CN1563982A - Bolting and identifying method for microbial cross antigen - Google Patents
Bolting and identifying method for microbial cross antigen Download PDFInfo
- Publication number
- CN1563982A CN1563982A CN 200410033282 CN200410033282A CN1563982A CN 1563982 A CN1563982 A CN 1563982A CN 200410033282 CN200410033282 CN 200410033282 CN 200410033282 A CN200410033282 A CN 200410033282A CN 1563982 A CN1563982 A CN 1563982A
- Authority
- CN
- China
- Prior art keywords
- microorganism
- cross
- protein
- antibody
- antiserum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The method includes following steps: two-dimensional electrophoresis separates protein of microbe, after immunization transfer printing, multiantiserum is as first antibody, enzyme marked antibody as second antibody, after color development, checking positive; analyzing immunogenic cross degree and range; determining the nature of immunogen and protein category of antigen. The invention determines all proteins possessing cross immunogenicity of a microbe rapidly. Features are high efficiency, fast, economic and practical. The invented authenticating method is applicable to bacteria, fungus and virus.
Description
Technical field
The present invention relates to the method for a kind of high flux screening and Identifying micro-organisms cross-reacting antigen.
Background technology
Proved already that adopting vaccine inoculation was the effective way of prophylaxis of microbial disease.Owing to pathogen is more, all pathogens are inoculated one by one have certain difficulty.This shows very outstandingly in the prevention of cultivated animals microorganism property disease.Because in actual production, as all common pathogens at cultivated animals carry out one by one immunity economically with the operation on be impossible.Therefore, the development new generation vaccine is a urgent task with control diseases related, particularly cultivated animals disease.There is the scholar to find that lipopolysaccharides (LPS) and cAg between different bacterium have cross-protection.Recently, we find to have tangible cross-protection between Aeromonas hydrophila and the Aeromonas sobria, and adopt the protection of proteomics binding immunoassay to attack malicious technology and found 2 polyvaccine target position from the external membrane protein of Aeromonas hydrophila.There is cross-neutralization originality gene in these presentation of results between bacterium.Therefore, development genetic engineering multivalent vaccine will become the most important aspect of vaccine development from now on.The polyvaccine target position is from the immunogene with cross reaction, but the employing conventional method identifies that from whole microproteins all protein with cross immunity originality are very difficult.At present former discovery is assert from the observed cross immunity phenomenon of a kind of chance often to cross immunity, or individual gene determined that therefore, efficient is not high.Proteomics (proteomics) is a kind of technology of high flux screening, it is the important platform of functional genome research, by by proteomics and immune protein group (immunoproteomics) technology that Western blotting (Western blotting) technology combines and grows up, in the patients serum, identified the immunogenic protein of streptococcus albus (Candida albicans) and sand holes Chlamydia (Chlamydia trachomatis).These immunity antigens can be used as a kind of index of monitoring of diseases progress, will become the object of development novel vaccine, help to break away from the constraint of traditional vaccine development, accelerate the development of novel vaccine.But existing immune protein group learns a skill and only can determine to have immunogenic protein, and can't determine whether this protein has cross immunity originality.
Summary of the invention
The present invention aims to provide the method for a kind of high flux screening and Identifying micro-organisms cross-reacting antigen, its technical scheme is to utilize dielectrophoresis (2-DE) technology that microprotein is showed in one plane, adopt the immune protein group of multiple antiserum (alloantiserum and heterologous antiserum) to learn a skill again, determine to have the protein of cross immunity originality, adopt the mass-spectrometric technique analysis at last and identify these immunogenes.
The screening of the said microorganism cross-reacting antigen of the present invention and the concrete steps of authentication method are as follows:
1) with dielectrophoresis method separate microorganism protein: behind microorganism-collecting, by quality than microorganism: ultrasonic damping fluid=1: 2～4 add ultrasonic damping fluid, and ultrasonic disruption carries out dielectrophoresis then.
2) behind the dielectrophoresis, carry out immunoblotting, adopt multiple antiserum as first antibody respectively, enzyme labelled antibody is a second antibody, with benzidine (DAB) or electrochemiluminescence reagent (ECL) colour developing, when the antiserum as first antibody has more than 2 kinds when positive, this point is the protein with cross immunity originality.
3) analyze these immunogenic intersection degree and scopes again, positive antiserum is many more, and the cross reaction scope is big more; Positive colour developing is strong more, and the cross reaction degree is good more.
4) immunogenic qualitative: take out all protein with cross immunity originality, the mass spectrum sample disposal route is carried out specimen preparation routinely, carries out mass spectrum and analysis of biological information then, to determine the kinds of protein of antigen.
Said microorganism is a bacterium, fungi, virus.
Said ultrasonic damping fluid can be selected Tris-HCl or ethylene dinitrilotetra-acetic acid salt (EDTA) etc. for use.
The output power that is adopted during ultrasonication, time, number of times and the time interval etc. can be adjusted by research object, generally can adopt each 10～20 seconds, and 20～40 seconds at interval, ultrasonic 2～4 times.
Said multiple antiserum is selected the specific antisera of anti-microorganism of the same race and xenogenesis microorganism for use.
The present invention utilizes bidirectional electrophoresis technique that bacterium or virus protein are showed in one plane earlier, the Western blotting that adopts of the same race and foreign sera again utilizes mass-spectrometric technique and analysis of biological information to determine the kind of these cross immunity originality protein to determine having immunogenic protein at last.Thereby set up and a kind ofly adopted multiple antiserum to learn a skill, be used for high flux screening and have the protein of cross immunity originality with candidate's target position as polyvaccine as the immune protein group of first antibody.According to immunogenic intersection degree and scope, can determine that it is as cross immunity former possibility and reach thereof.The present invention can determine all protein with cross immunity originality of certain microorganism rapidly, thereby reaches efficient, quick, the economic, practical former purpose of preferred cross immunity.The present invention can high flux screening has and intersects the vaccine candidate site of originality, for determining to have established solid foundation as the gene of polyvaccine target position, has improved the efficient of preparation polyvaccine target spot.Since all microorganisms as antigen-immunized animal after, body all can produce the specific antibody at its antigen, these antibody all can be used as first antibody and carry out Western blotting test, it is former to be used to screen cross immunity.So principle of the present invention and method have ubiquity, not only can be used for bacterium, but also can be used for the evaluation of microorganism cross-reacting antigens such as fungi and virus.
The present invention has further developed the immune protein group and has learned a skill, and promptly sets up a kind ofly to adopt multiple antiserum to learn a skill as the immune protein group of first antibody, thereby can determine with more than one antiserums immunoreactive protein to take place.Because this design is based on the proteomic techniques of high flux screening, thus not only can determine that fast the cross immunity that is had in certain microorganism is former, and can learn these immunogenic crossover range.According to these immunogenic intersection degree and scope, can determine possibility and reach as the polyvaccine target position.
Description of drawings
Fig. 1 is the immunoblotting result of the dielectrophoresis of vibrio parahaemolytious and himself and heterologous antiserum.Wherein, the 2-DE collection of illustrative plates of A, vibrio parahaemolyticus outer membrane protein matter; The 2-DEWestern-blot result of B, vibrio parahaemolytious antiserum and vibrio parahaemolyticus outer membrane protein matter; The 2-DEWestern-blot result of C, Aeromonas hydrophila antiserum and vibrio parahaemolyticus outer membrane protein matter; The 2-DE Western-blot result of D, Vibrio flurialis antiserum and vibrio parahaemolyticus outer membrane protein matter; The 2-DE Western-blot result of E, vibrio alginolyticus antiserum and vibrio parahaemolytious adventitia.
Fig. 2 is the Western blotting result of colibacillary 2-DE and himself and heterologous antiserum.Wherein, the 2-DE collection of illustrative plates of A, escherichia coli outer membrane protein matter; The 2-DEWestern-blot result of B, Aeromonas sobria antiserum and escherichia coli outer membrane protein matter; The 2-DE Western-blot result of C, Escherichia coli antiserum and escherichia coli outer membrane protein matter; The 2-DE Western-blot result of D, vibrio parahaemolytious antiserum and escherichia coli outer membrane protein matter; The 2-DE Western-blot result of E, Vibrio flurialis antiserum and escherichia coli outer membrane protein matter.
Embodiment
Following examples will be described further processing step of the present invention and outstanding effect thereof in conjunction with the accompanying drawings.
Embodiment 1
1, microbe growth, counting, collection and fragmentation: microorganism is adopted vibrio parahaemolytious, for preserving bacterial classification in this chamber.Vibrio parahaemolytious is inoculated in the LB nutrient culture media, and 28 ℃ of shaking tables are cultivated 18h, as kind of a daughter bacteria.Then (bacterium liquid: nutrient culture media) (volume ratio) ratio was inoculated in above-mentioned nutrient culture media of the same race, and 28 ℃ of shaking tables are cultivated 18h with 1: 50.After cultivating end, get 2mL bacterium liquid and carry out plate count.All the other nutrient solutions are in the centrifugal 10min of 4000rpm, collect thalline, with physiological saline washing thalline 3 times, claim thalline heavy, again in order to the ratio of 1: 3 (mass ratio) add the ultrasonication damping fluid (50mmol/L Tris-HCl, 1mmol/LEDTA).With the ultrasonication of ultrasonic cell disintegration instrument.Output power 40% is adopted in ultrasonication, and each 10～20 seconds, 20～40 seconds at interval, ultrasonic 2～4 times.
2, the sero-fast preparation of anti-microbial pathogen: cultivate vibrio parahaemolytious, vibrio alginolyticus, Aeromonas hydrophila, the Vibrio flurialis of 18h, the centrifugal 10min of 4000rpm; Suspend with 0.65% physiological saline, the blood counting chamber counting is diluted to 1 * 108/mL with bacterium liquid; Inject mouse respectively, every 0.1mL, control group injecting normal saline; Every 1 week injection 1 time; Put to death mouse in the 10th day after the 3rd all immunity and get blood, leave standstill; The centrifugal 10min of 4000rpm takes out serum, packing ,-20 ℃ of preservations.
3, the extraction of outer membrane protein: the vibrio parahaemolytious liquid after the ultrasonication, 2500g, 4 ℃ of centrifugal 10min collect supernatant.Supernatant 100,000g, 4 ℃ of centrifugal 40min.Abandon supernatant, precipitation (is assigned in 50mmol/L Tris-HCl, pH7.4) washing, room temperature placement 20min with 2% lauroyl propylhomoserin sodium.100,000g, 4 ℃ of centrifugal 40min.Precipitation is dissolved in the ultrasonic damping fluid of certain volume.With protein concentration in the Bradford method working sample.
4, dielectrophoresis method operation steps: first to prerunning 200V 15min, 300V 30min, 400V 2h, last sample (sample is a vibrio parahaemolytious liquid) back voltage 400V, take out adhesive tape after the 18h isoelectric focusing rapidly, move to 10% dodecyl sodium sulfonate after the balance and receive and continue electrophoresis on polyacrylamide gel electrophoresis (SDS-PAGE) glue, the one end outside adds standard protein, 1% agarose sealing.Electrophoresis finishes the back with Coomassie brilliant blue R-250 dyeing, scans the output photo.Referring to Fig. 1-A, the result shows, surplus the more clearly protein site 40.
5, Western-blotting operation steps: after dielectrophoresis finishes, carry out electricity and change, constant voltage 40V electricity turns over night; After electricity changes end, detect the effect that electricity changes with Ponceaux dyeing; The flush away Ponceaux adds confining liquid (skimmed milk power is formulated as 5% final concentration with TTBS) sealing, incubated at room 1h; Trishydroxymethylaminomethane-hydrochloric acid-polysorbas20 damping fluid (TTBS) is washed film, and 3 times, each 5min; Add mouse-anti vibrio parahaemolytious, vibrio alginolyticus, Aeromonas hydrophila, Vibrio flurialis antiserum (preparing) respectively, incubated at room 1h with confining liquid; Take out film, TTBS washes film 3 times, each 10min; Add horseradish peroxide labelled goat anti-mouse antibody (preparing), incubated at room 1h with confining liquid; Take out film, TTBS washes film 3 times, each 10min; 50mmol/LpH7.4 Tris-HCl washes film 5min; The DAB colour developing; Cessation reaction, film is sealed in scanning, preserves.Western-blotting analyzes, and the result has 8 antigens association reactions to occur significantly intersecting (to see Fig. 1-B～E), be numbered 1～8 respectively.
6, the preparation of mass spectrum sample: downcut the destination protein point with a clean scalper, downcut a roughly the same blob of viscose from no protein contamination district, blob of viscose is cut into about 1mm for contrast
3Size places the 0.5ml test tube, then carries out according to the following steps: wash blob of viscose to take off Coomassie brilliant blue (2～3 times * 15min), abandon 50%ACN with 50% acetonitrile (ACN); 100%ACN covers blob of viscose to white, abandons ACN; Then use 0.1mol/L NH
4HCO
3ACN/0.1mol/LNH is used in swelling then repeatedly
4HCO
3Be washed till colourless, the traditional vacuum drying.With 10mmol/L dithiothreitol (DTT) (DTT)/0.1mol/L NH
4HCO
3Behind 56 ℃ of reduction 45min, use the 55mmol/L iodoacetamide/0.1mol/LNH of fresh configuration again
4HCO
3Alkylation, lucifuge 30min.It is clean to decolouring to repeat above-mentioned steps.Add pancreatin (Trypsin) (12.5 μ g/mL) after the traditional vacuum drying and hatch 45min on ice, sop up and add no enzymic digestion liquid behind the unnecessary enzyme liquid, 37 ℃ are spent the night.Use 5% trifluoroacetic acid (TFA) and 50%ACN/2.5%TFA extracting 1～2 time repeatedly successively, merge supernatant, the traditional vacuum drying.Add 1～2 μ L 0.1%TFA sample dissolution before the point sample.
7, the analysis and the data base querying of peptide quality fingerprinting collection of illustrative plates sample: mass spectrum sample uses the ground substance assistant laser desorption ionization (ReFlex of German BRUKER company
TMIII MALDI-TOF) mass spectrometer is analyzed, reflective-mode, and ion gun accelerating potential 1 is 20kv, accelerating potential 2 is 23kv, N
2Optical maser wavelength 337nm, pulse width is 3ns, ion postpones to extract 2000ns, vacuum tightness 1.4 * 10
-7Torr, mass signal single sweep operation add up 50 times, and proofread and correct mass spectra peak with standard protein molecule peak as external standard, and the positive ion spectrum is measured, and obtain peptide quality fingerprinting collection of illustrative plates.
By Mascot (
Http:// www.matrixscience.com.) database selection NCBI, kind selection otherproteobacteria.Peptident (
Http:// www.expasy.org/tools/peptident.htmL). database is selected Swiss-prot, and kind is selected other bacterium (other bacteria). inquire about with the Peptident software (http://www.expasy.org/tools/peptident.html) that ExPASy Molecular Biology Server website provides.Querying condition: the fragments of peptides quality control in the peptide quality fingerprinting collection of illustrative plates is 800～3500, the error range of apparent molecular weight is ± 20%, apparent pI value is not done requirement, fragments of peptides molecular weight margin of error scope is ± 0.5Da, each peptide allows 2 incomplete cracking sites, the source of species selecting bacteria, ion is selected [M+H]
+(option in the database, no Chinese name) and single isotope (monoisotopic), minimum coupling fragments of peptides number is defined as 4, and aminothiopropionic acid is that iodoacetamide is handled.Qualification result sees Table 1.
The MALDI-TOF/MS of the cross-reacting protein matter of table 1 vibrio parahaemolytious and self and heterologous antiserum identifies
| Protein site | Anti-secondary haemolysis serum | Anti-molten alginic acid serum | Resist and have a liking for aqueous vapor serum | Anti-Vibrio flurialis | Qualification result |
| ????1 | ????+ | ????+ | ????+ | ????+ | Surface antigen (Surface antigen) |
| ????2 | ????+ | ????+ | ????+ | Outer membrane protein TolC (Outer membrane protein TolC) | |
| ????3 | ????+ | Long-chain fatty acid transfer protein (long-chain fatty acid transport protein) | |||
| ????4 | ????+ | ????+ | ????+ | ????+ | Outer membrane protein A (Outer membrane protein A) |
| ????5 | ????+ | Putative protein VP1475 (hypothetical protein VP1475) | |||
| ????6 | ????+ | ????+ | ????+ | ????+ | Outer membrane protein A (Outer membrane protein A) |
| ????7 | ????+ | ????+ | ????+ | ????+ | The peptide glycan lipoprotein (peptidoglycan-associated lipoprotein) of being correlated with |
| ????8 | ????+ | ????+ | ????+ | Lipoprotein (Lipoprotein) |
Embodiment 2 is except that bacterium changes Escherichia coli into, and all the other are all identical with embodiment 1.The result is as follows:
1, the 2-DE collection of illustrative plates of Bacillus coli cells adventitia
Adopt the 2-DE technology, colibacillary epicyte is analyzed, found that about 40 protein sites, see Fig. 2-A.
2, immunogenic protein is determined
Adopt Western blotting technology, be first and second antibody with mouse-anti Aeromonas sobria, Escherichia coli, vibrio parahaemolytious, Vibrio flurialis antiserum and the anti-mouse of HRP-rabbit respectively, carrying out Western blotting by test routine analyzes, the result has 6 antigens tangible association reaction to occur, is numbered 1～6 respectively.The Western blotting of Bacillus coli cells adventitia the results are shown in Figure 2-B～E.
3, MALDI-TOF peptide quality fingerprinting analysis of spectrum
Adopt biological mass spectrometry that 6 protein sites with cross immunity originality are analyzed, obtain peptide quality fingerprinting collection of illustrative plates separately respectively.
According to peptide dactylogram data and molecular weight ranges and some other parameters of obtaining, by the protein that matches in the Peptident software inquiry SWISS-PROT database, Search Results is as shown in table 2.
The MALDI-TOF/MS of the cross-reacting protein matter of table 2. Escherichia coli and self and heterologous antiserum identifies
| Protein site | Chinese People's Anti-Japanese Military and Political College enterobacteria | Temperature resistance and Aeromonas | Anti-Vibrio flurialis | Anti-vibrio parahaemolytious | Qualification result |
| ????1 | ????+ | ????+ | ????+ | Mucopeptide GLP (Murein lipoprotein) | |
| ????2 | ????+ | ????+ | ????+ | ????+ | Outer membrane protein X (Outer membrane protein X) |
| ????3 | ????+ | ????+ | Adjusting of starvation conditions (global regulator, starvation conditions) | ||
| ????4 | ????+ | ????+ | ????+ | Outer membrane protein A (Outer membrane protein A) | |
| ????5 | ????+ | ????+ | ????+ | Outer membrane protein 3a (Outer membrane protein 3a) | |
| ????6 | ????+ | ????+ | ????+ | ????+ | Outer membrane protein tolC precursor (Outer membrane protein tolC precursor) |
Claims (5)
1, the screening of microorganism cross-reacting antigen and authentication method is characterized in that its step is as follows:
1) with dielectrophoresis method separate microorganism protein: behind microorganism-collecting, by quality than microorganism: ultrasonic damping fluid=1: 2～4 add ultrasonic damping fluid, and ultrasonic disruption carries out dielectrophoresis then;
2) behind the dielectrophoresis, carry out immunoblotting, adopt multiple antiserum as first antibody respectively, enzyme labelled antibody is a second antibody, with benzidine or electrochemiluminescence reagent colour development, when the antiserum as first antibody has more than 2 kinds when positive, this point is the protein with cross immunity originality;
3) analyze these immunogenic intersection degree and scopes again, positive antiserum is many more, and the cross reaction scope is big more; Positive colour developing is strong more, and the cross reaction degree is good more;
4) immunogenic qualitative: take out all protein with cross immunity originality, the mass spectrum sample disposal route is carried out specimen preparation routinely, carries out mass spectrum and analysis of biological information then, to determine the kinds of protein of antigen.
2, the screening of microorganism cross-reacting antigen as claimed in claim 1 and authentication method is characterized in that said microorganism is a bacterium, fungi, virus.
3, the screening of microorganism cross-reacting antigen as claimed in claim 1 and authentication method is characterized in that said ultrasonic damping fluid selects Tris-HCl or ethylene dinitrilotetra-acetic acid salt for use.
4, the screening of microorganism cross-reacting antigen as claimed in claim 1 and authentication method, when it is characterized in that ultrasonication each 10～20 seconds, 20～40 seconds at interval, ultrasonic 2～4 times.
5, the screening of microorganism cross-reacting antigen as claimed in claim 1 and authentication method is characterized in that said multiple antiserum selects the specific antisera of anti-microorganism of the same race and xenogenesis microorganism for use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410033282 CN1247795C (en) | 2004-04-14 | 2004-04-14 | Bolting and identifying method for microbial cross antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410033282 CN1247795C (en) | 2004-04-14 | 2004-04-14 | Bolting and identifying method for microbial cross antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1563982A true CN1563982A (en) | 2005-01-12 |
| CN1247795C CN1247795C (en) | 2006-03-29 |
Family
ID=34481332
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200410033282 Expired - Fee Related CN1247795C (en) | 2004-04-14 | 2004-04-14 | Bolting and identifying method for microbial cross antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1247795C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103616434A (en) * | 2013-12-12 | 2014-03-05 | 佟雪梅 | Method for identifying microorganisms by adopting mass spectrum |
| CN103917873A (en) * | 2011-07-28 | 2014-07-09 | 图宾根埃伯哈德·卡尔斯大学 | Method for detecting biomolecules |
| CN104634979A (en) * | 2013-11-15 | 2015-05-20 | 汕头大学 | Method for screening and identifying microbial cell surface antigen |
| CN110628796A (en) * | 2019-05-20 | 2019-12-31 | 广东海洋大学深圳研究院 | Fish Nocardia disease common antigen DNA vaccine and preparation and application thereof |
| CN113388033A (en) * | 2021-05-26 | 2021-09-14 | 浙江理工大学 | Identification method of largemouth bass-derived aeromonas hydrophila immunogenic outer membrane protein |
| CN113820382A (en) * | 2021-09-18 | 2021-12-21 | 中国医学科学院医学实验动物研究所 | Novel screening method of mammal heterologous antigen and application thereof |
-
2004
- 2004-04-14 CN CN 200410033282 patent/CN1247795C/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103917873A (en) * | 2011-07-28 | 2014-07-09 | 图宾根埃伯哈德·卡尔斯大学 | Method for detecting biomolecules |
| US10274485B2 (en) | 2011-07-28 | 2019-04-30 | NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen | Method for detecting biomolecules |
| CN104634979A (en) * | 2013-11-15 | 2015-05-20 | 汕头大学 | Method for screening and identifying microbial cell surface antigen |
| CN103616434A (en) * | 2013-12-12 | 2014-03-05 | 佟雪梅 | Method for identifying microorganisms by adopting mass spectrum |
| CN103616434B (en) * | 2013-12-12 | 2015-08-19 | 佟雪梅 | Mass Spectrometric Identification method of microorganism |
| CN110628796A (en) * | 2019-05-20 | 2019-12-31 | 广东海洋大学深圳研究院 | Fish Nocardia disease common antigen DNA vaccine and preparation and application thereof |
| CN110628796B (en) * | 2019-05-20 | 2023-03-24 | 广东海洋大学深圳研究院 | Fish Nocardia disease common antigen DNA vaccine and preparation and application thereof |
| CN113388033A (en) * | 2021-05-26 | 2021-09-14 | 浙江理工大学 | Identification method of largemouth bass-derived aeromonas hydrophila immunogenic outer membrane protein |
| CN113820382A (en) * | 2021-09-18 | 2021-12-21 | 中国医学科学院医学实验动物研究所 | Novel screening method of mammal heterologous antigen and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1247795C (en) | 2006-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Croxatto et al. | Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology | |
| Cargile et al. | Identification of bacteriophage MS2 coat protein from E. coli lysates via ion trap collisional activation of intact protein ions | |
| Nishikawa et al. | Distribution of microbes producing antimicrobial ε-poly-L-lysine polymers in soil microflora determined by a novel method | |
| US9273339B2 (en) | Methods for identifying bacteria | |
| Amitai et al. | Distribution and function of new bacterial intein‐like protein domains | |
| US10144946B2 (en) | Mass spectrometric rapid detection of Salmonella | |
| CN102253110A (en) | Method for auxiliary identification of comma bacillus by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) | |
| CN105829544B (en) | Identification of microorganisms by mass spectrometry and infrared spectroscopy | |
| US20100057372A1 (en) | Rapid identification of proteins and their corresponding source organisms by gas phase fragmentation and identification of protein biomarkers | |
| WO2010100612A1 (en) | Method for identifying germs in a liquid medium | |
| Jabbour et al. | Identification of Yersinia pestis and Escherichia coli strains by whole cell and outer membrane protein extracts with mass spectrometry-based proteomics | |
| CN102520055A (en) | Construction method for MALDI-TOF-MS database of common pathogenic bacteria in food and animal products | |
| US8412464B1 (en) | Methods for detection and identification of cell type | |
| CN1247795C (en) | Bolting and identifying method for microbial cross antigen | |
| Vithanage et al. | Species-level discrimination of psychrotrophic pathogenic and spoilage gram-negative raw milk isolates using a combined MALDI-TOF MS proteomics–bioinformatics-based approach | |
| CN106093404A (en) | A kind of detection method of bacillus perfringens disease alpha toxin Envision SABC | |
| Kowalczewska et al. | Proteomics paves the way for Q fever diagnostics | |
| Kooken et al. | Assessment of marker proteins identified in whole cell extracts for bacterial speciation using liquid chromatography electrospray ionization tandem mass spectrometry | |
| CN1283995C (en) | Method for screening and identifying microbiological highly effective neutralization antigens | |
| Karlsson et al. | Proteotyping: Tandem mass spectrometry shotgun proteomic characterization and typing of pathogenic microorganisms | |
| Afanas’ ev et al. | MALDI-ToF mass spectrometric analysis for the identification of plague, cholera, and tularemia causative agents | |
| WO2021244001A1 (en) | Peptide fragment-based targeted proteome accurate quantification method | |
| Jabbour et al. | Extracellular protein biomarkers for the characterization of enterohemorrhagic and enteroaggregative Escherichia coli strains | |
| Shah et al. | Microbiological identification using MALDI-TOF and tandem mass spectrometry: industrial and environmental applications | |
| Marshall et al. | Immunochemical identification of Fusarium moniliforme ribosomes from diseased corn (Zea mays L.) stalk tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20060329 |