CN110540966B - Human haemophilus influenzae surface protein monoclonal antibody and antigen capture ELISA kit - Google Patents
Human haemophilus influenzae surface protein monoclonal antibody and antigen capture ELISA kit Download PDFInfo
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- CN110540966B CN110540966B CN201811564882.9A CN201811564882A CN110540966B CN 110540966 B CN110540966 B CN 110540966B CN 201811564882 A CN201811564882 A CN 201811564882A CN 110540966 B CN110540966 B CN 110540966B
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- haemophilus influenzae
- surface protein
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- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a human haemophilus influenzae surface protein monoclonal antibody and an antigen capture ELISA kit. The human haemophilus influenzae surface protein monoclonal antibody is prepared from the following components in percentage by mass: the hybridoma cell line of C2017218 was produced by secretion. The human haemophilus influenzae surface protein monoclonal antibody can be used for detecting human haemophilus influenzae. The invention also discloses a human haemophilus influenzae surface protein capture ELISA kit based on the monoclonal antibody.
Description
Technical Field
The invention belongs to the field of immunology, and relates to a human influenza haemophilus surface protein monoclonal antibody, a hybridoma cell strain and an antigen capture ELISA kit.
Background
Haemophilus influenzae (Haemophilus influenzae, hi) is abbreviated as influenza bacillus and belongs to the genus haemophilus. The bacteria of this genus must be grown by adding fresh blood or blood components during artificial culture, and are called haemophilus. There are generally six strains of haemophilus influenzae, designated as type a, type b (also known as type b), type c, type d, type e and type f. Diseases naturally occurring by haemophilus influenzae only occur in humans. In infants and young children, haemophilus influenzae b causes bacteremia and acute bacterial meningitis. Occasionally it causes cellulitis, osteomyelitis and joint infections. Since 1990, the prevalence of HiB-conjugated vaccines in the united states has been reduced to 1.3 childhood infections per hundred thousand children. However, hiB remains the leading cause of lower respiratory tract disease in infants and children in developing countries. In addition, non-subtype haemophilus influenzae without capsule also causes ear infections (e.g. otitis media), eye infections (conjunctivitis) and sinusitis in children and associated pneumonia, which strain has no corresponding vaccine application.
The existing method for detecting pathogenic microorganisms in respiratory tract mainly comprises the traditional method, namely a separation and identification method, wherein the method needs 2-3 days generally, and the method is difficult to meet the requirement of rapid identification; the PCR technology developed in recent years is a quick, sensitive and specific technology, but the technology still depends on the pre-enrichment step of the traditional method, and PCR inhibitors are often added in the enrichment liquid, so that the amplification effect is affected. Meanwhile, the technology also needs professional detection equipment, and is not suitable for bedside detection. Antibody-based immunological detection has become an indispensable important technical means for detecting pathogenic microorganisms in human body. Various specific immunoassay techniques have been developed such as Radioimmunoassay (RIA), enzyme Immunoassay (EIA), fluorescent Immunoassay (FIA), chemiluminescent Immunoassay (CIA), immunoprecipitation, immunoagglutination, ELISA assay kits, immune colloidal gold test strips, immune latex assay reagents, etc. The ELISA detection kit, immune colloidal gold test paper strip and other immunological detection techniques based on antibodies are important means for detecting pathogenic microorganisms. Therefore, research and development of antibodies against pathogenic microorganisms having independent intellectual property are the basis for development of ELISA detection methods, colloidal gold detection methods, and the like, which possess independent intellectual property.
The choice of the antigen component is critical for the specificity of the assay. In 1982, barenkamp et al, when typing 35 NTHi isolated from different specimens by SDS-PAGE, found that each strain had its own protein pattern, but a band with a molecular weight of about 16KD was present in all strains. In 1983 Munson RJ found that 3 Hib outer membrane proteins showed the same protein band spectrum of the main band after gel electrophoresis, and the 6 th main band of 16KD with the minimum molecular weight was named as P6.RFLP analysis result, which proves that the P6 gene and the adjacent fragments thereof are highly conserved. Studies have shown that the P6 protein is 100% homologous in amino acid and 97% -99% homologous in nucleotide. Its good conservation and surface accessibility make it a good target for antigen detection.
The research selects surface protein P6 with interspecific specificity as antigen, prepares monoclonal antibody with good specificity, and applies the monoclonal antibody to prepare human haemophilus influenzae ELISA detection kit.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides a human haemophilus influenzae surface protein monoclonal antibody and an antigen capture ELISA kit which have good specificity and have no cross reaction with other common pathogens of respiratory tract.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a hybridoma cell line producing a monoclonal antibody to a surface protein of haemophilus influenzae, comprising: the hybridoma cell strain for producing the human influenza haemophilus surface protein monoclonal antibody is preserved by China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: hybridoma cell line Hi-7# of C2017218.
A method for preparing a hybridoma cell strain for producing a human hemophilus influenzae surface protein monoclonal antibody, which is characterized by comprising the following steps: the preparation method comprises the following steps:
1) Taking recombinant human influenza haemophilus surface protein P6 as an antigen, immunizing a BALB/c mouse with the age of 8 weeks, and detecting the titer of antiserum by adopting an indirect ELISA method;
2) Resuscitating and culturing SP2/0 myeloma cells;
3) Preparing feeder cells;
4) Preparing an immune spleen cell suspension;
5) Preparing SP2/0 myeloma cell suspension;
6) Cell fusion;
7) Screening positive clones and cloning culture, taking human haemophilus influenzae thalli as coating antigen, and detecting by adopting an indirect ELISA method; determination of OD with an enzyme-linked detector 450 The P/N value is more than 2.1 and positive; selecting continuous positive cloning holes for 2-3 times of subcloning, and screening monoclonal hybridoma cells; performing amplification culture on the monoclonal hybridoma cells to obtain a cell culture solution supernatant containing the monoclonal antibodies; ELISA detection is carried out on the specificity of the screened antibodies by respectively coating the ELISA plates with respiratory tract pathogenic bacteria, and the ELISA plates are eliminatedMonoclonal antibodies that react positively with these pathogens, screening for acceptable cell lines; the respiratory tract pathogenic bacteria comprise human mycoplasma pneumoniae, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, legionella pneumophila, streptococcus pyogenes, staphylococcus aureus, human streptococcus pneumoniae, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida.
Preferably, the preparation process of the recombinant human haemophilus influenzae surface protein P6 in step 1) is as follows:
1.1 Clone expression of human haemophilus influenzae P6 gene
Bioinformatics analysis is carried out on the haemophilus influenzae surface protein P6 gene, the GC content, the codon preference, the mRNA secondary structure, the RNA unstable motif and the mRNA free energy stability are combined, the DNA coding sequence of the haemophilus influenzae surface protein P6 gene is optimized, meanwhile, an enzyme cutting site NdeI is introduced into the 5' of the haemophilus influenzae surface protein P6 gene, a termination signal TAA and an enzyme cutting site XhoI are introduced into the 3' end of the haemophilus influenzae surface protein P6 gene, and then the whole gene sequence is chemically synthesized and then connected to a vector pUC57, and the result is marked as P6'; carrying out double enzyme digestion on a vector pUC57 containing the artificially synthesized DNA fragment by NdeI and XhoI, and then recovering the target fragment for later use by a conventional method; simultaneously, ndeI and XhoI are adopted to carry out double enzyme digestion on a vector pET-28a (+) and the P6 'gene obtained after double enzyme digestion is connected into the pET-28a (+) vector according to a conventional method, and the E.coli TOP10 is transformed to construct a pET-P6' expression vector; enzyme digestion and sequence determination prove that the construction of the expression vector is correct; the vector expresses recombinant P6-His fusion protein;
1.2 Purification of human haemophilus influenzae P6 protein
The plasmid is extracted after the identification of correct positive clone bacteria is cultivated, and is transferred into competent E.coli BL21 (DE 3) plysS according to the conventional technology, after the transformation is completed, bacterial liquid is coated on LB plate containing 50 mug/mL kanamycin, and after the expression bacterial strain is screened according to the conventional method, the expression bacterial strain is cultivated and purified.
A human haemophilus influenzae surface protein monoclonal antibody, characterized in that: the human hemophilus influenzae surface protein monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell strain Hi-7# of claim 1.
A preparation method of a human haemophilus influenzae surface protein monoclonal antibody is characterized by comprising the following steps: the method comprises the following steps:
performing amplification culture on the positive monoclonal hybridoma cells obtained by screening; monoclonal antibody ascites is prepared by a conventional ascites in-vivo induction method, collected and purified by Protein A affinity chromatography to obtain the human haemophilus influenzae surface Protein monoclonal antibody Hi-7#.
The application of the human haemophilus influenzae surface protein monoclonal antibody in detecting human haemophilus influenzae, in particular to the application in preparing a reagent for detecting human haemophilus influenzae, and most particularly to the application in preparing a ELISA kit for detecting human haemophilus influenzae surface protein capture.
An ELISA kit for detecting human haemophilus influenzae surface protein capture, which is characterized in that: the ELISA kit for detecting the human haemophilus influenzae surface protein comprises an ELISA plate and a human haemophilus influenzae surface protein monoclonal antibody Hi-7#; the ELISA plate is a solid phase carrier coated with polyclonal antibody of human hemophilus influenza surface protein.
The preparation method of the human haemophilus influenzae surface protein polyclonal antibody comprises the following steps:
a) Immunizing New Zealand pure rabbit with recombinant human Haemophilus influenzae surface protein P6, and then detecting serum antibody level by indirect ELISA;
b) Purifying and extracting the polyclonal antibody by using a GE-HiTrap Protein A HP pre-packed column to prepare the polyclonal antibody of the human haemophilus influenzae surface protein.
The invention has the following advantages and effects:
firstly, the invention adopts the modes of gene optimization, low-temperature induced expression and the like to successfully obtain the soluble recombinant P6 protein, and the protein has natural structure and is a good material for preparing antibodies by being used as an immune source.
Secondly, the monoclonal antibody obtained by the invention can specifically identify the extracellular region of the bacterial P6 protein, and has no cross reaction with other common respiratory pathogens.
Finally, the results of the specificity experiments performed with 7 strains of haemophilus influenzae and 17 standard strains of haemophilus influenzae that are non-human (comprising most common pathogens of the respiratory tract) show that the kit of the invention has good specificity and stability, and can detect all tested strains of haemophilus influenzae without cross-reaction with all standard strains of haemophilus influenzae that are non-human. Secondly, the sensitivity test result shows that the detection sensitivity of the kit is 1 multiplied by 10 3 CFU/hole is obviously higher than the traditional detection method of microorganism, and has advantages of rapidness, high efficiency and the like.
Detailed Description
The methods used in the examples described below are conventional methods unless otherwise specified.
EXAMPLE 1 preparation of recombinant human Haemophilus influenzae surface protein P6
1) Cloning expression of human haemophilus influenzae P6 gene
The bioinformatics analysis of the human haemophilus influenzae surface protein P6 (access number in NCBI protein database is wp_ 005652235), the DNA coding sequence thereof was optimized in view of GC content, codon preference, mRNA secondary structure, RNA instability motif, mRNA free energy stability, etc., and the complete gene sequence was chemically synthesized after introducing the cleavage site NdeI at 5' and cleavage site XhoI at 3' end thereof (complete sequence synthesis was completed by kusnezoff biotechnology, inc., and the artificially synthesized gene fragment was linked to vector pUC57 at delivery), designated P6'. The whole sequence of the gene and the coded amino acid sequence are shown in a sequence table. The vector pUC57 containing the artificially synthesized DNA fragment was digested with NdeI and XhoI, and the target fragment was recovered by a conventional method for use. Meanwhile, ndeI and XhoI are adopted to carry out double enzyme digestion on the vector pET-28a (+) and the P6 'gene obtained after double enzyme digestion is connected into the pET-28a (+) vector according to a conventional method, and the vector is converted into escherichia coli TOP10 to construct a pET-P6' expression vector. The construction of the expression vector is proved to be correct by enzyme digestion and sequence determination. The vector expresses recombinant P6-His fusion protein.
2) Purification of human haemophilus influenzae P6 protein
The plasmid is extracted after the identification of the correct positive clone is cultivated, and is transferred into competent E.coli BL21 (DE 3) plysS according to the conventional technology, and after the transformation is finished, bacterial liquid is coated on an LB plate containing 50 mug/mL kanamycin, and the expression strain is screened according to the conventional method. Single colonies transformed with pET-P6 and having the ability to express the foreign protein were picked up and inoculated into 100mL of LB medium, and cultured overnight at 37 ℃. After the bacterial liquid is taken out, the following steps are carried out according to the following ratio of 1:100 inoculated in 100mL of LB medium containing 50. Mu.g/mL kanamycin, cultured at 30℃to OD 600 At=0.6, 1mol/L IPTG was added to a final concentration of 0.6mmol/L, and shake culture was performed at 18 ℃ to induce fusion protein expression. After 12h of induction, the cells were collected by centrifugation at 8000r/min for 10 min. The cells were treated with 50mL Buffer A (50 mM Na 3 PO 4 0.5M NaCl; pH 7.4) was washed 3 times and with 50mL of loading buffer (50 mM Na) 3 PO 4 0.5M NaCl;5mM imidazole, pH 7.4) was resuspended and sonicated under the following operating conditions: the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30min. After the completion of the sonication, the sediment and the supernatant were collected by centrifugation at 12000g for 15min, respectively, and then subjected to electrophoresis detection. Recombinant P6 protein was found to be present in the bacterial cells in solubilized form. Thin-layer scanning shows that the recombinant protein accounts for more than 30% of the total protein of bacteria, which indicates that the gene is optimized to obtain higher expression quantity. The sonicated supernatant obtained above was filtered through a 0.45 μm filter and then purified using His Trap affinity columns (GE healthcare Co.) according to the method described in the specification. The specific method comprises the following steps:
connecting a chromatography system, wherein the system comprises a sample inlet pipe, a peristaltic pump, a chromatography column and an ultraviolet detector (Shanghai Xie analytical instrument factory, model HD 1), the volume of the column is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable; proofreading T%: adjusting the brightness knob to display 100%; the sensitivity is rotated to a proper position, and generally 0.2A is used; the chromatography system was equilibrated with loading buffer and rotated "zeroed" to display "000" after the reading was stable; collecting penetrating fluid after protein sample is applied and the flow rate is controlled within 5 ml/min; washing unbound protein with a loading buffer, recording the reading until the reading is no longer changing, and collecting the eluate; eluting with Buffer A+10mM imidazole, and collecting eluting peak; eluting with Buffer A+20mM imidazole, and collecting eluting peak; eluting with Buffer A+40mM imidazole, and collecting eluting peaks; eluting with Buffer A+100mM imidazole, and collecting eluting peaks; eluting with Buffer A+150mM imidazole, and collecting eluting peaks; taking 100ul of each eluting peak sample, and performing SDS-PAGE electrophoresis; as a result, it was found that the target protein was eluted at 100mM imidazole, the purity was 90% or higher, and the concentration was adjusted to 0.2mg/mL for use after the protein concentration was measured by using the bradford kit. Thus, the recombinant human haemophilus influenzae surface protein P6 is prepared.
Example 2 preparation of polyclonal antibody to human Haemophilus influenzae surface protein
1) Immunization of New Zealand pure line rabbits
The recombinant P6 protein prepared in example 1 was mixed with Freund's complete adjuvant, emulsified and used as an immunogen to immunize 2 male New Zealand rabbits, and the total amount of the recombinant P6 protein per rabbit was 2ml by subcutaneous injection, and the total amount of the antigen was 2 mg/rabbit. And then the vaccine is immunized once by using an emulsion formed by the recombinant P6 protein and the Freund's incomplete adjuvant every two weeks, the total immunization is 5 times, and the antigen dosage is the same as that of the initial immunization. Cardiac massive blood taking is carried out 3-5 days after the five-day period, the blood is placed at 37 ℃ for 1 hour, then the blood is placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.
2) Determination of polyclonal antibody titers
The recombinant P6 protein is used as coating antigen, the coating concentration is 10 mug/ml, 100 mu l of each hole is coated, and the serum antibody level is detected by an indirect ELISA method. The serum dilution of the experimental group is 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1:102400, 1:204800; ELISA plates were coated with bovine serum albumin as a negative control and tested for OD450 by ELISA, satisfying P/N values > 2.1 as positive. The results showed that serum antibody titers of 2 rabbits all reached 1:102400, which shows that the immune effect is better.
3) Extraction of polyclonal antibodies
The antibody was purified by the GE-HiTrap Protein A HP pre-packed column according to the instructions as follows:
1.5 mL of antiserum was taken, 0.5mL of 1M Tris (pH 8.0) was added to adjust to pH8.0, and 20000g was centrifuged for 20min to remove the precipitate.
2. After loading onto the column, the column was washed with 10 volumes of buffer A (100 mM Tris-Cl, pH 8.0) and then with 10 volumes of buffer B (10 mM Tris-Cl, pH 8.0).
3. IgG was eluted with approximately three column volumes of IgG elution buffer (100mM glycine,pH3.0). (0.1 mL of IgG-neutralizing buffer (1M Tris-Cl, pH 8.0) was preloaded into the collection tube, and 0.9mL of eluent was packed per tube)
4. Dialysis was performed with 50 volumes of Tris (10 mM Tris-Cl, pH 8.0).
5. After ultrafiltration concentration, the concentration is adjusted to 1mg/ml, and the mixture is preserved at the temperature of-70 ℃ for standby. Thus, the polyclonal antibody of the human haemophilus influenzae surface protein is prepared.
EXAMPLE 3 preparation of human Haemophilus influenzae surface protein monoclonal antibody Hi-7#
1) Immunization of animals
Using the recombinant human haemophilus influenzae surface protein P6 prepared in example 1 as antigen, 5 BALB/c mice 8 weeks old were immunized, and 2 mice were not immunized as negative control. After the primary immunization antigen was fully emulsified with an equal amount of Freund's complete adjuvant, mice were immunized by subcutaneous multi-point injection at the back, 100. Mu.g/mouse. The second immunization was then performed by intraperitoneal injection after three weeks apart with the same dose of antigen fully emulsified with Freund's incomplete adjuvant, and the third immunization was performed by intraperitoneal injection after 2 weeks apart with the same dose of antigen fully emulsified with Freund's incomplete adjuvant. And blood is collected from tail vein 15 days after the third immunization, and the antiserum titer is detected.
2) Antiserum potency assay
The antiserum titers were measured by indirect ELISA: PBS (8.5 g/L NaCl,1.4g/L Na) 2 HPO 4 ，0.2g/L NaH 2 PO 4 pH 7.4) was diluted to an antigen concentration of 3. Mu.g/mL, added to a 96-well plate, 100. Mu.L/well, and incubated at 37℃for 2 hours or overnight at 4 ℃. With PBST (8.5 g/L NaCl,1.4g/L Na) 2 HPO 4 ，0.2g/L NaH 2 PO 4 Plates were beaten after 3 washes with 0.5% (v/v) Tween-20, pH 7.4). 1% bovine serum albumin in PBS solution250. Mu.L/well, blocking at 37℃for 1h or overnight at 4 ℃. Plates were beaten after washing 3 times with PBST. 4 immunized mouse serum is diluted by PBS gradient and added into corresponding holes, 100 mu L/hole, a blank control is PBS solution, and a negative control is a plate washed after the serum of the immunized mouse is coated for 1h at 37 ℃. HRP-labeled goat anti-mouse IgG at 1: dilution at 3000 times, 100. Mu.L/well, coating at 37℃for 1h and washing the plate. Adding 100 μl of freshly prepared TMB chromogenic substrate solution into each well, reacting at 37deg.C for 10min, adding 1M hydrochloric acid into each well to terminate the reaction, and measuring OD with enzyme-linked detector 450 nm values, readings and observations. The highest titer (titer 51200) was selected, the immunization was boosted one month after the third immunization interval, and the spleen cells of the mice were taken 5 days later for cell fusion.
3) Resuscitation and culture of SP2/0 myeloma cells
Resuscitating frozen myeloma cells (SP 2/0) in advance, rapidly taking out the frozen myeloma cells, slightly shaking in a water bath at 37deg.C to melt the frozen myeloma cells, taking out the frozen myeloma cells, transferring the frozen myeloma cells into a 24-well plate containing 2ml of RPMI-1640 complete medium (RPMI-1640 medium containing 20% fetal bovine serum obtained from Shanxi Runssen biological materials Co., ltd.) without contamination, placing the frozen myeloma cells into a water bath at 37deg.C and 5% CO 2 Culturing in an incubator for half an hour, changing the liquid in time when the cells are grown on the wall, and then carrying out passage once every 3 days, adjusting the cells to be in the optimal growth density, and preparing to fuse when the cells reach a certain activity and count. Cells were passaged 1 to 4 days prior to cell fusion and the cell concentration per bottle was adjusted to 1X 10 with fresh medium 5 /ml。
4) Preparation of feeder cells
4.1 BALB/c mice were sacrificed by pulling their necks after blood was taken, completely immersed in 75% alcohol for 5min, and transferred to a dish on a super clean bench with their abdomen facing upward.
4.2 Lifting the mouse chest and abdomen skin with forceps, cutting a small opening with scissors, tearing the skin with two forceps to open a larger opening, lifting the mouse peritoneum, cutting to find the mouse spleen, carefully taking the spleen out with scissors with forceps, placing in a disposable plate, carefully peeling off fat, connective tissue and the like attached to the spleen, adding 5ml of RPMI-1640 medium (purchased from Hyclone, cat# SH 30809.01), puncturing the spleen with a needle of a syringe having 5ml of RPMI-1640 medium absorbed therein, carefully washing out spleen cells, sieving, adding the spleen cell suspension into a 10ml centrifuge tube, centrifuging at 1100rpm for 5min, discarding supernatant, and washing twice with RPMI-1640 medium.
4.3 Lightly resuspending and mixing the cells with 5ml HAT medium, counting, and adding HAT medium to a cell concentration of 1×10 5 /ml。
4.4 Dripping the cell suspension into 96-well cell culture plate at 130 μl/well, and placing into 37 deg.C and 5% CO 2 Culturing in an incubator.
5) Preparation of immune spleen cell suspension
5.1 5 days after booster immunization, BALB/c mice with highest serum titers were selected, eyeballs were removed, bloodletting was performed, and serum was collected and isolated as a positive control for antibody detection.
5.2 After the mice were sacrificed by cervical fracture, the mice were immersed in 75% alcohol for 5min, and the mice were taken out and placed in a dish of a sterile ultra-clean bench with their abdomen facing upward.
5.3 Lifting the mouse chest and abdomen skin with forceps, cutting a small opening with scissors, tearing the skin with two forceps to form a larger opening, then lifting the mouse peritoneum with new forceps again, cutting, finding the mouse spleen, carefully taking the spleen out, placing in a disposable plate, and carefully removing fat and connective tissues.
5.4 After washing with RPMI-1640 wash, fresh RPMI-1640 wash was added, spleen was carefully washed out of spleen cells by puncturing with a needle of a syringe with 5ml of RPMI-1640 medium, and then sieving, so that the spleen cells were pressed into solution as much as possible through the mesh, the spleen cell suspension was transferred into a centrifuge tube, centrifuged at 1100rpm for 5min, the supernatant was discarded, and washed by centrifugation twice.
5.5 The spleen cells were gently resuspended in RPMI-1640 medium and counted for further use.
6) Preparation of SP2/0 myeloma cell suspension
6.1 2 bottles of T75 flasks were taken and cultured myeloma cells (the day before fusion, the cells should be in logarithmic growth phase) were collected in 50ml centrifuge tubes.
6.2 Centrifugation at 1000rpm for 5 minutes, and the supernatant was discarded.
6.3 30ml of RPMI-1640 wash solution was added to the pellet, gently resuspended, and the mixture was centrifuged again in the same manner.
6.4 The spleen cells were gently resuspended and mixed uniformly with 10ml of RPMI-1640 medium and counted for further use.
7) Cell fusion
7.1 Will contain 1X 10 8 Suspension of individual spleen cells and 1X 10 containing 7 The suspension of SP2/0 myeloma cells was mixed in a 50ml centrifuge tube, and medium was added to 40ml, followed by thorough mixing.
7.2 1200rpm for 5 minutes, the supernatant was discarded, and the supernatant was removed as much as possible.
7.3 Lightly flicking the bottom of the centrifuge tube by hand to loosen and uniformly paste the cell mass.
7.4 Taking out the prepared 50% PEG (MW 1450) and RPMI-1640 lotion from the refrigerator at the temperature of 4 ℃, placing the lotion in a water bath at the temperature of 37 ℃ and preheating for standby.
7.5 50% PEG (MW 1450) 0.8ml was pipetted into a 1ml pipette and slowly added to the centrifuge tube with stirring for 60 seconds.
7.6 Then 40ml of pre-warmed RPMI-1640 wash was gradually added over a period of 60 seconds to dilute the PEG and lose the fusogenic effect.
7.7 Centrifugation at 1000rpm for 5min, and discarding the supernatant.
7.8 400mL of HAT medium (purchased from Sigma, cat# H0262) was added, and the pelleted cells were gently aspirated and resuspended.
7.9 Adding the fused cell suspension into 96-well culture plate containing feeder cells, 50 μl/well, spreading 20 culture plates together, and placing the culture plates at 37deg.C and 5% CO 2 Is cultured in an incubator of (a).
8) Screening of Positive clones and cloning culture
Starting on day 3 after fusion, cells were observed daily for growth in each well and immediately treated with sodium azide if contaminated. After fusion, the HT broth was changed completely (HT, 50×, purchased from Sigma, cat No. H0137) at 7 d. The next day after the liquid is changed,well supernatants from which clones appeared were aspirated for specific detection. Haemophilus influenzae cells (ATCC 49247, 1X 10) 8 Individual cells/wells) are coated antigens, and are detected by an indirect ELISA method. Determination of OD with an enzyme-linked detector 450 And the positive value that the P/N value is more than 2.1 is satisfied. And (3) completely replacing the positive holes with HT culture medium again, detecting positive holes by ELISA again the next day, selecting continuous positive cloning holes for 2-3 times, and screening monoclonal hybridoma cells. Subcloning specifically comprises the following steps: (1) blowing off and mixing the positive fusion hole hybridoma cells, and measuring the cell concentration; (2) feeder cells were prepared in advance and suspended in HT medium, 130 ul/well, plated into 96 well plates, and the plates were then incubated at 37℃with 5% CO 2 Is reserved in an incubator; (3) taking positive fusion hole hybridoma cells, and dispersing the positive fusion hole hybridoma cells into the 96-well plate in the step (2) on average; (4) placing the culture plate at 37deg.C and 5% CO 2 Culturing for 7-8d in an incubator; (5) screening positive single colony holes by ELISA, and subcloning again; (6) after 2-3 times of continuous subcloning, the colony holes of the subcloned cells are positive and have similar values, so that the monoclonal hybridoma cells are obtained; and performing amplification culture on the monoclonal hybridoma cells to obtain a cell culture solution supernatant containing the monoclonal antibodies. By Mycoplasma pneumoniae (ATCC 15531, 1X 10) 8 Individual cells/well), pseudomonas aeruginosa (ATCC 27853, 1X 10) 8 Individual cells/well), moraxella catarrhalis (ATCC 25240, 1X 10) 8 Individual cells/well), acinetobacter baumannii (ATCC 19606, 1X 10) 8 Individual cells/well), haemophilus parainfluenza (ATCC 7901, 1X 10) 8 Individual cells/well), legionella pneumophila (ATCC 33152, 1X 10) 8 Individual cells/well), streptococcus pyogenes (ATCC 19615, 1X 10) 8 Individual cells/well), staphylococcus aureus (ATCC 25923,1×10) 8 Individual cells/well), human Streptococcus pneumoniae (ATCC 49619, 1X 10) 8 Individual cells/well), klebsiella pneumoniae (ATCC 700603,1 ×10) 8 Individual cells/well), enterobacter cloacae (ATCC 13047, 1X 10) 8 Individual cells/well), escherichia coli (ATCC 25922, 1X 10) 8 Individual cells/well), candida (ATCC 10231,1×10) 8 Individual cells/holes) and other respiratory tract pathogenic bacteria are respectively coated with an ELISA plate, ELISA detection is carried out on the specificity of the screened antibodies, and the screening and the elimination are carried outMonoclonal antibodies that respond positively to these pathogens. In this step 8 qualified cell lines were screened out.
9) Ascites preparation and cell preservation
And performing expansion culture on the finally screened 8 positive monoclonal hybridoma cells. The monoclonal antibody ascites is prepared by a conventional ascites in-vivo induction method. (1) Western blot detection of mAb: the ascites is determined to be 1 by adopting a conventional Western blot experiment method: the specificity at 2000 dilutions shows that all of the 8 antibodies only bind to the 16KD molecular weight P6 protein in human hemophilus influenzae whole cell proteins. (2) antibody purification and potency determination: the ascites was purified by Protein A affinity chromatography as follows:
the antibody was purified by the GE-HiTrap Protein A HP pre-packed column according to the instructions as follows:
a. 5mL of the hybridoma supernatant was taken, and 0.5mL of 1M Tris (pH 8.0) was added thereto to adjust the pH to 8.0, and the mixture was centrifuged at 20000g for 20min to remove the precipitate.
b. After loading onto the column, the column was washed with 10 volumes of buffer A (100 mM Tris-Cl, pH 8.0) and then with 10 volumes of buffer B (10 mM Tris-Cl, pH 8.0).
c. IgG was eluted with approximately three column volumes of IgG elution buffer (100mM glycine,pH3.0). (0.1 mL of IgG-neutralizing buffer (1M Tris-Cl, pH 8.0) was preloaded into the collection tube, and 0.9mL of eluent was loaded per tube);
d. with 50 times the volume of PBS (8.5 g/L NaCl,1.4g/L Na) 2 HPO 4 ，0.2g/L NaH 2 PO 4 pH 7.4) was dialyzed.
e. After ultrafiltration concentration, PBS was used to adjust the concentration to 1mg/ml and stored at-70℃until use.
The titers of the 8 purified antibodies were determined by indirect ELISA after dilution by a double ratio. The purity of the antibody is over 95% by SDS-PAGE analysis, and the ELISA titer is 1:1000000 or more. Purified antibody was adjusted to a concentration of 1mg/ml and stored at-70℃until use.
The 8 antibodies are respectively used as labeled antibodies for ELISA detection, and monoclonal antibody-polyclonal antibody pairing experiments are carried out to screen out the optimal combination. Finally, through a plurality of experiments, it was determined that the monoclonal antibody secreted by the hybridoma cell line designated Hi-7# is preferably a labeled antibody. This antibody was designated monoclonal antibody Hi-7#.
Hybridoma cell lines secreting the antibody are sent to China center for type culture collection for preservation in 10 months and 15 days of 2018, and are named after classification: hybridoma cell strain Hi-7#, and preservation number CCTCC NO: c2017218; address: university of martial arts in chinese.
Example 4 human Haemophilus influenzae surface protein Capture ELISA kit
1) Human haemophilus influenzae surface protein capture ELISA kit composition
The solid phase carrier coated with the polyclonal antibody of the surface protein of the human influenza haemophilus is an ELISA plate, the monoclonal antibody Hi-7# of the surface protein of the human influenza haemophilus, goat anti-mouse IgG marked by horseradish peroxidase, substrate reaction liquid of enzyme, positive and negative control, washing liquid and reaction stopping liquid together form the ELISA kit for capturing the surface protein of the human influenza haemophilus.
ELISA plate coated polyclonal antibody
Human haemophilus influenzae surface protein polyclonal antibodies (prepared as described in example 2) were diluted to a concentration of 10 μg/mL with PBS buffer, coated with 96-well EIA high-efficiency binding elisa plate, 50 μl/well, for 2 hours at 37 ℃. And (5) taking out, washing the plate with a washing liquid for three times, and spin-drying. The plate was blocked with a wash solution containing 1% BSA as blocking solution at 250. Mu.L/well for 1 hour at 37 ℃. Taking out, washing the plate with the washing liquid for 3 times, each time for one minute, spin-drying, and sealing and preserving after drying.
Wherein, PBS buffer: 1.4g of disodium hydrogen phosphate, 0.2g of sodium dihydrogen phosphate, 8.5g of sodium chloride and pH7.4 to 1000mL; washing solution (PBST): PBS aqueous solution containing 0.01% Tween-20, pH 7.4; sealing liquid: the aqueous washing solution containing 1% BSA had a pH of 7.4.
2) Human haemophilus influenzae surface protein monoclonal antibody Hi-7#, and preparation method thereof
Human haemophilus influenzae surface protein monoclonal antibody Hi-7# at a concentration of 1mg/mL as described in example 3 was split at 20 μl each and diluted 1000-fold with PBS buffer for use.
3) Horseradish peroxidase marked goat anti-mouse IgG
Horseradish peroxidase-labeled goat anti-mouse IgG is a common commercial product, which in this example was purchased from beijing-sai biotechnology limited, product number 030006-G, diluted 3000-fold with PBS buffer when used.
4) Substrate reaction solution for enzyme
The substrate reaction solution of the enzyme is prepared as follows:
and (3) solution A: configuration quantity 1L)
1. 3.14g of citric acid (containing 1 molecule of crystal water and having a molecular weight of 210.14 g) and 11.56g of sodium acetate (containing 3 molecules of crystal water and having a molecular weight of 136.0) were dissolved in 970mL of double distilled water to prepare an aqueous sodium acetate solution having a pH of 5.0.
2. 0.08g of phenacetin was weighed into 30mL double distilled water and heated to 100deg.C, and added to the solution of the first step after dissolution was complete.
3. Adding 0.5g of carbamide peroxide, and uniformly mixing.
And (2) liquid B: configuration quantity 1L)
500mL of methanol was added to the 2L beaker, followed by 1.27g of 3, 5-tetramethylbenzidine TMB (SIGMA), and after dissolution by heating at 60℃was added, 500mL of glycerol was added.
A, B liquid 1:1, mixing to prepare a substrate reaction solution of the enzyme.
5) Positive and negative controls
Positive control: recombinant human Haemophilus influenzae surface protein P6, 10 μg/mL was extracted as described in example 1.
Negative control: normal mouse serum of the non-immunized recombinant human haemophilus influenzae surface protein P6 was diluted 200-fold with PBS buffer.
6) The washing solution, PBST solution, was prepared as described in (1).
7) The reaction was stopped and a 1M HCL solution prepared with double distilled water was used.
Example 5 method of Using human Haemophilus influenzae surface protein Capture ELISA kit
1) Treatment of samples to be examined
The throat swab of the subject was obtained by a conventional method, and was inserted into a soft plastic tube containing 500. Mu.L of a washing solution (PBST), and the plastic tube wall was squeezed to sufficiently dissolve the sample on the swab. Ultrasonic crushing (Ningbo new sesame JY96-IIN type ultrasonic cell crusher, 30% power, 15 min) is carried out on the sample to obtain crushing liquid.
2) Adding control sample and sample to be detected
Taking 100 mu L of a crushed liquid sample to be detected, adding the crushed liquid sample into a corresponding enzyme-labeled hole, adding 1 hole of a positive control (100 mu L/hole), 4 holes of a negative control (100 mu L/hole), incubating for 1 hour at 37 ℃, washing the plate 3 times by using 250 mu L of PBST washing liquid, and spin-drying.
3) Adding monoclonal antibody Hi-7#, and
50. Mu.L/well of human hemophilus influenzae surface protein monoclonal antibody Hi-7# working solution (blocking solution 1:1000 dilution) described in example 4 was added, incubated at 37℃for 1 hour, and the plates were washed 3 times with 250. Mu.L of PBST wash and spun dry.
4) Adding enzyme-labeled antibody
Horseradish peroxidase-labeled goat anti-mouse IgG (described in example 4) working solution (blocking solution 1:3000 dilution) was added at 50. Mu.L/well, incubated at 37℃for 1 hour, and after washing the plate 3 times with 250. Mu.L of PBST wash, it was spun dry.
5) Adding substrate for color development
50. Mu.L/well of the freshly prepared enzyme substrate reaction solution described in example 4 was added and after complete color development (15 minutes).
6) Adding reaction stopping solution
The reaction was terminated by adding 50. Mu.L of a reaction termination solution to each well.
7) OD measurement 450 nm value
The ELISA plate is placed in an ELISA instrument to measure OD 450 nm value.
8) Result determination
OD of 4-hole negative quality control and 1-hole positive quality control samples are respectively read 450 nm value; OD of 4-well negative quality control sample 450 The sum of the average value of nm readings and 3 times of standard deviation is the CUT-OFF value; detection OD of the human throat swab sample 450 If the nm value is larger than the CUT-OFF value, judging that the human influenza haemophilus antigen in the clinical throat swab is positive, otherwise, judging that the human influenza haemophilus antigen in the human throat swab sampleNegative; if the OD of the positive quality control sample 450 A nm value less than the CUT-OFF value indicates that the kit is not functional.
Example 6 specificity and sensitivity determination of human Haemophilus influenzae surface protein Capture ELISA kit
1) Specificity assay
To verify the specificity of the human haemophilus influenzae surface protein capture ELISA kit of the invention, 7 human haemophilus influenzae strains and 17 non-human haemophilus influenzae standard strains were tested according to the kit compositions and methods described in example 4 and example 5, see table 1. The results show that the kit provided by the invention has positive detection results on all 7 human haemophilus influenzae strains, and has negative detection results on other 17 common pathogenic microorganisms in respiratory tract. The kit shows good specificity.
TABLE 1
2) Sensitivity determination
Inoculating Haemophilus influenzae ATCC49247 strain into sheep blood chocolate culture medium, culturing at 37deg.C for 24 hr, and performing gradient dilution with physiological saline 10 times while plate counting to obtain thallus with concentration of 10 8 -10 3 The CFU/mL cell solution was then added dropwise to the ELISA plate at 100. Mu.L, and the detection was performed according to the kit composition and method described in example 4 and example 5. The result shows that the detection sensitivity of the kit is 10 3 CFU/mL。
Sequence listing
<110> Hubei cloud celluloid bioengineering Co., ltd
<120> human influenza haemophilus surface protein monoclonal antibody and antigen capturing ELISA kit
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 471
<212> DNA
<213> P6' Gene sequence (Artificial Sequence)
<400> 1
catatgaaca aattcgttaa atcacttctt gttgctggtt cagttgctgc tcttgctgct 60
tgttcatcat caaacaacga cgctgctggt aacggtgctg ctcaaacttt cggtggttac 120
tcagttgctg accttcaaca acgttacaac actgtttact tcggtttcga caaatacgac 180
atcactggtg aatacgttca aatccttgac gctcacgctg cttaccttaa cgctactcca 240
gctgctaaag ttcttgttga aggtaacact gacgaacgtg gtactccaga atacaacatc 300
gctcttggtc aacgtcgtgc tgacgctgtt aaaggttacc ttgctggtaa aggtgttgac 360
gctggtaaac ttggtactgt ttcatacggt gaagaaaaac cagctgttct tggtcacgac 420
gaagctgctt actcaaaaaa ccgtcgtgct gttcttgctt actaactcga g 471
<210> 2
<211> 153
<212> PRT
<213> P6' protein sequence (Artificial Sequence)
<400> 2
Met Asn Lys Phe Val Lys Ser Leu Leu Val Ala Gly Ser Val Ala Ala
1 5 10 15
Leu Ala Ala Cys Ser Ser Ser Asn Asn Asp Ala Ala Gly Asn Gly Ala
20 25 30
Ala Gln Thr Phe Gly Gly Tyr Ser Val Ala Asp Leu Gln Gln Arg Tyr
35 40 45
Asn Thr Val Tyr Phe Gly Phe Asp Lys Tyr Asp Ile Thr Gly Glu Tyr
50 55 60
Val Gln Ile Leu Asp Ala His Ala Ala Tyr Leu Asn Ala Thr Pro Ala
65 70 75 80
Ala Lys Val Leu Val Glu Gly Asn Thr Asp Glu Arg Gly Thr Pro Glu
85 90 95
Tyr Asn Ile Ala Leu Gly Gln Arg Arg Ala Asp Ala Val Lys Gly Tyr
100 105 110
Leu Ala Gly Lys Gly Val Asp Ala Gly Lys Leu Gly Thr Val Ser Tyr
115 120 125
Gly Glu Glu Lys Pro Ala Val Leu Gly His Asp Glu Ala Ala Tyr Ser
130 135 140
Lys Asn Arg Arg Ala Val Leu Ala Tyr
145 150
Claims (7)
1. A hybridoma cell line producing a monoclonal antibody to a surface protein of haemophilus influenzae, comprising: the hybridoma cell strain for producing the human hemophilus influenzae surface protein monoclonal antibody is preserved by China Center for Type Culture Collection (CCTCC) NO: hybridoma cell line Hi-7# of C2017218;
the preparation method of the hybridoma cell strain Hi-7# comprises the following steps:
1) After 8-week-old BALB/c mice are immunized by taking recombinant human hemophilus influenzae surface protein P6 as an antigen, an indirect ELISA method is adopted to detect the antiserum titer:
1.1 Clone expression of human haemophilus influenzae P6 gene
Bioinformatics analysis is carried out on the haemophilus influenzae surface protein P6 gene, the GC content, the codon preference, the mRNA secondary structure, the RNA unstable motif and the mRNA free energy stability are combined, the DNA coding sequence of the haemophilus influenzae surface protein P6 gene is optimized, meanwhile, an enzyme cutting site NdeI is introduced into the 5' of the haemophilus influenzae surface protein P6 gene, a termination signal TAA and an enzyme cutting site XhoI are introduced into the 3' end of the haemophilus influenzae surface protein P6 gene, and then the whole gene sequence is chemically synthesized and then connected to a vector pUC57, and the result is marked as P6'; carrying out double enzyme digestion on a vector pUC57 containing the chemically synthesized complete gene sequence by NdeI and XhoI, and then recovering a target fragment for later use by a conventional method; simultaneously, ndeI and XhoI are adopted to carry out double enzyme digestion on a vector pET-28a (+) and the P6 'gene obtained after double enzyme digestion is connected into the pET-28a (+) vector according to a conventional method, and the E.coli TOP10 is transformed to construct a pET-P6' expression vector; enzyme digestion and sequence determination prove that the construction of the expression vector is correct; the vector expresses recombinant P6-His fusion protein;
1.2 Purification of human haemophilus influenzae P6 protein
The plasmid is extracted after the identification of the correct positive clone bacteria is cultivated, and is transferred into competent E.coliBL21 (DE 3) plysS according to the conventional technology, bacterial liquid is coated on an LB plate containing 50 mug/mL kanamycin after the transformation is finished, and the expression bacterial strain is screened according to the conventional method, cultivated and purified;
2) Resuscitating and culturing SP2/0 myeloma cells;
3) Preparing feeder cells;
4) Preparing an immune spleen cell suspension;
5) Preparing SP2/0 myeloma cell suspension;
6) Cell fusion;
7) Screening positive clones and cloning culture, taking human haemophilus influenzae thalli as coating antigen, and detecting by adopting an indirect ELISA method; determination of OD with an enzyme-linked detector 450 The P/N value is more than 2.1 and positive; selecting continuous positive cloning holes for 2-3 times of subcloning, and screening monoclonal hybridoma cells; performing amplification culture on the monoclonal hybridoma cells to obtain a cell culture solution supernatant containing the monoclonal antibodies; coating ELISA plates with respiratory tract pathogenic bacteria respectively, performing ELISA detection on the specificity of the screened antibodies, eliminating monoclonal antibodies with positive reaction with the pathogens, and screening qualified cell strains; the respiratory tract pathogenic bacteria comprise human lungMycoplasma, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, legionella pneumophila, streptococcus pyogenes, staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida.
2. A human haemophilus influenzae surface protein monoclonal antibody, characterized in that: the human hemophilus influenzae surface protein monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell strain Hi-7# of claim 1.
3. A method for preparing a monoclonal antibody based on the human haemophilus influenzae surface protein according to claim 2, which is characterized in that: the method comprises the following steps:
performing amplification culture on the positive monoclonal hybridoma cells obtained by screening; monoclonal antibody ascites is prepared by a conventional ascites in-vivo induction method, collected and purified by Protein A affinity chromatography to obtain the human haemophilus influenzae surface Protein monoclonal antibody Hi-7#.
4. Use of the monoclonal antibody of human haemophilus influenzae surface protein according to claim 2 in the preparation of a reagent for detecting human haemophilus influenzae.
5. Use of the monoclonal antibody of human haemophilus influenzae surface protein according to claim 2 for preparing a detection of human haemophilus influenzae surface protein capture ELISA kit.
6. An ELISA kit for detecting human haemophilus influenzae surface protein capture, which is characterized in that: the ELISA kit for detecting human haemophilus influenzae surface protein comprises an ELISA plate and a human haemophilus influenzae surface protein monoclonal antibody Hi-7# according to claim 2; the ELISA plate is a solid phase carrier coated with polyclonal antibody of human hemophilus influenza surface protein.
7. The detection of human haemophilus influenzae surface protein capture ELISA kit according to claim 6, characterized in that: the preparation method of the human haemophilus influenzae surface protein polyclonal antibody comprises the following steps:
a) Immunizing New Zealand pure rabbit with recombinant human Haemophilus influenzae surface protein P6, and then detecting serum antibody level by indirect ELISA;
b) Purifying and extracting the polyclonal antibody by using a GE-HiTrap Protein A HP pre-packed column to prepare the polyclonal antibody of the human haemophilus influenzae surface protein.
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