clear liquid, and cleaning for 2 times in total;

3. add 38. mu.L NHS (50 mg/ml) and 19. mu.L EDC (50 mg/ml), mix the suspended beads well with a vortex mixer, rotate with a bead mixer for 1h at room temperature;

4. and (3) placing the trace thick-wall reaction bottle on a magnetic separation frame for about 1min, carefully removing clear liquid, adding 1 mL of connection buffer solution, fully and uniformly mixing suspended magnetic beads by using a vortex mixer, and cleaning for 3 times in total to obtain activated magnetic beads.

(II) Cross-linking of activated magnetic beads with S-RBD

1. Adding 60ug S-RBD into activated magnetic beads, blowing, mixing, diluting the activated magnetic beads to about 10 mg/mL with MES buffer solution, mixing with vortex mixer, and shaking to coat in dark for 16-20 hr to obtain coated magnetic beads;

2. placing the coated magnetic beads on a magnetic separation frame for about 1min, carefully removing clear liquid, adding 200ul of TBST magnetic bead preservation solution, fully and uniformly mixing by using a vortex mixer, and cleaning for 4 times in total to obtain a magnetic bead-S-RBD compound;

3. diluting the magnetic bead-S-RBD compound to 500 mu g/mL by using TBST magnetic bead preservation solution, totally 4mL, and storing at 2-8 ℃ in a dark place.

Biotin cross-linked mouse anti-human IgG

Desalting treatment of mouse anti-human IgG

1. The tail of the column was broken off and the cap was released and the column was placed in a 1.5-2.0ml centrifuge tube.

2.1500Xg for 1min, the stored liquid was removed.

3. A mark is made on the tube wall at the inclined top end of the compacted resin. All subsequent centrifugation steps were performed with the column labeled out into the centrifuge.

4. 300ul PBS buffer was added to the column, centrifuged at 1500Xg for 1min, and the buffer removed.

5. Repeat step 4 2 times and discard the buffer from the tube.

6. The column was placed in a fresh centrifuge tube, the lid removed, and 780ug of mouse anti-human IgG solution was added to the compacted resin core.

7.1500Xg for 2min, and collecting the bottom liquid to obtain desalted mouse anti-human IgG.

(II) antibody-crosslinked biotin

1. Dissolving 5mg NHS-biotin in 100ul DMSO, adding 15ul NHS-biotin liquid into the desalted mouse anti-human IgG, and carrying out shake reaction for 5 hours at room temperature in a dark place;

2. after the reaction is finished, redundant NHS-biotin and reaction products are removed by a desalting column to obtain a mouse anti-human IgG-biotin compound.

[ second embodiment ]

The kit obtained in the first example was used to test clinical specimens.

Clinical sample detection process

Flow cytometer setup: the PE channel voltage of the flow cytometer was adjusted to suitable parameters such that the fluorescence detection value of the mixture of 1ul of magnetic bead-S-RBD complex and 150ul of wash solution was in the range of 1400-. Taking the beckmann DxFLEX flow cytometer as an example, the voltage of the PE channel is adjusted to 50.

Before detection, the magnetic bead-S-RBD compound is shaken vigorously and mixed uniformly before use, and is rapidly mixed with other liquid reagents for 2-3 seconds.

The detection steps are as follows:

adding 1ul of magnetic bead-S-RBD compound, 45ul of diluent and 1ul of plasma or serum into the hole respectively, and oscillating for reaction for 10 minutes at room temperature. After the reaction was completed, the reaction mixture was magnetically adsorbed for 1 minute, and the supernatant was removed as much as possible.

② 40ul of mouse anti-human IgG-biotin compound is added into each hole, and the reaction is carried out for 10 minutes under the condition of shaking at room temperature.

③ after the reaction, 40ul of PE-SA is added into each hole, and the reaction is carried out for 7 minutes at room temperature by shaking.

Fourthly, after the reaction is finished, the mixture is magnetically adsorbed for 1 minute, and the supernatant is removed. 150ul of cleaning solution is added into each hole, and the machine is used for detection.

(II) detection results and analysis

In the present example, 217 clinical negative samples and 139 clinical positive samples were tested, and table 1 shows partial test data of the clinical negative samples.

TABLE 1

See fig. 2, which is the fluorescence intensity value of the negative sample 1 of this example.

Table 2 shows the data of 3 of the clinical positive samples.

TABLE 2

Wherein the positive sample 1 shows weak positive, see fig. 3, which is the fluorescence intensity value of the positive sample 1. Referring to fig. 4, the results of the detection of the three samples by using the colloidal gold test paper of a certain company are shown in the figure, and the detection results of the weak positive sample and the positive sample are not obviously different, so that the comparison shows that the kit provided by the embodiment of the invention is more intuitive and accurate.

The detection method provided by the embodiment judges whether the clinical sample is negative or positive according to the cutoff value, and the clinical sample is positive when the fluorescence intensity value of the clinical sample is larger than or equal to the cutoff value; the fluorescence intensity value of the clinical sample is less than the cutoff value, and the clinical sample is negative; wherein the cutoff value =1ul of fluorescence value x 2.5 for a mixture of magnetic bead-S-RBD complex and 150ul of wash. Referring to fig. 5, the vertical line in the graph represents the cutoff value, the left peak represents the fluorescence intensity value of the negative sample 1, and the right peak represents the fluorescence intensity value of the positive sample 1.

The results of the sample testing of this example were counted as shown in table 3.

TABLE 3

The results of the measurements in Table 3 were analyzed, as shown in Table 4.

TABLE 4

The detection method provided by the invention has the following advantages:

1. the required sample amount is small, namely 1ul of blood plasma or serum;

2. the sensitivity and the precision of detection are high;

3. the detection method is adopted to carry out batch-to-batch experiments, the Coefficient of Variation (CV) of the batch experiments is not more than 10%, and the CV of the batch experiments is not more than 15%.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting a novel coronavirus neutralizing antibody is characterized in that a kit is adopted, the kit comprises a magnetic bead-S-RBD compound and a mouse anti-human IgG-biotin compound, and the detection method comprises the following steps:

s30: adding the magnetic bead-S-RBD compound, a sample and a diluent into a 96-well plate for immunoreaction, and capturing a neutralizing antibody in the sample by the magnetic bead-S-RBD compound to form a magnetic bead-S-RBD-neutralizing antibody compound;

2. The detection method according to claim 1, wherein the detection data is the fluorescence intensity value of the magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin-SA-PE complex.

3. The detection method according to claim 2, wherein the cutoff value is set to a fluorescence value of a mixture of 1ul of the magnetic bead-S-RBD complex and 150ul of the washing solution x 2.5;

and when the fluorescence intensity value of the magnetic bead-S-RBD-neutralizing antibody-mouse anti-human IgG-biotin-SA-PE compound is greater than or equal to the cutoff value, judging the compound to be positive, otherwise, judging the compound to be negative.

4. A kit comprising magnetic bead-S-RBD complex and mouse anti-human IgG-biotin complex, wherein the kit is for the detection of novel coronavirus neutralizing antibodies using the detection method of any one of claims 1 to 3.

5. A method for preparing a kit according to claim 4, comprising the steps of:

6. The method according to claim 5, wherein the S10 includes:

7. The method according to claim 6, wherein the mass ratio of NHS to EDC is 2: 1.

8. the method for preparing a composite material according to claim 5, wherein the step S20 includes the steps of:

9. The method according to claim 8, wherein the solvent in the NHS-biotin solution is DMSO.