CN117214434B - HBV PreS1+PreS2 region protein-based typing kit and preparation method and application thereof - Google Patents
HBV PreS1+PreS2 region protein-based typing kit and preparation method and application thereof Download PDFInfo
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention provides an HBV typing kit, a preparation method and application thereof. The kit comprises a B19 antibody which is a detection antibody of the B genotype of the hepatitis B virus and/or a C04 antibody which is a detection antibody of the C genotype, wherein the variable region sequence of the B19 antibody is SEQ ID NO. 1 and SEQ ID NO. 2; the variable region sequences of the C04 antibody are SEQ ID NO. 3 and SEQ ID NO. 4; the preparation method of the kit comprises the preparation of B19 antibody and/or C04 antibody; the application is HBV B genotype and HBV C genotype identification. The kit provided by the invention can directly use a serum sample for detection, simplifies the procedure of hepatitis B virus typing diagnosis, and has high detection accuracy.
Description
Technical Field
The invention belongs to the field of biomedical inspection, and particularly relates to a HBV PreS1+PreS2 region protein typing kit, a preparation method and application thereof.
Background
Hepatitis b is a disease caused by Hepatitis B Virus (HBV) infection. Studies show that the effect of antiviral treatment on hepatitis B patients is closely related to the genotype of the virus in the infected person. HBV genotypes are associated with viral replication, variation, clinical disease profile, disease prognosis and antiviral efficacy. The genotyping of the HBV infected person can effectively guide the clinical accurate medication and forecast the antiviral treatment effect of the patient. HBV is divided into nine genotypes A-I according to the variability of the hepatitis B virus gene sequence, and the genotypes of the hepatitis B virus infected by different regional groups are different. With the development of modern society, the population migration frequency and the activity range are continuously enlarged, the cross infection trend is increased, the medical drug can not judge the genotype by the geographic position alone, and certain medical detection means are applied to reagent diagnosis.
The main method for detecting HBV genotypes at the present stage is based on a PCR amplification technology, and one is to utilize a common PCR technology to carry out full-length sequencing after amplifying HBV genomes, and determine the genotypes of HBV to be detected by comparing the HBV genotypes with known sequences, and is also a gold standard for current genotype judgment. The HBV genotyping technology based on PCR amplification is characterized in that viral nucleic acid is extracted from serum/plasma of an infected person, then PCR reaction liquid is prepared for amplifying HBV sequences, sequencing is carried out by sending the amplified genes to a sequencing company for sequencing after a common PCR method, and the genotype of the virus can be determined after comparison. The other HBV genotype detection method is a probe fluorescence PCR-based method, and HBV with different genotypes is distinguished by setting HBV genotype specific primers and probes to amplify HBV with specific genotypes. The fluorescent quantitative PCR detection by the probe method can directly judge the genotype according to the CT value of an amplification curve, but the operations such as centrifugal boiling and the like are involved in the nucleic acid extraction process, and special equipment such as a clean workbench, a PCR amplification instrument and the like are used, so that detection pollution of different samples is extremely easy to cause, false positive results are generated, and 8 hours are required from the extraction of a nucleic acid sample to the acquisition of the detection results.
The two typing methods depend on quantitative PCR or a common PCR instrument and sequencing equipment, and because the PCR amplification sensitivity is high, the base sequence is easily amplified by mistake, the accurate typing result cannot be obtained, and the judgment of the B genotype and C genotype co-infection condition by the PCR amplification technology cannot be realized, so that the difficulty of judging the result is increased. In addition, the PCR detection method has the requirement on the DNA load of the sample, the minimum detection limit is 1X 10 3 IU/mL, and the sample with the DNA load smaller than 1X 10 3 IU/mL cannot be accurately detected, so that the HBV typing detection is greatly limited from the DNA level. Although the antibody method has also been proposed, the 6H3 antibody (obtained based on HBV PreS2 region) used therein lacks specificity for C genotype recognition, and the antibody also exhibits a certain recognition ability for HBV D genotype, but the poor specificity makes the detection method and discrimination method complicated and error-prone. Therefore, developing a hepatitis B virus typing method which is simple and convenient to operate, short in time consumption and low in cost has a necessary significance for assisting clinical diagnosis.
The Chinese patent with publication number CN101560576A discloses a specific probe for identifying HBV genotype and a method and a kit for detecting HBV genotype by using the specific probe, and the process comprises the steps of preparing specific primers and specific probes, extracting PCR templates, carrying out PCR, carrying out fluorescent PCR reaction, judging results and the like, which relate to operations such as PCR and the like, and use special equipment such as a clean workbench, a PCR amplification instrument and the like, and the steps are complicated, time-consuming and labor-consuming.
Chinese patent publication No. CN107475444a discloses a PCR primer, kit and method for selectively amplifying RNA from total nucleic acid of hepatitis b virus. Comprises PCR preparation of HBV virus RNA standard, extraction of HBV total nucleic acid from patient serum comprising DNA and RNA, quantitative PCR detection. The invention extracts DNA and RNA from serum of patient, uses multiple PCR amplification, and the PCR amplification has the defects or disorder of amplified sequences under normal conditions, and the multiple PCR amplification is time-consuming and labor-consuming, and affects the accuracy of result measurement.
The serological ELISA detection method for detecting pathogenic microorganism antigens and antibodies in serum is a common detection method, is simple and convenient to operate, can directly use a serum sample of a person to be detected for detection, and can finish detection within 2.5h, and the HBV genotyping accuracy reaches more than 95%. Development of antibodies for serological enzyme-linked immunosorbent assay is helpful to promote clinical application thereof.
Disclosure of Invention
In order to solve the problems, the technology for carrying out HBV genotyping identification based on an ELISA method is developed, a monoclonal antibody which specifically recognizes the genotype of the hepatitis B virus B, C is obtained through screening, and an ELISA kit is prepared by utilizing the monoclonal antibody with high specificity and sensitivity, so that the detection by directly utilizing a serum sample of a patient is realized. Compared with nucleic acid detection, the method omits the extraction process, greatly simplifies experimental operation and avoids pollution possibly caused in the nucleic acid extraction process. Meanwhile, the kit can be directly implemented simultaneously with the existing five hepatitis B detection, and reduces the workload of clinical detection. The success of the kit simplifies the procedure of hepatitis B virus typing diagnosis, increases the willingness of doctors to typing detection, enables the doctors to timely and comprehensively know the condition of patients, and promotes the accurate medication of hepatitis B treatment.
The invention provides an HBV typing kit, a preparation method and application thereof.
In one aspect, the invention provides an application of a hepatitis B virus B genotype detection antibody and/or a hepatitis B virus C genotype detection antibody in preparing an HBV protein typing kit.
Specifically, the hepatitis B virus B genotype detection antibody is a B19 antibody, the heavy chain variable region of the B19 antibody comprises SEQ ID NO. 1, and the light chain variable region comprises SEQ ID NO. 2; the hepatitis B virus C genotype detection antibody is a C04 antibody, the heavy chain variable region of the C04 antibody comprises SEQ ID NO. 3, and the light chain variable region comprises SEQ ID NO. 4.
Specifically, the HBV protein typing is based on HBV PreS1+PreS2 regional protein typing, and the detection step comprises the following steps:
s1, diluting a coated antibody with PBS, adding the diluted coated antibody into an ELISA plate 1, and incubating;
s2, adding serum of a sample to be detected into the ELISA plate 1, incubating and washing to obtain an ELISA plate 2;
S3, coupling the B19 antibody and/or the C04 antibody with HRP, adding the HRP into the ELISA plate 2, incubating and washing to obtain an ELISA plate 3;
S4, adding TMB solution into the ELISA plate 3, and stopping the reaction after incubation to obtain the ELISA plate 4, and detecting the OD value of each hole of the ELISA plate 4 at the dual wavelengths of 450nm and 630 nm.
Preferably, the coated antibody comprises either or both of a 16D12 antibody or a 6H3 antibody.
Specifically, the detection concentration of the B19 antibody is 2-4 mug/mL, and the detection concentration of the C04 antibody is 1-3 mug/mL.
Preferably, the detection concentration of the B19 antibody is 3 mug/mL, and the detection concentration of the C04 antibody is 2 mug/mL.
Specifically, the coating concentration of the 16D12 antibody is 3-5 mug/mL; the coating concentration of the 6H3 antibody is 2-4 mug/mL.
Preferably, the coating concentration of the 16D12 antibody is 4 mug/mL; the coating concentration of the 6H3 antibody is 3 mug/mL.
Specifically, the detection sample is serum.
In another aspect, the invention provides a HBV protein typing detection kit.
Specifically, the HBV protein typing detection kit comprises: a hepatitis B virus B genotype detection antibody and/or a hepatitis B virus C genotype detection antibody; the hepatitis B virus B genotype detection antibody is a B19 antibody, the heavy chain variable region of the B19 antibody comprises SEQ ID NO. 1, and the light chain variable region comprises SEQ ID NO. 2; the hepatitis B virus C genotype detection antibody is a C04 antibody, the heavy chain variable region of the C04 antibody comprises SEQ ID NO. 3, and the light chain variable region comprises SEQ ID NO. 4.
Specifically, the B19 antibody and the C04 antibody are labeled with peroxidase. Preferably, the peroxidase comprises horseradish peroxidase.
Specifically, either one or both of the 16D12 antibody or the 6H3 antibody is also included as a coating antibody.
Specifically, the kit also comprises a sample diluent, a concentrated washing solution, a stop solution, a quality control product or a standard product.
Specifically, the quality control product or standard product comprises fusion proteins, wherein the fusion proteins are HBV B genotype and C genotype fusion proteins.
Specifically, the kit detection sample is serum.
In a further aspect, the invention provides the use of a kit as described above.
Specifically, the HBV protein typing detection kit is used for HBV B genotype and HBV C genotype identification.
In yet another aspect, the invention provides a method of preparing the above kit.
Specifically, the preparation of B19 antibody and/or the preparation of C04 antibody are included.
In yet another aspect, the invention provides a hepatitis B virus B genotype detection antibody.
Specifically, the hepatitis B virus B genotype detection antibody is a B19 antibody, and the heavy chain variable region of the B19 antibody comprises SEQ ID NO. 1; the light chain variable region of the B19 antibody comprises SEQ ID NO. 2.
In yet another aspect, the invention provides a hepatitis b virus genotype C detection antibody.
Specifically, the hepatitis B virus C genotype detection antibody is a C04 antibody, and the heavy chain variable region of the C04 antibody comprises SEQ ID NO. 3; the light chain variable region of the C04 antibody comprises SEQ ID NO. 4.
The invention has the beneficial effects that:
the invention provides an HBV typing kit, a preparation method and application thereof. The kit provided by the invention can directly use a serum sample for detection, simplifies the procedure of hepatitis B virus typing diagnosis, and has high detection accuracy.
Drawings
FIG. 1 is an electrophoresis chart of an extracted DNA template.
FIG. 2 is a pET-28a-HBV BPreS1+PreS2+S recombinant plasmid map.
FIG. 3 is a map of pET-28a-HBV CPreS1+PreS2+S recombinant plasmid.
FIG. 4 shows the enzyme digestion electrophoresis.
FIG. 5 is a SDS-PAGE pattern of HBV genotype B and genotype C proteins after purification.
FIG. 6 is a diagram showing HBV genotype B/genotype C protein-specific assay WB.
FIG. 7 is a diagram showing alignment of HBV C genotype and D genotype amino acid sequences.
FIG. 8 is a flow chart of development and preparation of a protein typing kit and performance evaluation thereof.
FIG. 9 is an ELISA plate coating diagram.
Fig. 10 is a loading layout.
FIG. 11 is a graph showing the results of blood sample testing.
FIG. 12 shows the results of three typing methods.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
Main instrument, consumable: full-automatic enzyme-labeled plate washer (model: YF-SS-097), vacuum packaging machine (model: SC-SS-014), peristaltic pump (split charging machine) (model: SC-SS-017), dehumidifier (model: SC-SS-013), electronic precision balance (model: FM-SB-001), incubator (model: YF-SS-088) single-function enzyme-labeled instrument (model: cmax Plus), electronic constant-speed stirrer (model: BM-SB-062), electrothermal constant-temperature culture (DHP-9032B), ETC811 PCR instrument purchased from Soviet, kyowa scientific instruments, high-speed centrifuge purchased from Hunan instrument laboratory instrument development Co, DYY D electrophoresis instrument purchased from Beijing six biotechnology Co., bio-Rad gel imaging analysis system purchased from Bio-Rad, refrigerator, 10 μL-1000 μL model pipettor 30-300 μL 8 channel pipettor the like; 96-hole ELISA plate, cover plate film, packing tube, packing bottle, label paper, packing box, etc. Coli DH5 alpha and BL21 (DE 3) were purchased from Optimazaceae, and the expression vector pET-28a was purchased from Ding Guo Changchun Biotechnology responsibility Co., ltd (model MCV 012) and stored in Beijing An Bi Qie Biotechnology Co., ltd. Viral genome DNA/RNA extraction kit, DNA gel recovery kit (DNA Gel Extraction Kit), QIAPREP SPIN MINIPREP KIT (50) centrifugation plasmid miniextraction kit, restriction enzymes purchased from root (beijing, china). 2X Hieff. HotStart PCR Genotyping Master Mix (With Dye) PCR premix solutions were purchased from the Aleurites. 10X Cutsmart Buffer and 2X Ligation solution were purchased from Beauzebo. Horseradish peroxidase-HRP was purchased from the loyang baioto experimental materials center. Agarose, kanamycin, acrylamide, bisacrylamide, TEMED and isopropyl beta-D1-thiogalactoside (IPTG) were purchased from Sigma (St.Louis, MO, USA). DNA MARKER protein markers were purchased from soribao. Sodium chloride, potassium chloride, disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, tween-20, proclin-300, sucrose, concentrated sulfuric acid, coomassie brilliant blue, 0.2 μm filter, 0.45 μm filter were purchased from national pharmaceutical chemicals company (Sinopharm Chemical reagent co., ltd). Horse serum was purchased from israel BI company. Bovine serum albumin was purchased from Bovogen Biologicals Pty Ltd. Carmine was purchased from Beijing precision chemical Co. TMB is available from Mo De Biotechnology Inc. of Peking Meinaceae.
The 16D12 antibody and the 6H3 antibody are purchased from Abelmoschus manihot and have the product numbers M10513 and M10514 respectively.
Example 1 construction and preparation of quality control
1. Preparation of DNA fragment of target Gene
1.1 Primer design
DNA templates are extracted from serum of hepatitis B genotype B patients by using a DNA extraction kit, and amplification is carried out.
According to the sequence in Genbank: AB819611.1 design HBV B genotype upstream primer FB (SEQ ID NO: 9): CGCCATATGGCGATGGGGACAAATCTTTCTGTCCCC (the underlined part is the cleavage site, nedI endonuclease is used, the italic part is the protecting base, and the rest is the matable primer);
According to the sequence in Genbank: AB298720.1 design HBV C genotype upstream primer FC (SEQ ID NO: 10): CGCCATATGGCGATGGGGACGAATCTTTCTGTTCC (the underlined part is the cleavage site, nedI endonuclease is used, the italic part is the protecting base, and the rest is the matable primer);
Through sequence alignment, a downstream primer R (SEQ ID NO: 11) comprising HBV B genotype and HBV C genotype of the same base sequence was designed: CAAGCTTGAAGCCCTACGAACCACTGAACA (HindIII endonuclease is used in the underlined part, protective base in the italic part, and matable primer in the rest).
1.2 Hepatitis B virus DNA extraction
The test method extracts the viral genome DNA in the serum of the hepatitis B patient according to the use instruction of the DNA extraction kit. Taking 50 mu L of serum, respectively adding 200 mu L of Buffer VGB, 20 mu L of protease K and 1.0 mu LCARRIER RNA, fully mixing, and carrying out water bath at 56 ℃ for 10min; 200 mu L absolute ethyl alcohol is added into the lysate, and the mixture is fully sucked and uniformly mixed. Placing adsorption Column (Spin Column) in Collection Tube (Collection Tube), transferring the solution into adsorption Column, centrifuging at 12000rpm for 2min, and discarding the filtrate; adding 500 μl Buffer RWA, centrifuging at 12000rpm for 1min, and discarding the filtrate; adding 700 μl Buffer RWB, centrifuging at 12000rpm for 1min, and discarding the filtrate; placing the adsorption column on a collecting pipe, and centrifuging at 12000rpm for 2min; placing the adsorption column in new 1.5mL RNase free ddH 2 O, standing at room temperature for 5min; centrifuging at 12000rpm for 2min, and eluting DNA.
1.3 Target fragment amplification
PCR conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 1min, renaturation at 55℃for 30s, extension at 72℃for 1min, 36 cycles of amplification; extending at 72deg.C for 3min, and storing the product at 4deg.C for detection. The PCR reaction system is shown in Table 1. After PCR amplification, 5. Mu.L of the PCR product was taken and the success or failure of PCR amplification was verified by agarose gel electrophoresis at 12 g/L. The amplified DNA was subjected to gel electrophoresis and photographed as shown in FIG. 1.
TABLE 1 PCR reaction system
Construction and verification of pET-28a-HBV BPres1+Pres2+S recombinant plasmid and pET-28a-HBV CPres1+Pres2+S recombinant plasmid
2.1 Double enzyme cutting connection of target fragment and carrier fragment
The target gene obtained is cut and recovered 1 from agarose gel by using a gel recovery kit, and the target fragment is recovered by performing double enzyme digestion with NedI and HindIII overnight and running gel; extracting the pET-28a vector by using a radix angelicae fast plasmid small extraction kit, referring to a kit instruction, carrying out double enzyme digestion on NedI and HindIII for 16 ℃ overnight, carrying out gel running to recover a target vector fragment, mixing and connecting the target fragment and the pET-28a target vector fragment for 16 ℃ overnight, wherein the molar ratio of the target fragment to the pET-28a target vector fragment is 3:1. the double cleavage system and ligation system are shown in tables 2 and 3. The plasmid map is shown in FIGS. 2 and 3.
Table 2 double enzyme digestion System
Table 3 connection system
2.2 Cloning transformation of recombinant plasmids
The pET-28a-HBV B PreS1+PreS2+S recombinant plasmid and pET-28a-HBV C PreS1+PreS2+S recombinant plasmid are respectively transformed into E.coli Ecoli DH5 alpha-coated plate for culture, and single colony is selected and inoculated into LB solid medium containing 50mg/L Kanamycin (KANAMYCIN) for culture overnight at 37 ℃. The activated single colony is picked from the LB plate and inoculated into 3-5mL LB liquid medium, and the culture is carried out for about 12 hours at 37 ℃ in a shaking way until the late logarithmic growth phase. The bacterial suspension was mixed with 1:100 were inoculated into 100mL of LB liquid medium, and cultured at 37℃for 3 hours with shaking until OD600 = 0.5. Centrifuging at 4 ℃,2700rmp for 10min, discarding the supernatant, and collecting bacterial precipitate.
2.3 Sequencing and enzyme digestion identification of products
The bacteria obtained in 2.2 were extracted using a plasmid extraction kit, and PCR was performed using the plasmid as a template, with the primer removing the protecting base and the cleavage site, and the reaction system and conditions were the same as those in 1. Sequencing the PCR product in Shanghai, and the result shows that the obtained DNA sequence has 98% consistency with HBV BPreS1+PreS2+ SDNA and HBV CPreS1+PreS2+ SDNA reported in GenBank; nedI and HindIII, FIG. 4 shows an electrophoresis diagram of double digestion identification (in FIG. 4, M represents a protein Marker; B represents pET-28a-HBV BPreS1+PreS2+S recombinant plasmid digestion; C represents HBV C pET-28a-HBV BPreS1+PreS2+S recombinant plasmid digestion), the vector part is about 5000bp, and the target genes are about 1050bp respectively, which indicates that enzyme digestion sites are successfully introduced to both ends of HBV BPreS1+PreS2+S fragments, and the recombinant plasmid construction is successful.
3. Protein-induced purification and specificity identification
3.1 Protein-induced purification
The recombinant plasmid verified by DNA sequencing is transformed into escherichia coli BL21 (DE 3), a positive single colony containing the recombinant prokaryotic expression vector is selected and inoculated into LB liquid medium containing kanamycin (containing 50mg/L kanamycin), and the culture is carried out at 37 ℃ overnight. 1% of the culture medium was inoculated into fresh LB liquid medium, cultured at 37℃under shaking at 200r/min until the OD600 was about 1.0, and then added with lactose IPTG to a final concentration of 0.3mM, followed by induction at 16℃for 9 hours. Centrifugation is carried out at 4 ℃ and 10000r/min for 10min, bacterial precipitation is collected, SDS-PAGE gel electrophoresis is carried out to identify coomassie brilliant blue staining, ni 2+ -NTA affinity chromatography purification is carried out on fusion proteins, HBV BPreS1+PreS2+S-His fusion proteins and HBV CPreS1+PreS2+S-His fusion proteins which are successfully expressed are purified, and the results are shown in a graph in FIG. 5 (in FIG. 5, M represents a protein Marker;1 represents a protein before purification; B represents purified HBV BPreS1+PreS2+S-His fusion proteins; C represents purified HBV CPreS1+PreS2+S-His fusion proteins). In FIG. 5, the protein size was around 42KD, and the protein expression was successful.
3.2 Specificity assay of fusion proteins
The HBV BPreS1+PreS2+S-His fusion protein and HBV CPreS1+PreS2+S-His fusion protein separated by PAGE are taken as antigens, transferred onto a solid phase carrier, after skim milk is blocked, secondary antibodies (product number: se 131) purchased from Sorebate are added after 12H washing treatment of incubation at 6H3,4 ℃ for identifying HBV B genotype coated antibody 16D12 and C genotype coated antibody, and the result is shown in FIG. 6 (A) for 16D12 antibody incubation, (B) for 6H3 antibody incubation, M for protein Marker, B for B genotype coated protein, C for C genotype coated protein, and FIG. 6 shows that the B genotype and C genotype coated proteins are respectively combined with 16D12 antibody and 6H3 antibody, and the fusion protein specificity is good.
The HBV B genotype fusion protein antigen DNA sequence is SEQ ID NO. 12; the HBV C genotype fusion protein antigen DNA sequence is SEQ ID NO. 13; the HBV B genotype fusion protein antigen amino acid sequence is SEQ ID NO. 14; the HBV C genotype fusion protein antigen amino acid sequence is SEQ ID NO. 15.
Example 2 detection antibody preparation and screening
1. Preparation of detection antibodies
Monoclonal antibodies were prepared by immunizing mice with the key epitope P94 peptide fragment as detection antibodies. P94B and P94C genotype peptides (SEQ ID NO:16 PASTNRQSGRQPTPLSPPLRDTHP;SEQ ID NO:17 PASSNRQSGKQPTPISPPLRDSHP, respectively) were synthesized, followed by coupling of KLH and BSA, respectively. P94B-KLH and P94C-KLH polypeptides conjugated to P94B and P94C of KLH will be used to immunize two groups of animals, respectively, while BSA conjugated complexes will be used to screen to exclude antibody responses against KLH, eventually obtaining 10B-genotype antibodies and 6C-genotype antibodies.
2. Screening of detection antibodies:
The 16D12 antibody is selected as a coating antibody of the B genotype fusion protein, and the 6H3 antibody is selected as a coating antibody of the C genotype fusion protein. HBV B genotype and C genotype fusion proteins were added separately and then detected with 10B genotype antibodies and 6C genotype antibodies, respectively. The results of the coated antibody and detection antibody paired screening experiments are shown in Table 4. Finally, B19 antibody (the heavy chain variable region sequence is SEQ ID NO. 1, the light chain variable region sequence is SEQ ID NO. 2, the heavy chain variable region gene sequence is SEQ ID NO. 5, and the light chain variable region gene sequence is SEQ ID NO. 6) is selected as a B genotype detection antibody; the C04 antibody (the heavy chain variable region sequence is SEQ ID NO:3, the light chain variable region sequence is SEQ ID NO:4, the heavy chain variable region gene sequence is SEQ ID NO:7, and the light chain variable region gene sequence is SEQ ID NO: 8) is used as a C genotype detecting antibody.
TABLE 4 screening table for coated antibodies paired with detection antibodies
| B genotype detection antibody | OD value | C genotype detection antibody | OD value |
| B01 | 2.093 | C01 | 1.392 |
| B02 | 1.449 | C02 | 1.033 |
| B04 | 1.435 | C04 | 2.432 |
| B06 | 1.673 | C05 | 1.543 |
| B07 | 1.739 | C06 | 0.621 |
| B09 | 1.773 | C08 | 2.115 |
| B19 | 1.845 | ||
| B27 | 1.582 | ||
| B28 | 1.421 | ||
| B32 | 1.684 |
The HBV C genotype detecting antibody was prepared in this laboratory using HBV PreS1 region (94-117) in which the amino acid sequences of the C genotype and the D genotype were greatly different, as shown in FIG. 7. The paired screening experiment results show that the prepared antibody has better specificity, and overcomes the defect of the capability of 6H3 for identifying HBV C genotype specificity.
Example 3 development and preparation of kits
1. Main solution of finished product kit
The research and development preparation of the protein typing kit and the performance evaluation flow chart are shown in figure 8, and the optimal conditions are obtained through multiple experimental screening in each step of flow. The specific components are prepared as follows:
1) And (3) preparing a coating liquid: 8.0g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium dihydrogen phosphate and 0.2g of potassium chloride were weighed, dissolved to a constant volume of 1L by using ddH 2 O to form a1 XPBS coating buffer, and the coated antibodies 16D12 and 6H3 were diluted to 4. Mu.g/mL to 3. Mu.g/mL by using 1 XPBS, respectively, to form a coating solution.
2) Sealing liquid: 8.0g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 2.0g of BSA, 80.0mL of horse serum, 1.0mL of Tween-20, 300.0 mL of Proclin and 40g of sucrose are fully dissolved by ddH 2 O, and the volume is fixed to 1L to form a sealing liquid.
3) Sample dilution: the solution was sufficiently dissolved with ddH 2 O to a volume of 1L to form a sample diluent from 11.0g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium dihydrogen phosphate, 0.2g of potassium chloride, 2.0g of BSA, 40.0mL of horse serum, 1.0mL of Tween-20, 300.0 mL of Proclin, and 0.02g of phenol red.
4) Enzyme-labeled detection antibody dilution: is formed by fully dissolving 11.0g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of monopotassium phosphate, 0.2g of potassium chloride, 2.0g of BSA, 80.0mL of horse serum, 1.0mL of Tween-20 and 1.0mL of Proclin300 by ddH 2 O, and fixing the volume to 1L.
5) 10 X concentrated wash: a10 Xconcentrated washing solution was prepared from 80g of sodium chloride, 29g of disodium hydrogen phosphate dodecahydrate, 2g of potassium dihydrogen phosphate, 2g of potassium chloride, 5.0mL of Tween-20 and the like, and was dissolved thoroughly with ddH 2 O to a volume of 1L.
6) TMB color development liquid: the TMB solution is purchased from Beijing Mei Kemo De Biotechnology Co., ltd.
7) Stop solution: dilute sulfuric acid from concentrated sulfuric acid with ddH 2 O to 2M.
2. Preparation flow of kit
1) Preparation of a pre-coated ELISA plate (HBV B/C reaction plate): the coating liquid of the step 1 is added into 96-well ELISA plates at intervals of 100 mu L/well, incubated overnight at 4 ℃, the ELISA plates are shown in figure 9 in a coating layout, and the coating plates are detachable ELISA plates, and can be optionally selected from one of 2 layouts shown in figure 9 according to the needs of operators.
2) Washing an ELISA plate: 10 Xconcentrated washing solution, 9 times ddH 2 O was added to dilute to 1 Xwashing solution, 350. Mu.L of washing solution was added to each well of 1 Xwashing solution, washing was repeated 3 times, and drying was performed.
3) And (3) sealing the ELISA plate: the blocking solution prepared by the method is added with 180 mu L of blocking solution according to each hole, and incubated for 2 hours at 37 ℃ to block the ELISA plate.
4) Washing an ELISA plate: the method steps are the same as 2).
5) Drying and vacuum packaging the ELISA plate: placing the washed ELISA plate in an incubator with humidity less than 30%, and sufficiently drying at 22-28deg.C for 12-16 hr; after drying, placing the dried product into a tin foil bag, vacuumizing the tin foil bag by a vacuum packaging machine, sealing and packaging, and storing the packaged ELISA plate at 2-8 ℃.
6) Sample diluent, concentrated washing solution, detection antibody, substrate reaction solution, and stop solution are prepared and split-packed:
① And subpackaging the sample diluent according to 6.0mL per bottle to form a finished diluent.
② HBV genotype B/genotype C enzyme conjugate: coupling HRP with detection antibodies B19 and C04 of HBV B genotype and C genotype screened in example 2 to obtain B19-HRP and C04-HRP, diluting B19-HRP to 3 μg/mL with detection antibody diluent, diluting C04-HRP to 2 μg/mL, and sub-packaging according to 6 mL/bottle respectively.
③ 10X concentrated washing solution according to 50.0 mL/bottle split charging 10X concentrated washing solution.
④ Substrate solution: the color-developing solution TMB purchased by Beijing Mei Kemo De Biotechnology Co., ltd was prepared by sub-packaging 12.0 mL/bottle.
⑤ Stop solution: after diluted to 2M with ddH 2 O, the mixture was dispensed in 6.0 mL/bottle.
⑥ HBV genotype B and genotype C quality control solutions (HBV type B and type C positive controls): the HBV B genotype and C genotype fusion protein antigen purified in example 1 was diluted to 5. Mu.g/mL with the sample dilution, and dispensed into the cryopreservation tube at 0.6 mL/tube.
7) And (3) assembling a kit: the pre-coated ELISA plates (HBV B/C type reaction plates), sample dilutions, HBV B genotype and C genotype enzyme conjugates, HBV B genotype and C genotype quality control solutions (HBV B type and C type positive controls), concentrated washes (10X), substrate solutions, stop solutions, and quantitative sets were assembled into complete kits as shown in Table 5.
TABLE 5 major composition of kit
Example 4 sample detection
1) Reagent preparation: diluting the 10X concentrated washing solution with ddH 2 O to 1X washing solution for later use; the HBV B/C type reaction plate and the rest of the solution except HBV B/C genotype enzyme conjugate in the kit were taken out from the refrigerator at 4℃to equilibrate to room temperature.
2) Loading: serum samples of 10 HBV B genotype B patients (numbered P1-P10) and 10 HBV C genotype B patients (numbered P11-P20) which had been subjected to PCR sequencing were examined. Adding HBV B genotype and C genotype quality control solutions (HBV B type and C type positive control) to B, C genotype pre-coated ELISA plates according to 100 μl, repeating the double-hole sample application as shown in figure 10 (B and C in the transverse direction respectively represent B, C genotype antibody; the first row in the longitudinal direction only represents serial number; QB represents B genotype quality control; QC represents C genotype quality control; P+ number represents sample serial number; black represents blank), adding 50 μl sample diluent to the rest of each hole, respectively taking 50 μl of serum samples (serial numbers P1-P20) to be tested, sequentially adding into HBV B/C type reaction plates, and incubating at 37deg.C for 60min.
3) Washing an ELISA plate: after the incubation of the sample, the sample was washed 5 times repeatedly with 1 Xof the washing solution at 350. Mu.L of the washing solution per well, and then the sample was dried by pipetting.
4) Incubation of enzyme-labeled detection antibody: HBV genotype B enzyme conjugate and HBV genotype C enzyme conjugate were added to the corresponding B, C enzyme-labeled strip at 100. Mu.L/well, respectively, and incubated at 37℃for 60min.
5) Washing an ELISA plate: step 3).
6) TMB color development: TMB solution was added to all wells of the microplate in an amount of 100. Mu.L/well and incubated at 37℃for 10min.
7) Terminating the reaction: after TMB development, 2M dilute sulfuric acid was added to the wells of the enzyme-labeled plate in an amount of 50. Mu.L/well, and the mixture was gently mixed by shaking to terminate the substrate reaction.
8) Results determination: after the reaction was terminated, the OD value of each well of the post-termination ELISA plate was measured using a dual wavelength of 450nm and a reference of 630 nm. The HBV B genotype quality control and the HBV C genotype quality control both show good specificity, and 20 serum results are shown in FIG. 11, and the typing is successful.
9) Analysis and interpretation of results:
① And (3) quality control of the kit: the detection OD value of the HBV B genotype quality control product on the B genotype enzyme label strip and the C genotype enzyme label plate is larger than 1.0 and smaller than 0.10 respectively, and the detection OD value of the HBV C genotype quality control product on the B genotype enzyme label strip and the C genotype enzyme label plate is smaller than 0.10 and larger than 1.0 respectively, otherwise, the detection is ineffective.
② When the detection OD value of the serum sample to be detected on the B genotype strip is greater than 0.15, the sample to be detected is judged to be infected with HBV B genotype virus; when the detection OD value of the serum sample to be detected on the C genotype strip is greater than 0.15, the sample to be detected is judged to be infected with HBV C genotype virus; if B, C lath detection values are simultaneously larger than 0.15, the sample to be detected is HBV B and C genotype mixed infection.
Comparative example 1
The serum of 135 patients suffering from hepatitis B is detected by three methods of gold standard sequencing typing, protein typing kit typing and Shanghai river PCR typing kit typing, 56 serum is obtained from Fuzhou infectious disease hospital, 79 serum is obtained from Jilin university white-board first hospital.
At present, the quantitative detection result of HBV surface antigen (HbsAg) is positive with the DNA loading of >50 IU/mL. The detection limit of the Shanghai river kit is 1000IU/mL.
After grouping 135 samples, three methods were used simultaneously for typing, and the typing results are shown in Table 6.
Table 6 results of serotyping 135 hepatitis B patients
For 77 samples with DNA load of >1000IU/mL and HBV HbsAg of >250IU/mL, the protein typing kit has higher sensitivity, the detection rate reaches 98.70 percent (76/77), the gold standard sequencing typing detection rate is 93.51 percent (72/77), and the detection rate of the Shanghai river PCR typing kit is 92.22 percent (71/77). For samples with DNA loading of 50-1000IU/mL, the two methods of gold standard sequencing typing based on PCR amplification and the Shanghai river PCR typing kit typing obviously show defects, the typing detection rate is only 36.36% (8/22) and 27.27% (6/22), and the detection rate of the protein typing kit reaches 100%; for 17 samples with DNA loading <50IU/mL, the detection rate of the protein typing kit reaches 88.24% (15/17), and the other two methods completely fail.
For 77 samples with DNA loading of >1000IU/mL and HBV HbsAg of >250IU/mL, the results of the three typing methods are shown in FIG. 12, and the B genotypes are 25, 28 and 27 cases and the C genotypes are 46, 44 and 38 cases respectively, which are measured by gold standard sequencing typing, protein typing kit typing and Shanghai river PCR typing kit typing. The golden standard sequencing typing can not detect B, C genotype mixed infection patient serum, the protein typing kit detects 4 cases B, C genotype mixed infection patient serum, the Shanghai river typing kit detects 6 cases B, C genotype mixed infection patient serum, and the high proportion of mixed infection can be caused by the fact that the DNA extraction step of the Shanghai river typing kit is easy to be interfered by external environment and easy to cause false positive. The reason for this analysis may be that the typing probes are designed to have a certain degree of similarity, which may lead to the possibility of multiple amplifications of DNA at the time of amplification.
The three parting consistencies are analyzed in the ICC group through SPSS software, and the consistency correlation coefficient is 0.775, so that the consistency is higher. The protein parting kit can be used as the supplement of two PCR parting methods and applied to HBV parting work to assist diagnosis and treatment of patients suffering from hepatitis B.
Claims (6)
1. The application of the hepatitis B virus B genotype detection antibody and/or the hepatitis B virus C genotype detection antibody in preparing HBV protein typing kit is characterized in that the hepatitis B virus B genotype detection antibody is a B19 antibody, the heavy chain variable region sequence of the B19 antibody is SEQ ID NO. 1, and the light chain variable region sequence of the B19 antibody is SEQ ID NO. 2; the hepatitis B virus C genotype detection antibody is a C04 antibody, the heavy chain variable region sequence of the C04 antibody is SEQ ID NO. 3, and the light chain variable region sequence of the C04 antibody is SEQ ID NO. 4.
2. The use according to claim 1, wherein said HBV protein typing is based on HBV pres1+pres2 region protein typing, the detection step comprising the steps of:
s1, diluting a coated antibody with PBS, adding the diluted coated antibody into an ELISA plate 1, and incubating;
S2, adding serum of a sample to be detected into the ELISA plate 1, and washing after incubation to obtain an ELISA plate 2;
s3, coupling the B19 antibody and/or the C04 antibody with HRP, adding the HRP into the ELISA plate 2, incubating and washing to obtain an ELISA plate 3;
S4, adding TMB solution into the ELISA plate 3, and stopping the reaction after incubation to obtain an ELISA plate 4, and detecting the OD value of each hole of the ELISA plate 4 at two wavelengths of 450nm and 630 nm;
The coating antibody comprises any one or two of a 16D12 antibody or a 6H3 antibody; the detection concentration of the B19 antibody is 2-4 mug/mL, and the detection concentration of the C04 antibody is 1-3 mug/mL; the coating concentration of the 16D12 antibody is 3-5 mug/mL, and the coating concentration of the 6H3 antibody is 2-4 mug/mL.
3. An HBV protein typing assay kit comprising: a hepatitis B virus B genotype detection antibody and/or a hepatitis B virus C genotype detection antibody; the hepatitis B virus B genotype detection antibody is a B19 antibody, the heavy chain variable region sequence of the B19 antibody is SEQ ID NO.1, and the light chain variable region sequence of the B19 antibody is SEQ ID NO. 2; the hepatitis B virus C genotype detection antibody is a C04 antibody, the heavy chain variable region sequence of the C04 antibody is SEQ ID NO. 3, and the light chain variable region sequence of the C04 antibody is SEQ ID NO. 4.
4. A method of preparing a kit according to claim 3, comprising the preparation of said B19 antibody and/or the preparation of said C04 antibody.
5. The hepatitis B virus B genotype detection antibody is characterized in that the hepatitis B virus B genotype detection antibody is a B19 antibody, the heavy chain variable region sequence of the B19 antibody is SEQ ID NO. 1, and the light chain variable region sequence of the B19 antibody is SEQ ID NO. 2.
6. The hepatitis B virus C genotype detection antibody is characterized in that the hepatitis B virus C genotype detection antibody is a C04 antibody, the heavy chain variable region sequence of the C04 antibody is SEQ ID NO. 3, and the light chain variable region sequence of the C04 antibody is SEQ ID NO. 4.
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| WO2008003236A1 (en) * | 2006-06-27 | 2008-01-10 | Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. | Method of jointly detecting hbv pres1 antigen and core antigen, test kit, solid substrate and virus lysis solution |
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| CN101586170A (en) * | 2009-07-06 | 2009-11-25 | 重庆医科大学 | Method and kits for detecting genotype of hepatitis B virus |
| CN112779290A (en) * | 2021-01-27 | 2021-05-11 | 北京安必奇生物科技有限公司 | Gene tandem recombinant plasmid of pre-S1 region of hepatitis B virus B, C genotype and construction method and application thereof |
| CN115160433A (en) * | 2022-06-28 | 2022-10-11 | 吉林大学 | Humanized HBV B and C genotype pre-S1 protein antibody and application thereof |
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