CN114316039B

CN114316039B - Kit for rapidly detecting viruses and preparation method thereof

Info

Publication number: CN114316039B
Application number: CN202210038830.8A
Authority: CN
Inventors: 陈卫国; 林宣伟
Original assignee: Baiding Bioengineering Beijing Co ltd
Current assignee: Baiding Bioengineering Beijing Co ltd
Priority date: 2022-01-13
Filing date: 2022-01-13
Publication date: 2024-04-12
Anticipated expiration: 2042-01-13
Also published as: CN114316039A

Abstract

The invention discloses a kit for rapidly detecting viruses and a preparation method thereof. In one aspect, the invention screens a monoclonal antibody capable of specifically recognizing the Hantavirus NP protein, wherein the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3. The NP protein prepared by combining the monoclonal antibody and the polyclonal antibody are used for preparing two colloidal gold detection test strips (antigen detection or antibody detection), the test strips have good specificity and sensitivity, and the two are combined for use, so that the detection accuracy can be further improved, and the method is suitable for large-scale popularization and use.

Description

Kit for rapidly detecting viruses and preparation method thereof

Technical Field

The invention belongs to the technical field of diagnosis of viral epidemic diseases, and particularly relates to a kit for rapidly detecting viruses and a preparation method thereof.

Background

Hantavirus, also known as hemorrhagic fever with renal syndrome virus (HRSV), was first successfully isolated from black streaked mice in 1978. Hantavirus can be divided into 6 serotypes around the world, namely, a jia murine, a domestic murine or a rat, a brown dorsal, a field murine, a Huang Gengji murine and a mouse or a mouse domestic murine. A variety of wild rodents such as black-line mice are natural hosts of viruses that are devoid of any clinical symptoms, but which are transmitted to humans by faeces and secretions. The main clinical manifestation is that after the premenstrual symptoms such as fever, headache and the like about 4 days, acute respiratory failure characterized by non-cardiac pulmonary edema and high fatality rate (52.4% -78.0%) occurs, the serious death occurs for 3-7 days, and survivors recover quickly without sequelae. The pathogenesis is mainly the direct pathogenic effect of the virus, kidneys are early primary damaged organs, and viruses are a direct factor in kidney injury.

Hantavirus belongs to the bunyaviridae family, is an enveloped segmented negative-strand RNA virus, and comprises L, M, S segments in genome, wherein the segments respectively encode L polymerase protein, G1 glycoprotein, G2 glycoprotein and Nucleoprotein (NP). The NP is a main structural protein of virus, has strong immunogenicity, high antibody titer and long maintenance time for stimulating the organism to produce, plays an important role in antiviral immunity, has conserved amino acid sequences of different strains and types of virus NPs, all carries T cell and B cell antigenic determinants, can induce the organism to produce specific humoral immune response and cellular immune response, can be directly combined with RNA of in vitro transcription virus and organelles of infected cells, plays a role in regulating the replication of the virus, and is commonly used as a diagnostic protein after HV infection.

JiroArikawa et al used baculovirus expressed viral nucleoprotein for ELISA and could be used for serological monitoring of Hantavirus infection. The recombinant nucleoprotein and N-terminal deleted nucleoprotein are used for ELISA and IFA, and provide a rapid, sensitive and safe diagnosis method for hantavirus infection.

Disclosure of Invention

In view of the defects of the prior art, the invention mainly aims at 2 points, namely, researching a monoclonal antibody of hantavirus nucleoprotein and a preparation method thereof; secondly, a detection method for preparing hantavirus by using the NP protein and/or monoclonal antibody prepared by the invention and a kit thereof are researched. To achieve the object of the present invention, the present invention is implemented according to the following technical solutions:

firstly, the invention prepares a monoclonal antibody aiming at Hantavirus NP, and the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3.

Further, the invention further analyzes the sequence of the prepared monoclonal antibody, wherein the heavy chain variable region of the monoclonal antibody comprises CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6. The light chain variable region of the monoclonal antibody comprises CDR1, CDR2 and CDR3; the amino acid sequences of the CDR1, the CDR2 and the CDR3 are respectively shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

Secondly, the invention develops a kit for detecting hantavirus based on the prepared monoclonal antibody, wherein the kit comprises an effective amount of the monoclonal antibody of claim 1. The kit is a colloidal gold detection test strip, and the test strip comprises a latex pad prepared from polyclonal antibodies, a nitrocellulose membrane, absorbent paper and a PVC backboard; the nitrocellulose membrane comprises a detection line prepared from monoclonal antibodies and a quality control line prepared from goat anti-rabbit IgG.

The invention further develops a Hantavirus antibody detection kit, which comprises an effective amount of the monoclonal antibody of claim 1 and Hantavirus NP protein. The kit is a colloidal gold detection test strip, and the test strip comprises a latex pad prepared from Hantavirus NP protein, a nitrocellulose membrane, absorbent paper and a PVC backboard; the nitrocellulose membrane comprises a detection line prepared from Hantavirus NP protein and a quality control line prepared from monoclonal antibodies.

And finally, the two developed kits are combined to obtain the kit for double detection of the hantavirus by the antigen-antibody. The combined detection kit has higher accuracy than the single detection.

Finally, the invention also discloses application of the monoclonal antibody in preparation of hantavirus detection reagents. The application comprises, but is not limited to, IFA detection of Hantavirus, colloidal gold detection test strips of Hantavirus, and Hantavirus antibody detection test strips.

The NP protein prepared by the invention is the most basic prokaryotic expression, but the protein is also suitable for preparing different Hantavirus diagnostic reagents, such as detection kits of colloidal gold, chemiluminescence and the like.

The monoclonal antibody prepared by the invention has good specificity and sensitivity, is suitable for preparing detection reagents of different hantaviruses, not only comprises the colloidal gold detection test strip, but also can be an ELISA detection kit and a chemiluminescent detection kit, and can be applied to detection of IFA, wersterin blot and the like in scientific research.

The two colloidal gold detection test strips prepared by the invention can be simultaneously applied to detection of antigens and antibodies of hantaviruses, have strong specificity, high sensitivity, good stability and high detection speed, have higher combined detection accuracy, and can be used for early screening and field screening of hantaviruses.

Drawings

FIG. 1 shows SDS-PAGE of purified Hantavirus Nucleoprotein (NP), wherein 1 is Marker and 2 is purified NP protein.

FIG. 2 is a SDS-PAGE diagram of two purified monoclonal antibodies, wherein 1 is Marker and 2 is purified monoclonal antibody.

FIG. 3IFA test results.

The test result of the test strip in FIG. 4, wherein 1, 2 and 3 are adenovirus inactivated virus liquid, influenza A inactivated virus liquid and influenza B inactivated virus liquid respectively.

The test strip of FIG. 5 shows the detection results, wherein 1, 2 and 3 are adenovirus virus positive serum (after inactivation), influenza A virus positive serum (after inactivation) and influenza B virus positive serum (after inactivation) respectively.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.

Reagents and materials used in the following examples are commercially available unless otherwise specified.

Example 1: construction of prokaryotic expression vector for expressing hantavirus Nucleoprotein (NP)

The nucleotide sequence of the CDS sequence of the NP protein of GenBank AF159370.1 is subjected to codon optimization, the nucleotide sequence of the NP protein after the codon optimization (shown as SEQ ID NO. 1) is cloned into a prokaryotic expression vector (PET 28 a), and 6 His labels are added at the C end. The work entrusts the completion of the Huada gene.

Example 2: expression and purification of hantavirus Nucleoprotein (NP)

Culturing induced genetically engineered bacteria (PET 28 a-NP) expressing NP protein at 37 ℃ for 6 hours by shaking at 220rpm, harvesting the thalli, collecting the thalli by a 50 mL-specification centrifuge tube, centrifuging the thalli for 10 minutes at 4 ℃ at 5000rpm, and discarding the supernatant to retain the thalli; the bacterial body is re-suspended and washed in 20mL PBS solution, and centrifuged for 10min at 12000rpm at 4 ℃, and the supernatant is discarded to retain bacterial body; suspending the strain in 20mL of washing solution, performing ultrasonic crushing in ice bath, performing ultrasonic treatment at ultrasonic frequency of 40%, performing ultrasonic treatment for 3s, stopping for 5s, and performing ultrasonic treatment for 8min; the cells were resuspended in 20ml of a washing solution containing a small amount of urea by centrifugation at 12000rpm for 10min at 4℃and the supernatant was discarded to retain the cells by centrifugation at 12000rpm for 10min at 4 ℃. The recombinant protein was purified using the GE company nickel column HIS purification system, and the purification operation was briefly described as follows: re-suspending the precipitate with 20ml of solution, centrifuging at 12000rpm at 4deg.C for 10min, and transferring the supernatant into another centrifuge tube; 1ml of 50% NI-NTA resin was added to 4ml of lysate, mixed well and incubated overnight at 4℃in a shaker; adding the mixture of the lysate and the NI-NTA resin suspension into a hollow column with a closed lower end, and collecting effluent; adding 12ml of renaturation solution containing urea, placing the mixture on a shaking table, incubating the mixture in an ice bath for 6 hours to renaturate the target protein, and finally flowing the renaturation solution; eluting with eluent containing urea for 3 times (12 ml, 6 ml), placing into ice bath at 4deg.C for 30min on a shaking table for the first time, placing into ice bath at 4deg.C for 15min on a shaking table for the other two times, eluting target protein, and collecting eluent; concentrating the collected eluent to 3-5 ml by using an ultrafiltration tube, dialyzing the eluent in PBS buffer solution, replacing the eluent, subpackaging, and storing at-20 ℃ for later use.

10 μl of purified NP protein was subjected to electrophoresis using SDS-PAGE protein gel, and a clear band was present at about 49kDa, with a purity of 90% or more (FIG. 1). Protein content was measured using BCA kit at a protein concentration of 1.02mg/ml.

Example 3: preparation and purification of hybridoma cells expressing NP protein monoclonal antibodies

Immunization of mice: 8-10 week old female BALB/c mice were immunized with purified NP protein as immunogen. The immunization method is as follows: first, each mouse was mixed with 100 μg np protein and equal amount FCA to make an emulsifier, and injected subcutaneously and intraperitoneally at the nuchal; second, 2 weeks after the first, FCA was changed to FICA, and the doses and methods were the same; three-way, 2 weeks after two-way, the dose and method are the same as two-way. The boost was performed 1 week prior to cell fusion by intraperitoneal injection of 100 μg of purified NP protein.

Cell fusion: cell fusion was performed 3-5 days after mouse booster immunization. The procedure for cell fusion is briefly described as follows: neck fracturePositive mice were sacrificed, spleen cells thereof were aseptically taken, fused with PEG4000 at a ratio of 5:1 of spleen cells to SP2/0 myeloma cells, the fusion was terminated by adding RPMI 1640 in a lump after standing for 1min, centrifuging at room temperature at 1000rpm for 8min, discarding the supernatant, adding 50ml of complete RPMI 1640 containing 20% of HAT neonatal bovine serum, and adding the fused cells into 96-well plates at 100. Mu.l per well. Placing the culture plate at 37deg.C and 5% CO ₂ Culturing in incubator for 15 days, and changing to complete RPMI 1640 containing HT 20% calf serum.

Cell strain selection: subcloning the positive hybridoma obtained by screening, diluting the original hole cells with HT culture medium, and dividing the original hole cells into 96 hole cell plates, wherein one original hole cell is divided into one plate. After subcloning was completed, the cell number and status of each well were observed. The secondary subcloning was performed in wells where the secreted antibodies were stable after subcloning and were as single clone as possible. The positive rate of the detection result of the subcloned plate which is originally monoclonal after three subcloning should reach 100%. 1 hybridoma cell line capable of stably secreting a monoclonal antibody specific for the NP protein was obtained.

Example 4: preparation and detection of NP protein monoclonal antibody

Preparing ascites: 8-10 weeks old healthy BALB/c mice were injected intraperitoneally with liquid paraffin, 0.5ml each, 10 daily i.p.) ⁶ And (3) extracting ascites when the abdomen of the mouse is extremely swelled after 7-10 days, extracting once every 2 days, centrifuging the extracted ascites at 2-8 ℃ for 10 minutes at 1000rpm, removing upper-layer grease and sediment, sub-packaging the supernatant (5 ml/tube), and preserving at-20 ℃ or-70 ℃ for later use.

Antibody purification: for the caprylic acid-ammonium sulfate precipitation method (see, in particular, zhou Yu, etc., research on a method for purifying monoclonal antibodies of the mouse ascites IgG class, 10 th 2006 in Heilongjiang animal doctor), after purification, the purified antibodies were subjected to purity test by SDS-PAGE, and the results show that the SDS-PAGE purity of the monoclonal antibodies is greater than 90%; the purified antibodies were subjected to concentration detection using BCA kit, and the detection results were 5.82mg/ml, respectively.

Antibody titer determination: will be pureThe functionalized NP protein was added to the ELISA plate at 0.1. Mu.g/well and left overnight at 4 ℃. After 3 washes, 200. Mu.L of blocking solution was added to each well, and the mixture was left at 37℃for 1 hour, followed by 3 washes. The prepared antibody is serially diluted by 10 times with PBS, 100 mu L/hole is added on a closed ELISA plate, and PBS is used as a blank control; incubation for 1h at 37℃in an incubator, washing 3 times followed by addition of HRP-labeled goat anti-mouse (1:5000) antibody, 100. Mu.L/well, incubation for 0.5h at 37℃in an incubator, washing 3 times followed by addition of freshly prepared substrate solution 100. Mu.L/well, incubation for 10min at 37℃in an incubator protected from light, and addition of 50. Mu.L/Kong Zhongzhi solution, OD450 was determined with P/N > 2.1 as positive determination criterion (P is the sample OD450 value, N is the blank OD450 value). The test results (Table 1) show that the monoclonal antibody has a titer of 10 ⁷ 。

TABLE 1 results of antibody titer determinations

Dilution factor	10 ³	10 ⁴	10 ⁵	10 ⁶	10 ⁷	10 ⁸	10 ⁹
								OD450 value	1.443	1.354	0.765	0.454	0.201	0.054	0.061
P/N value	21.54	20.21	11.42	6.78	3.00	0.81	0.91

Note that: the blank OD450 value was 0.067.

Monoclonal antibody subclass identification: the identification is carried out by adopting a mouse monoclonal antibody parting gold-labeled test paper kit of IsoStrip company, and the specific steps are operated according to the instruction of the kit. The monoclonal antibody subclass is IgG after detection ₁ 。

Determination of monoclonal antibody sequences: the determination of the heavy chain variable region and the light chain variable region was performed on the monoclonal antibody prepared in the method of example 7 of chinese patent (202110793081.5). Through detection and sequencing, the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3; analysis of the determined sequence shows that the amino acid sequences of 3 CDRs of the heavy chain variable region of the monoclonal antibody are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the amino acid sequences of 3 CDRs of the light chain variable region are respectively shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO. 9.

Example 5: FITC labeled monoclonal antibody and detection thereof

For a specific test method, see invention patent 201710054599.0, briefly described below: concentrating monoclonal antibody to 10mg/ml, adjusting pH to 9.0,4 deg.C, and storing; 1mg of FITC is dissolved in 0.2ml of DMSO, slowly added into the antibody in a dropwise manner, reacted for 2 hours at room temperature in a dark place, and purified by a Sephadex G25 column after the reaction. The purified monoclonal antibody was diluted 1:100 with PBS containing 1% FBS, and IFA was performed on hantavirus-infected murine lung tissue (identified by IFA, PCR and sequencing). The results show (FIG. 3) that dense green fluorescent particles are visible in infected murine lung tissue under fluorescence microscopy, demonstrating good specificity and sensitivity of the monoclonal antibodies prepared according to the invention.

Example 6: preparation and detection of colloidal gold detection test strip (antigen)

Preparation of polyclonal antibodies: the NP protein prepared by the invention is used for preparing vaccines (the mass ratio of antigen to adjuvant is emulsified and the like, 100 mug/ml) by using an ISA 201 adjuvant, and the back of the NP protein is injected with New Zealand white rabbits in a multipoint way, and the volume of the NP protein is 1 ml/ml; the same dose and route boost once after 2 weeks interval; boosting the immunization once again with the same dose after 2 weeks interval; after 7 days of three-phase, rabbit blood is collected, centrifuged at 3000rpm for 10min, serum is separated, and the obtained product is preserved at-20 ℃ for standby. Purifying the isolated serum with a protein G pre-cartridge, concentrating, detecting the concentration of the antibody with BCA at 2.12mg/ml, and detecting the antibody titer with the method of the antibody titer determination of example 4 at a titer of 10 ⁵ Packaging, and storing at-20deg.C.

Preparing a test strip: taking 1ml of carboxyl modified latex microspheres (Shanghai Hui, 400 nm), centrifuging at a room temperature of 12000rpm for 30min, washing with 0.1mol/L MES solution for 3 times, adding 5mmol/L EDC and 2mmol/L NHS, performing room temperature activation reaction for 30min, adding 1mg of prepared polyclonal antibody after activation, performing reaction for 3h at 37 ℃, adding 2% BSA for blocking, and performing room temperature reaction for 30min; the labeled polyclonal antibody is centrifuged at 12000rpm for 30min, resuspended in PBS containing 1% BSA, evenly spread on the treated latex microsphere mat, and placed in a 37 ℃ oven overnight, dried at room temperature and stored for standby. The goat anti-rabbit IgG is diluted to 0.5mg/ml by PBS buffer, the monoclonal antibody is diluted to 1mg/ml by PBS buffer, the goat anti-rabbit IgG is sprayed on the position of a quality control line (C line), the monoclonal antibody is sprayed on the position of a detection line (T line) to prepare a nitrocellulose membrane, and the nitrocellulose membrane is placed in an oven at 37 ℃ for overnight and dried at room temperature for storage. And assembling the dried nitrocellulose membrane with a latex pad, absorbent paper and a PVC backboard, and cutting to obtain the quick detection test strip for detecting the Hantavirus. In detection, the sample to be detected is dripped into a detection hole (about 40 μl), and the equivalent sample diluent (PBS buffer solution) is immediately added; after the sample is added, the test strip is horizontally placed on a table top, the result is observed for about 8-10 minutes, and if the T line and the C line are both provided with the strip, the test strip is judged to be positive; if the C line has a strip, the T line has no strip, and the judgment is negative; if the C line has no stripe, whether the T line has a stripe or not is judged to be invalid.

And (3) test strip detection: and detecting by using the prepared test strip, and respectively detecting the adenovirus inactivated virus liquid, the influenza A inactivated virus liquid and the influenza B inactivated virus liquid which are negative reference products, wherein the detection results (shown in figure 4) show that the detection results of the negative reference products are negative. The NP protein at different concentrations was detected using the prepared test strip, and the results showed (Table 2) that the minimum detection limit of the test strip could reach 2ng/ml.

TABLE 2 detection results of NP protein at different concentrations

NP protein concentration	8ng/ml	4ng/ml	2ng/ml	1ng/ml
					Detection result	+	+	+	-

Note that: "+" indicates that the detection result is positive, and "-" indicates that the detection result is negative.

Example 7: preparation and detection of colloidal gold detection test strip (antibody)

Based on example 5, the latex-pad-labeled polyclonal antibody was exchanged for the NP protein prepared by the present invention; the T line (detection line) is also replaced by the NP protein prepared by the invention, and the C line (quality control line) is replaced by the monoclonal antibody prepared by the invention; the test strip for preparing the double antigen detection antibody is the same as that of example 5 except for the preparation process, the detection step and the detection result judgment.

And (3) test strip detection: and (3) detecting by using the prepared test strip, and respectively detecting adenovirus positive serum (after inactivation), influenza A virus positive serum (after inactivation) and influenza B virus positive serum (after inactivation) which are negative reference products, wherein the detection results (figure 5) show that the detection results of the negative reference products are negative. The test strips prepared are used for detecting the Hantavirus positive serum (after inactivation) with different dilutions, and the result shows that the lowest detection limit of the test strips can reach 1:3200 (table 3).

TABLE 3 results of Hantavirus positive serum (after inactivation) at different dilutions

Dilution degree

1:100

1:200

1:400

1:800

1:1600

1:3200

1:6400

Detection result

+

-

Example 8: combined detection of two colloidal gold detection test strips

The collected 232 clinical samples (189 negative samples and 43 positive samples, which are all inoculated with related vaccines, are derived from a certain hospital clinical laboratory in Guangdong) are tested, and the single test and the combined test (at least one of which is positive and both of which are negative) show that (table 4) the positive rate of the single test of the two test strips is above 90%, but the positive rate of the combined test is 97.7%, which is higher than that of the single test, so that the combined application of the two test strips can further improve the diagnosis accuracy.

TABLE 4 clinical sample test results

Test paper strip	Positive rate	Negative rate
			Test paper (antigen detection)	39/43＝90.1％	189/189＝100％
Test paper (antibody detection)	40/43＝93％	189/189＝100％
			Test paper (Simultaneous detection)	42/43＝97.7％	189/189＝100％

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

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Claims

1. The monoclonal antibody for hantavirus detection is characterized in that the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.2 and SEQ ID NO. 3.

2. A kit for detecting hantavirus, comprising an effective amount of the monoclonal antibody of claim 1.

3. The kit according to claim 2, wherein the kit is a colloidal gold detection test strip, and the test strip comprises a latex pad, a nitrocellulose membrane, a water absorbing paper and a PVC back plate which are prepared by polyclonal antibodies; the nitrocellulose membrane comprises a detection line prepared from monoclonal antibodies and a quality control line prepared from goat anti-rabbit IgG.

4. A kit for detecting hantavirus antibodies, which is characterized by comprising an effective amount of the monoclonal antibody of claim 1 and hantavirus NP protein.

5. The kit according to claim 4, wherein the kit is a colloidal gold detection test strip, and the test strip comprises a latex pad, a nitrocellulose membrane, a water absorbing paper and a PVC back plate which are prepared from Hantavirus NP protein; the nitrocellulose membrane comprises a detection line prepared from Hantavirus NP protein and a quality control line prepared from monoclonal antibodies.

6. A kit for dual detection of hantavirus by antigen-antibody, wherein the kit comprises the kit of claim 3 and the kit of claim 5.

7. Use of the monoclonal antibody of claim 1 for preparing a hantavirus detection reagent.