CN103645318A - Detection method and detection kit of antigen-specific IgG (immunoglobulin G) antibody of staphylococcus aureus SpA5 mutant - Google Patents
Detection method and detection kit of antigen-specific IgG (immunoglobulin G) antibody of staphylococcus aureus SpA5 mutant Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and relates to a detection method and a detection kit of an antigen-specific IgG (immunoglobulin G) antibody of a staphylococcus aureus (SA) SpA5 mutant. By adopting the method disclosed by the invention, the specificity of the method is improved, and the method is simple and fast in operation, and good in repeatability.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of staphylococcus aureus (SA) SpA5 mutant, more specifically, relate to detection method and the detection kit of the antigentic specificity IgG antibody of this mutant.
Background technology
Staphylococcus aureus (Staphylococcus aureus, SA), hereinafter to be referred as golden Portugal bacterium, has the another name of " having a liking for meat bacterium ".As the representative of gram-positive bacteria, it is a kind of important pathogenic bacteria that causes that hospital infection and community infect.That infection be take is acute, purulent inflammation is feature, and local infection can cause the pyogenic infection of skin and soft tissue etc., does not prolongedly heal; General infection can cause severe infections and the complication such as osteomyelitis, septic arthritis, endocarditis, pneumonia, septicopyemia, and mortality ratio is up to 20%.Meanwhile, the exotoxin of golden Portugal bacterium also can cause the whole body lethal infections such as food poisoning, scalded skin syndrome and TSS.Therefore, strengthen the immune protection research that Dui Jin Portugal bacterium infects, develop safe and effective novel golden Portugal bacterium recombinant vaccine and extensively infect and there is important strategy and realistic meaning the golden Portugal of effective control bacterium drug resistance being spread with clinical golden Portugal bacterium.
Along with microbiotic is used for a long time, widely, bacterial drug resistance problem becomes increasingly conspicuous, methicillin-resistant staphylococcus aureus (methicillin-resistant Staphylococcus aureus as Typical Representative, MRSA) from 1961, found first so far, become one of the Inner of hospital infection pathogen that the infection rates such as global ICU ward, postoperative infection, burn, war wound are the highest at present.Meanwhile, because of its pathogenic strong, route of transmission extensively, easy outbreak of epidemic, and be multidrug resistant sexual development and form as the difficult point for the treatment of clinically, be called as " the first superbacteria ".
Most SA clinical separation strains can be expressed SpA.SpA molecular weight is 55kDa, is positioned on cell membrane.SpA precursor comprises a N end signal peptide and C end screens signal so that it is covalently bonded on cell membrane.The Ig that comprises 5 56-61 amino acid residues binding structural domain is divided in the N end of mature peptide, is curled into three α helical bundles.These domains can be combined with the Fc of mammal IgG section, destroy the conditioning phagocytosis of antibody; Also can be combined with the B-cell receptor of VH3 type, make B cell death, destroy acquired and innate immune reaction.Thereby induction body produces the antibody for SpA, the Immune escaping mechanism of blocking-up MRSA is an important selection of SA vaccine.But because SpA possesses antibody binding capacity, the natural SpA of usining cannot realize re-set target as antigen, need to suddenly change and remove as far as possible its antibody binding activity it, retain its immunogenicity simultaneously.
Present inventor is through studying for a long period of time, five IgG domains of EDABC to the SpA albumen of MRSA252 bacterial strain suddenly change, develop a kind of biologically active higher than the SpA5 mutant (as shown in SEQ ID NO.1) of prior art, there is very strong immunogenicity, can be used as the candidate antigens of staphylococcus aureus vaccine.But this SpA5 mutant still can be partly in conjunction with the Fc section of mammal IgG antibody, thereby caused difficulty to the detection of SpA5 specific IgG.Inventor finds early stage in the detection to gained serum after SpA5 immune animal, SpA5 mutant coated elisa plate and not immune negative control group seroreaction, still be weak positive findings, Here it is due to due to IgG antibody Fc section in SpA5 non-specific binding serum.
Summary of the invention
Thereby the present invention aims to provide a kind of detection method and records SpA5 antigen-specific antibodies to remove nonspecific interference, the method can solve the Fc section non-specific binding problem of SpA5 and IgG, can the SpA5 antigentic specificity IgG antibody in different plant species after vaccine immunity be detected, for studying antigenicity and the immunogenicity of SpA5, lay the foundation.
Aspect first, the invention provides a kind of staphylococcus aureus SpA5 mutant antigentic specificity IgG antibody detection method, comprise: 1) obtain the animal blood serum after SpA5 protein immunization, with pepsin enzyme, cut the F (ab ') of the antibody acquisition antibody in serum
2fragment; 2) ELISA detects the specific F of SpA5 (ab ')
2fragmentation IgG antibody.
On the other hand, the invention provides a kind of kit of the SpA5 of detection specific IgG antibodies, described kit comprises: enzyme cutting buffering liquid, pepsin, ELISA reaction reagent.
In the present invention, the amino acid sequence of mentioned SpA5 albumen is as shown in SEQ ID NO.1.
The present invention utilizes pepsin enzyme to cut and enzyme linked immunosorbent assay detects the SpA5 specific IgG antibodies in the animal blood serum with SpA5 vaccine immunity, obtains tiring of Serum Antibody.This detection method has been got rid of SpA(SpA5) interference that causes of non-specific binding antibody Fc section, has improved the specificity of method.Simultaneously, the method is simple to operation, favorable repeatability, in the Serum Antibody Detection of different plant species (BALB/C mice, SD rat, New Zealand's large ear rabbit etc.), all obtain checking, can be used for immunogenicity and the antigenicity research of recombination staphylococcus aureus vaccine.
Accompanying drawing explanation
Fig. 1: each treated animal serum antibody geometric mean titer figure.
Embodiment
Aspect first, the detection method of SpA5 specific IgG antibodies of the present invention comprises: 1) obtain the animal blood serum after SpA5 protein immunization, cut the F (ab ') of the antibody acquisition antibody in serum with pepsin enzyme
2fragment; 2) ELISA detects the specific F of SpA5 (ab ')
2fragmentation IgG antibody.
The concrete steps of the inventive method are as follows:
1) antibody preparation: in 4 ℃ of placement 2h, 4 ℃ of centrifugal 10min of 8000rpm/min, draw upper serum by the blood sample gathering after SpA5 protein immune animal, and-20 ℃ of placements are standby;
2) preparation enzyme is cut solution: in the sodium acetate solution of 0.1-0.2M, add pepsin, making pepsic final active unit is 30-150IU/mL, is preferably 60IU/mL, regulates pH to 4.0～4.6;
3) use 2) enzyme of preparation is cut solution by 10 times of the serum dilutions of step 1) acquisition, mixes rear 37 ℃ of water-bath enzymes and cuts 6h, every 1h concussion, mixes 5～10min; By this step, adopt pepsin cutting antibody to obtain a F (ab ')
2fragment and some small fragment pFc ', avoid the interference of Fc fragment;
4) the SpA5 albumen that adopts purifying detects the specific F of SpA5 (ab ') by ELISA
2fragmentation IgG antibody titer; Preferably, with the coated elisa plate of the SpA5 albumen of 2 μ g/ml concentration, the sample after step 3) enzyme is cut is diluted to after 1:2000 with antibody diluent, by the method for doubling dilution, measures wherein the specific F of SpA5 (ab ')
2fragmentation IgG antibody titer.
On the other hand, the invention provides a kind of kit of the SpA5 of detection antigentic specificity IgG antibody, described kit comprises: enzyme cutting buffering liquid, pepsin, ELISA reaction reagent.Preferably, the sodium acetate solution that described enzyme cutting buffering liquid is 0.1-0.2M; Preferably, described ELISA reaction reagent comprises: coating buffer, antibody diluent, cleansing solution, confining liquid and stop buffer.Those skilled in the art according to this area routine techniques can be beyond all doubt know to need which reagent to carry out ELISA experiment.
Experiment material used in the present invention and main agents are as follows:
1, animal used as test
BALB/C mice (Beijing China Fukang), SD rat (Beijing Vital River Experimental Animals Technology Co., Ltd.), New Zealand's large ear rabbit (Third Military Medical University's Experimental Animal Center).
2, experiment material
SpA5(30 μ g/600 μ l) bacterin preparation, is prepared by present inventor oneself.
3, main agents
Glycocoll, pepsin are purchased from Shanghai Sheng Gong bioengineering company limited; Common and F (ab ')
2fragmentation goat anti-mouse, rat and rabbit igg two are anti-purchased from the Shanghai henry commerce and trade company limited of doing; Solubility single component substrate solution is acted on behalf of purchased from Tian Gen Chongqing, Beijing; Sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, polysorbate 20 are all purchased from Chemical Reagent Co., Ltd., Sinopharm Group; Potassium chloride is purchased from Chengdu Ke Long chemical reagent factory; PBS phosphate buffer is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge.
4, reagent preparation
1) enzyme is cut solution: the sodium acetate solution of 0.2M, adding pepsin to make its final active unit is 60IU/mL, regulates pH to 4.0-4.6.
2) ELISA reagent preparation
1. coating buffer: take Na on electronic balance
2cO
31.6g, NaHCO
32.9g, regulates pH to 9.6, and distilled water is settled to 1000mL;
2. antibody diluent: take NaCl8g on electronic balance, KH
2pO
40.2g, Na
2hPO
412H
2o2.9g, KCl0.2g, polysorbas20 0.5mL, regulates pH to 7.4, and adding distil water is settled to 1000mL;
3. cleansing solution: 0.05% polysorbas20-PBS(pH7.4);
Getting specification is that 1000mL/ bag PBS1 bag is dissolved in 1000mL pure water, adds 0.5mL polysorbas20;
4. confining liquid (now with the current):
The antibody diluent of 20ml, the ratio in 1% adds BSA, and 4 ℃ of placements are standby;
5. stop buffer (2mol/L sulfuric acid):
The 22.2mL concentrated sulphuric acid is added to 177.8mL ddH
2in O.
Embodiment 1: the immunity of animal and the acquisition of immune serum
1) animal grouping: according to random principle, animal is divided into vaccine immunity group and control group, grouping situation is as shown in table 1.
Table 1: immunogenicity research experiment animal grouping
2) immunity of animal used as test: vaccine group adopts the intramuscular injection of SpA5 albumen, one (600 μ L) of every animal injection; Control group adopts the physiological saline immunity of same volume, and immune programme for children is three immunity in 0 day, 14 days, 21 days.
3) blood sampling: last immunity finishes to get for latter 14 days blood, mouse is got blood by extracing eyeball, and rat adopts tail venous blood sampling, and rabbit adopts auricular vein to get blood.Blood is placed in 4 ℃ and hatches after 2 hours, the centrifugal 10min of 8000rpm, and separation of serum is also stored in-20 ℃, standby.
Embodiment 2: pepsin enzyme is cut the antibody in serum
Get the serum 20 μ l that embodiment 1 obtains, add the enzyme of 180 μ l to cut solution, after mixing, in 37 ℃ of water-bath enzymes, cut 6h, every 1h concussion, mix 5min.
Embodiment 3: the detection of anti-SpA5 specific antibody
Experimental technique:
1) coated: with SpA5 albumen to the 2 μ g/mL of coating buffer dilution purifying.100 μ L/ holes are coated with elisa plate, and fully concussion paving is even, and 4 ℃ of refrigerators are placed and spent the night or 37 ℃ of 2h.
2) sealing: cleansing solution is washed plate 4 times, each liquid feeding 300 μ L, vibrations 30s, imbibition 2.5s.With confining liquid 200 μ L/ hole sealase bracings, 4 ℃ of refrigerators are placed and are spent the night or 37 ℃ of 2h.
3) add primary antibodie: cleansing solution is washed plate 4 times, each liquid feeding 300 μ L, vibrations 30s, imbibition 2.5s.Serum after above-mentioned enzyme is cut dilutes 200 times again to 1:2000, then uses 7 gradients of 2 times of doubling dilutions of antibody diluent, and vibration shakes up.Sample after dilution is added to elisa plate with 100 μ L/ holes, 37 ℃ of incubation 40min.
4) add two to resist: cleansing solution is washed plate 4 times, each liquid feeding 300 μ L, vibrations 30s, imbibition 2.5s.By the F of HRP mark (ab ')
2the anti-antibody diluent 1:10000 that uses of fragmentation goat anti-mouse, rat and rabbit igg two, adds by 100 μ L/ holes, and vibration shakes up, 37 ℃ of incubation 40min.Adopt common goat anti-mouse, rat and rabbit igg two to resist contrasts simultaneously.
5) colour developing: wash plate 4 times with cleansing solution, each liquid feeding 300 μ L, vibrations 30s, imbibition 2.5s.By 100 μ L/ holes, add nitrite ion again, in dark place colour developing 5-10min.
6) cessation reaction: when colour developing finishes, 50 μ L/ holes add 2mol/L H
2sO
4cessation reaction.Adopt microplate reader to measure each hole OD value under 492nm.
7) statistical method: adopt A
sample/ A
negative valuethe positive standard of >2.1, obtains the high dilution of specific antibody in each serum.Adopt geometric mean titer to make histogram.
Experimental result:
As shown in table 2, ELISA testing result shows: if adopt common IgG bis-anti-in detection, because SpA5 albumen contains IgG binding structural domain, can be non-specifically in conjunction with the Fc section of mammal IgG antibody, vaccine immunity group is similar with control group testing result, does not have notable difference.But when adopting F (ab ')
2after fragmentation goat anti-mouse, rat and rabbit igg two are anti-, control group result is all negative, has got rid of nonspecific interference, can reflect more accurately the true horizon of SpA5 protein-specific IgG antibody in serum, and specificity is higher.Fig. 1, for asking geometric mean titer (GMT) by the antibody titer that each experimental mice serum of different plant species is detected, makes histogram analysis result.
Serum specific antibody bioactivity after table 2:SpA immunity different animals
By above embodiment; those skilled in the art utilize this area relevant knowledge that the detection method providing in the present invention can be provided apparently to prepare coherent detection kit, for diagnosing antigen-specific antibodies level and the immune protective effect evaluation after SpA5 mutant protein vaccine immunity.
Claims (10)
1. a method that detects SpA5 protein-specific IgG, comprises step: 1) obtain the animal blood serum after SpA5 protein immunization, cut the F (ab ') of the antibody acquisition antibody in serum with pepsin enzyme
2fragment; 2) ELISA detects the specific F of SpA5 (ab ')
2fragmentation IgG antibody; The amino acid sequence of described SpA5 albumen is as shown in SEQ ID NO:1.
2. method according to claim 1, wherein, is used enzyme to cut solution enzyme and cuts antibody in step 1), and described enzyme is cut the pepsin that the sodium acetate that solution comprises 0.1-0.2M, final active unit are 30-150IU/mL, pH4.0～4.6.
3. method according to claim 2, wherein, described pepsic final active unit is 60IU/mL.
4. for a kit for the method as described in claim 1～3 any one, described kit comprises: enzyme cutting buffering liquid, pepsin, ELISA reaction reagent.
5. kit according to claim 4, wherein, the sodium acetate solution that described enzyme cutting buffering liquid is 0.1-0.2M.
6. according to the kit described in claim 4 or 5, wherein, described ELISA reaction reagent comprises: coating buffer, antibody diluent, cleansing solution, confining liquid and stop buffer.
7. kit according to claim 6, wherein, coating buffer comprises described in 1000mL: Na
2cO
31.6g, NaHCO
32.9g, pH value is 9.6.
8. kit according to claim 6, wherein, antibody diluent comprises described in 1000mL: NaCl8g, KH
2pO
40.2g, Na
2hPO
412H
2o2.9g, KCl0.2g, polysorbas20 0.5mL, pH value is 7.4.
9. kit according to claim 6, wherein, described cleansing solution is 0.05% polysorbas20-PBS solution, pH value is 7.4.
10. kit according to claim 6, wherein, described confining liquid is: the ratio in 1% adds BSA to 20ml antibody diluent.
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Cited By (3)
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| WO2015144653A1 (en) * | 2014-03-26 | 2015-10-01 | Glaxosmithkline Biologicals S.A. | Mutant staphylococcal antigens |
| CN111484552A (en) * | 2019-12-26 | 2020-08-04 | 成都欧林生物科技股份有限公司 | Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody |
| CN112898387A (en) * | 2021-03-24 | 2021-06-04 | 成都欧林生物科技股份有限公司 | Purification method of recombinant staphylococcus aureus vaccine antigen protein |
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| BE1022744B1 (en) * | 2014-03-26 | 2016-08-29 | Glaxosmithkline Biologicals Sa | MUTANT STAPHYLOCOCCAL ANTIGENS |
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| KR20190110655A (en) * | 2014-03-26 | 2019-09-30 | 글락소스미스클라인 바이오로지칼즈 에스.에이. | Mutant staphylococcal antigens |
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| WO2015144655A1 (en) * | 2014-03-26 | 2015-10-01 | Glaxosmithkline Biologicals S.A. | Immunising against staphylococcus aureus |
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| WO2021128946A1 (en) * | 2019-12-26 | 2021-07-01 | 成都欧林生物科技股份有限公司 | Anti-spa5 protein monoclonal antibody, use thereof and kit containing same |
| CN111484552B (en) * | 2019-12-26 | 2022-03-04 | 成都欧林生物科技股份有限公司 | Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody |
| CN111484552A (en) * | 2019-12-26 | 2020-08-04 | 成都欧林生物科技股份有限公司 | Monoclonal antibody against SpA5 protein, application thereof and kit containing monoclonal antibody |
| CN112898387A (en) * | 2021-03-24 | 2021-06-04 | 成都欧林生物科技股份有限公司 | Purification method of recombinant staphylococcus aureus vaccine antigen protein |
| CN112898387B (en) * | 2021-03-24 | 2023-04-28 | 成都欧林生物科技股份有限公司 | Purification method of recombinant staphylococcus aureus vaccine antigen protein |
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