CN101535811B - For the method for in-vitro diagnosis PVL-producing staphylococcus aureus - Google Patents
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- CN101535811B CN101535811B CN200780038592.4A CN200780038592A CN101535811B CN 101535811 B CN101535811 B CN 101535811B CN 200780038592 A CN200780038592 A CN 200780038592A CN 101535811 B CN101535811 B CN 101535811B
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- staphylococcus aureus
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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 present invention relates to use that staphylococcus aureus is built group or the individual biological sample that infects carrys out in-vitro diagnosis and produce the method for the staphylococcus aureus of P-VL (PVL) from being easy to occur, wherein carry out described diagnosis by adopting conventional Radioimmunoassay of vascular endothelial growth to detect PVL, it is characterized in that described biological sample to carry out pretreatment to make PVL sex change.
Description
The field of the invention relates to existence and belongs to infection that the bacterium of staphylococcus is relevant and produce Pan with existingThe staphylococcus aureus of pause-Valentine's leukocidin (Panton-ValentineLeukocidin, PVL)(Staphylococcusaureus) relevant infection. More specifically, theme of the present invention is to use conventional immunologic assayMethod is determined the method for PVL-producing staphylococcus aureus in the biological sample that may contain staphylococcus aureus.
The infection that PVL-producing staphylococcus aureus causes mainly causes community to infect. Staphylococcus aureus is expressed multipleVirulence factor, one of them is P-VL (PVL), it is a kind of cell toxicant that causes disorganizationElement1. This toxin of PVL is common in Community acquired-methicillin resistant Staphylococcus aureus (CA-MRSA) bacterial strain, and the latter is entirelyThe world is widely distributed2。
PVL and other leukocidin are the protein that belongs to Synergohymenotropic toxin family. This familyAll toxin of family form by having synergistic two polypeptide fractions, are called S and F. Two vicinities that PVL is recorded by corotationGene is lukF-PV and lukS-PV coding. PVL is exotoxin, because no matter whether cleaved its of cell all secreted to extracellularIn medium, this is different from endotoxin or lipopolysaccharides, and they only just discharge in the time that their Gram-negative bacteria of secretion is destroyed.
PVL-producing staphylococcus aureus is gram-positive bacteria, its mankind cause relevant specific infection for example furuncle,The skin infection of abscess, cellulitis and myositis type or subcutaneous infection, bone joint infection and serious gangrenosum acne lungInflammation, the latter mainly involves children and youth, and the death rate is about 70%.
Although its pathogenesis it be unclear that, there are some evidence prompting PVL to cause PVL-producing staphylococcus aureusThe Pathological Physiology of infection in play a significant role:
I) between the staphylococcus aureus separator of synthetic PVL and the clinical manifestation of infection, there is EPDML strong passConnection property,
Ii) low leucocyte mass formed by blood stasis (one of known effect of PVL) occurrence rate is high,
Iii) respiratory tract gangrenosum acne pathology, similar with the necrosis of causing to rabbit intracutaneous injection PVL,
Iv) in lung, there is the PVL of target polymorphonuclear cell,
V) only have the bacterial strain of PVL-producing or the PVL of purifying could induce animal used as test to occur necrotizing pneumonia7。
What detection PVL-producing staphylococcus aureus adopted at present is the determination method that detects nucleic acid, and particularly passes through PCRDetect lukF-PV and lukS-PV3。
The defect of this type of determination method is that they are round-about ways, because they can not determine whether gene is to have functionAnd/or express, due to the special therefore somewhat expensive of equipment of needs, neither be very quick, and also it is large to implement difficulty. In addition,Just use PCR, may occur the problem of polluting.
Sometimes also detect PVL with polyclonal antibody by SRID4, but this technology is made way for gene inspection very soonSurvey, this is that therefore it does not become the conventional good tool adopting because SRID is difficult for implementing and being difficult to standardization.
The seriousness of some infection causing due to PVL-producing staphylococcus aureus, therefore need badly a kind of easy to implement andCan overcome the rapid test method of the defect of the determination method of currently used detection PVL-producing staphylococcus aureus.
Patent application EP597110A discloses by the routine immunization determination method by anti-PVL monoclonal antibodyDetect MRSA. But, although these determination methods can overcome above-mentioned defect, its detection specificity is not high enough.
Unexpectedly, the applicant now confirms, although PVL the same with other exotoxins to physical chemical factor asTemperature is very responsive5, but by biological sample being carried out to pretreatment to make PVL sex change, can improve and adopt conventional immunityLearn the specificity that determination method detects PVL-producing staphylococcus aureus.
Therefore, theme of the present invention is to use from being easy to occur the individual life that staphylococcus aureus is built group or infectsThe thing product in-vitro diagnosis that imitates produces the method for staphylococcus aureus of P-VL (PVL), wherein passes throughAdopt conventional Radioimmunoassay of vascular endothelial growth to detect PVL and carry out described diagnosis, it is characterized in that described biological sample to carry out in advanceProcess to make PVL sex change.
Term " PVL-producing staphylococcus aureus " refers to all staphylococcus aureus strains that can produce PVL, bothComprise that methicillin-sensitivity bacterial strain (also claiming MSSA) also comprises methicillin-resistant bacterial strain (also claiming MRSA).
Term " is easy to occur staphylococcus aureus and builds group's individuality " to refer to those to be had possibility and becomes this bacteriumThe individuality of healthy carrier's risk, for example medical personnel. Term " is easy to occur the individuality of infection of staphylococcus aureus " and refers toBe the patient who shows infection of staphylococcus aureus symptom.
Term " infection of staphylococcus aureus " refers to any infection because existing this bacterium to cause in organism, nothingWhat opinion infected is PVL-producing staphylococcus aureus or the staphylococcus aureus without this ability. Para-infectious as thisExample can be mentioned pyogenic infection, respiratory tract infection, central nervous system infection, urinary tract infections, the heart of skin and soft tissueInfection and muscle and bone infection in valve and blood vessel. This type of infection and relevant symptom thereof are that those skilled in the art are knownAnd can be specifically referring to Pr é cisdeBact é riologieClinique5[HandbookofClinicalBacteriology]。
Term " from being easy to occur the individual biological sample that staphylococcus aureus is built group or infects " refers to easilyIn any sample of the PVL that contains these bacteriums or secretion, for example purulence, respiratory tract sample, nose sample, urine or blood cultureThing.
Can be the sample of unmodified for the biological sample of method of the present invention, or its can be derived from described inThe culture of the bacterium of sample. Can be at solid-phase culture base for example in culture dish or cultivate in culture broth, canUnderstand, the colony of the staphylococcus aureus that can separate from described biological sample by described sample or in advance is inoculated in cultivationIn meat soup, the method that forms colony is well known in the art, for example, by inoculating in culture dish. Then can be directly solidPhase culture medium or carry out immunologic assay in meat soup. Interested bacterium or sample are cultivated about 24 hours conventionally. WantNote, no matter which kind of training method, culture medium will contain the factor that promotes that PVL produces, for example CCY culture medium (caseinHydrolysate and yeast extract medium), can use or not use the molecule that can make PVL greater amount produce, for example sub-pressing downThe OXA of concentration processed and bacitracin.
Term " Radioimmunoassay of vascular endothelial growth " refers to any immunology of the binding partners that use can specific binding PVLDetermination method, described PVL can be the LukS fragment of LukF fragment, PVL of PVL or both.
Term " conventional Radioimmunoassay of vascular endothelial growth " refers to any immunologic assay that is widely used in laboratory routine workMethod. For example, conventional Radioimmunoassay of vascular endothelial growth can be the folder of ELISA or immunochromatographic method (also claiming lateral flow) formHeart determination method and particle (granules of polystyrene) aggegation test. All these determination methods are all well known in the art. CanIn one or more steps, carry out sandwich assay, there is no washing step or have one or more washing step.
For example, PVL specific binding partner can be antibody, antibody fragment, acceptor, mimotope and any energyEnough molecules in conjunction with PVL.
Binding partners antibody is for example polyclonal antibody or monoclonal antibody.
Can use PVL, PVL fragment or PVL peptide immune animal also to collect subsequently the serum of described animal and obtain polycloneAntibody. Antibody for method of the present invention can be purifying or unpurified. The purifying of polyclonal antibody for example can pass throughGet the serum of described animal and described antibody separated and carried out with other serum composition, particularly being undertaken by affinity chromatography,Antigen, particularly PVL that described antibody can specific recognition wherein on chromatographic column, are adhered to.
Can obtain monoclonal antibody by hybridoma technology, below summarize its basic principle.
In the first step, with PVL, PVL fragment or PVL peptide immune animal, normally mouse is (if external immunity isThe cell of cultivating), the bone-marrow-derived lymphocyte of this mouse just can produce the antibody for this antigen thus. Subsequently these are produced anti-The lymphocyte of body and " immortality " myeloma cell (be mouse in embodiment) fusion, so that generation hybridoma. Then use asThis heterogeneous cell mixture obtaining screens and can produce specific antibodies cell that can infinite multiplication. Each hybridomaAll, with clone's form propagation, each clone produces the monoclonal antibody of a kind of specific recognition PVL. Institute in method of the present inventionWith antibody can be purifying or unpurified. Can be according to affinity chromatography technology monoclonal antibody purification as above.
Monoclonal antibody can also be the recombinant antibodies that adopts genetic engineering technology well known in the art to obtain.
Term " for antibody fragment of the present invention " refers to F (ab ') 2, Fab, Fab ' or the scFv matrix of natural antibodySection, they have retained the ability of specific binding PVL.
Preferably, adopt following characteristics to carry out method of the present invention, described feature can adopt separately or combination mutually:
It is sandwich method that-described routine immunization is learned determination method,
-use LukS-PV-specific binding partner or LukF-PV-specific binding partner,
-use antibody fragment and particularly F (ab ') 2 fragments.
With regard to using the sandwich assay of two kinds of PVL-specific binding partners, it can use for example two kinds of monoclonalsAntibody, two kinds of polyclonal antibodies or their fragment, or a kind of monoclonal antibody and a kind of polyclonal antibody or theyFragment. For particle agglutination determination method, can use single a kind of PVL-specific binding partner, for example monoclonal antibody orPolyclonal antibody, they can be or not be the forms of fragment.
According to a concrete embodiment, described Radioimmunoassay of vascular endothelial growth uses at least one anti-PVL monoclonal antibody.
For particle agglutination determination method, use the PVL-specific binding partner of acquisition mode. For sandwich assay,Use the PVL-specific binding partner of catching form and test format.
If use binding partners as detecting reagent, it will be labeled to find between PVL/ binding partnersIn conjunction with.
Term " mark binding partners " can directly or indirectly produce the label of detectable signal on referring to and connecting.The limiting examples of these labels comprises:
For example produce the enzyme of detectable signal by colorimetric method, fluorescence method or luminescence method, for example horseradish peroxidaseEnzyme, alkaline phosphatase, alpha-galactosidase or glucose-6-phosphate dehydrogenase,
Chromophore, for example fluorescence, luminous or dye composition,
Geigers, for example32P、35S or125I, and
Fluorescence molecule, for example Alexa or phycocyanin.
Also can use indirect detection system, for example can with the part of anti-ligand reaction. Part/anti-part is to being this areaPersonnel know, for example, can be following collocation: biotin/streptavidin, haptens/antibody, antigen/antibody, peptide/The complementary series of antibody, sugar/agglutinin, polynucleotides/these polynucleotides. In this case, part is loaded with binding partners.Can pass through the anti-part of aforesaid label direct-detection, or anti-part itself is detectable with the form of part/anti-part.
In some cases, indirect detection system can make signal be amplified. Amplification of signal technology is that those skilled in the art are ripeKnow, for example can be referring to J.Histochem.Cytochem6.45：481-491，1997。
According to type used, those skilled in the art can add reagent to develop the color to mark.
For " competition " method, the method mark PVL of mark binding partners as described above.
If what use is acquisition mode, can use methods known in the art by direct PVL-specific binding partnerOr be indirectly immobilized onto solid phase.
Can be any protein denaturation well known in the art to the pretreatment of the sample for method of the present inventionProcessing method. This processing can be passed through, and for example, changes pH, uses chemical denaturant as urea, lauryl sodium sulfate (SDS)Or guanidinium ion or contain and need the sample of denatured protein and carry out by heating.
According to an embodiment, the temperature that sample pretreatment is included between 60 to 100 DEG C heats at least 10 minutes. ExcellentSelection of land, the temperature between 80 to 100 DEG C heats, the more preferably heating of the temperature between 90 to 100 DEG C.
Certainly, learn and measure if carry out described routine immunization on sample, can carry out sample to described sample itself pre-Process, otherwise, if carry out described Radioimmunoassay of vascular endothelial growth on culture, culture is carried out to sample pretreatment.
Method of the present invention also can comprise the additional step that has staphylococcus aureus in the described biological sample of checking,This step can shift to an earlier date or carry out simultaneously. The method that detects these bacteriums is that those skilled in the art are known, for example, can makeWith containing, this bacterium is had to specific colour reagent, the patent application WO for example submitting to referring to one of applicant of the application02/079486。
Similarly, method of the present invention can comprise that the staphylococcus aureus of determining in described biological sample is resistance to methoxyThe additional step of XiLin bacterium (MRSA) or methicillin-sensitivity bacterium (MSSA), has formed concrete an enforcement of the present invention thusMode.
The step of described definite methicillin-sensitivity (MRSA or MSSA) can adopt method well known in the art to enterOK, for example Radioimmunoassay of vascular endothelial growth (as use PBP2 ' albumen is had to specific binding partners, this protein be only byThe protein that MRSA expresses), detect the determination method of nucleic acid or other microbiological assays (for example use contain antibiotic asOXA or cynnematin are as the culture medium of Cefoxitin).
Following non-limiting example and appended Fig. 1 to 3 will contribute to be expressly understood the present invention more, wherein:
-Fig. 1 has provided for detection of containing PVL-producing (PVL+) or the staphylococcus aureus strains of PVL-producing (PVL-) notBiological sample in the ELISA measurement result (OD of each bacterial strain) of PVL,
-Fig. 2 has provided for detection of containing PVL-producing (PVL+) or the staphylococcus aureus strains of PVL-producing (PVL-) notBiological sample in the ELISA measurement result (OD of each bacterial strain) of PVL, described sample through heating pretreatment to makePVL sex change, and
-Fig. 3 has provided for detection of containing PVL-producing staphylococcus aureus strains (LY990084, A92007, LUG855)Or PVL in the biological sample of the staphylococcus aureus strains of PVL-producing (LY990333, LY991321, RN6911) notELISA measurement result (OD of each bacterial strain), described sample is through heating pretreatment (processing 1), the pretreatment of use chemical denaturant(processing 2) or use denaturant and heating pretreatment (processing 3), state 0 is equivalent to sample not carried out to any pretreatment.
Embodiment 1: prepare anti-PVL antibody
1. produce restructuring PVL
LukSHis-Tag and LukFHis-Tag that sequence is respectively SEQIDNo.1 and SEQIDNo.2 are producedProtein, uses carrier pIVEX2.4d (Roche) to transform Escherichia coli (Escherichiacoli) bacterial strain BL21star(DE3) pLys (Invitrogen), 37 DEG C, 2 liters cultivating systems, shake; With 1mMIPTG (isopropyl-β-D-galactolipin sulphur pyrroleThe glucosides of muttering, Eurobio) 37 DEG C of inductions 3 hours, and then cultivate 5 hours. Culture is divided in 6 different pipes.
4000g removes supernatant in centrifugal 20 minutes, reclaims each cell mass. Use QIAexpressionist kit according to lifeThe explanation purification of recombinant proteins matter of business men (Qiagen). Use the non-sex change cracked solution (Qiagen) of 20ml thin 4 DEG C of crackingBorn of the same parents group, then spends the night in-80 DEG C of preservations. After freeze thawing, process and within 1 hour, continue cracking at 4 DEG C with 1mg/ml lysozyme (Eurobio),Use subsequently Vibracell (Bioblock) in 100 watts of ultrasonic processing 2 minutes, every circulation 6 seconds.
Solution centrifugal 30 minutes with 10000g, 4 DEG C. Supernatant is contacted 3 little with agarose-NiNTA (Qiagen) of 5mlTime, 4 DEG C, soft annular is stirred. Then agarose is placed in to (Biorad) on 20ml post. Use 200ml lavation buffer solution(Qiagen) column scrubber, then carries out wash-out using the imidazoles of 250mM as elution buffer. Finally at monoSP ion exchange column(Amersham) on, complete purifying.
At the upper concentrated recombinant protein of Centricon (Vivaspin), with 50mMMES (2-(N-morpholino) ethyl sulfonic acid)Buffer solution is dialysed, then with NaCl gradient (0 to 1M) wash-out. And then use without the sterilized water of pyrogen and dialyse.
2. produce monoclonal antibody
Obtain following monoclonal antibody:
-anti-LukF:10D1A10,16A10A3,6H10E5 and
-anti-LukS:2H2H12,3D9D12,7C1F9,7F8D7,18A4E10
Process is as follows:
Anti-LukF monoclonal antibody: 10D1A10,16A10A3,6H10E5
According to following scheme immune mouse: at the restructuring LukF albumen of the 0th day (D0) intraperitoneal injection 10 μ g and completely notFamily name's adjuvant. At the 14th day (D14), the 28th day (D28), the further restructuring LukF albumen of the same amount of intraperitoneal injection and not completeFull Freund's adjuvant. Merge first 4 days, intravenous injection is with the LukF antigen of 50 μ g of normal saline dilution.
By 1600 portions of supernatants of indirect ELISA technology screening. Be dissolved in dense in PBS buffer solution (pH7.2) with 100 μ lDegree is antigen (restructuring LukF albumen) " being coated with " culture plate of 1 μ g/ml. " coated " plate is the temperature incubation mistake of 18-22 DEG CNight. Plate spends the night and 2 DEG C of incubations of 37 ° of +/-1 hour with the PBS-1% milk powder of 200 μ l is saturated. Add 100 μ l to be diluted in PBS slowRush supernatant or the ascites of liquid-0.05% polysorbas20, then by plate 2 DEG C of incubations of 37 ° of +/-1 hour. Add 1/ of 100 μ l2000 are diluted in puting together in alkaline phosphatase (JacksonImmunoresearchref:115-in PBS buffer solution-1%BSAGoat anti-mouse Ig (H+L) polyclonal antibody 055-062), plate is subsequently 2 DEG C of incubations of 37 ° of +/-1 hour. Add 100 μ l denseWhat degree was 2mg/ml is dissolved in DEA-HCl (bioM é rieuxref.60002989) PNPP (bioM é rieux (pH=9.8)Ref.60002990). Plate was 2 DEG C of incubations of 37 ° of +/-30 minutes. By adding the 1NNaOH blocking reaction of 100 μ l. Each step itBetween with the washing of the PBS-0.05% polysorbas20 of 300 μ l 3 times. Before adding PNPP, also wash with distilled water.
Indirect ELISA is found 72 parts of positive supernatant, OD > 0.6. After specificity test, produce aforesaid 3 kinds and resistedBody.
Anti-LukS monoclonal antibody: 2H2H12,3D9D12,7C1F9,7F8D7,18A4E10
According to following scheme immune mouse: at the restructuring LukS albumen of the 0th day (D0) intraperitoneal injection 10 μ g and completely notFamily name's adjuvant. At D14, D28, further restructuring LukS albumen and the incomplete Freund's adjuvant of the same amount of intraperitoneal injection. Before fusion4 days, intravenous injection was with the LukS albumen of 50 μ g of normal saline dilution.
By 1800 portions of supernatants of indirect ELISA technology screening. Be dissolved in dense in PBS buffer solution (pH7.2) with 100 μ lDegree is that the restructuring LukS albumen of 1 μ g/ml " is coated with " culture plate. " being coated with " plate is incubated overnight the temperature of 18-22 DEG C. Plate is usedThe PBS-1% milk powder of 200 μ l is saturated to spend the night and 2 DEG C of incubations of 37 ° of +/-1 hour. Add 100 μ l be diluted in PBS buffer solution-The supernatant of 0.05% polysorbas20 or ascites, then by plate 2 DEG C of incubations of 37 ° of +/-1 hour. Add 1/2000 dilution of 100 μ lPuting together in alkaline phosphatase (JacksonImmunoresearchref:115-055-062) in PBS buffer solution-1%BSAGoat anti-mouse Ig (H+L) polyclonal antibody, plate is subsequently 2 DEG C of incubations of 37 ° of +/-1 hour. Adding 100 μ l concentration is 2mg/Ml is dissolved in DEA-HCl (bioM é rieuxref.60002989) PNPP (bioM é rieux (pH=9.8)Ref.60002990). Plate was 2 DEG C of incubations of 37 ° of +/-30 minutes. By adding the 1NNaOH blocking reaction of 100 μ l. Each step itBetween with the washing of the PBS-0.05% polysorbas20 of 300 μ l 3 times. Before adding PNPP, also wash with distilled water.
Indirect ELISA is found 51 parts of positive supernatant, OD > 0.6. After specificity test, produce aforesaid 5 kinds and resistedBody.
3. produce many grams and fall antibody
3.1.Produce anti-LukS polyclonal antibody
Obtained anti-LukS polyclonal antibody No.173/89 and 176/89, process is as follows:
At D0, give the LukS-PV synthetic peptide of rabbit (NewZealandWhite) intracutaneous injection 200 μ g, its sequence isCSGHDPNLFVGYKPYSQN (SEQIDNo.3), N coupling KLH (keyhole limpet hemocyanin (KeyholeLimpetHemocyanin)) (Agro-Bio). At D14, D28, D42 and D81, further give being coupled to of the same amount of rabbit skin hemostasisThe synthetic peptide of KLH. Get the serum of animal at D0, D49 and D89. By carrying out affinity chromatography and purifying D89 institute by described synthetic peptideThe rabbit anteserum of getting. For this reason, according to manufacturer's (Amersham-Pharmacia) explanation, described peptide is coupled to 1mlHi-TrapOn Ago-Gel post. Serum 50/50 is diluted in PBS buffer solution, and pH7.4 is then injected on post, and speed is 1ml/ minute.After PBS (pH7.4) washing with glycine/HCl mixture pH3 wash-out of 50mM, then immediately in and wash-out composition, use subsequently0.15MPBS (pH7.4) dialysis.
3.2.Produce anti-LukF polyclonal antibody
Obtained anti-LukF polyclonal antibody, process is as follows:
At D0, give the LukF-PV synthetic peptide of rabbit (NewZealandWhite) intracutaneous injection 200 μ g, its sequence isCNFNWIGNNYKDENRATHTS (SEQIDNo.4), N coupling KLH (keyhole limpet hemocyanin (KeyholeLimpetHemocyanin)) (Agro-Bio). At D14, D28, D42 and D81, further give being coupled to of the same amount of rabbit skin hemostasisThe synthetic peptide of KLH. Get the serum of animal at D0, D49 and D89. By carrying out affinity chromatography and purifying D89 institute by described synthetic peptideThe rabbit anteserum of getting. For this reason, according to manufacturer's (Amersham-Pharmacia) explanation, described peptide is coupled to 1mlHi-TrapOn Ago-Gel post. Serum 50/50 is diluted in PBS buffer solution, and pH7.4 is then injected on post, and speed is 1ml/ minute.After PBS (pH7.4) washing with glycine/HCl mixture pH3 wash-out of 50mM, then immediately in and wash-out composition, use subsequently0.15MPBS (pH7.4) dialysis.
4. the generation of antibody fragment and coupling
With 30 points of the anti-LukS polyclonal antibodies of the pepsin digestion that is incorporated into agarose 173/89 and 176/891 hourClock, pH3.5,37 DEG C, to remove its Fc fragment and obtain F (ab ') 2 fragments.
With these fragments of peroxidase labelling, process is as follows: with 2 of the carbonate buffer solution dialysis F of pH9.6 (ab ')Section, and with use in advance NaIO4The peroxidase (Roche) of oxidation was 18-25 DEG C of coupling 2 hours, and ratio is: the F of 1mol(ab ') 2: 2mol peroxidase. Use NaBH4Process 1 hour with blocking-up coupling reaction at 2-8 DEG C, product is with containing anticorrisive agentThe dialysis of PBS buffer solution.
Use subsequently biotin labeling, process is as follows: by the carbonate buffer solution dialysis F of pH8.3 (ab ') 2 fragments, and withBiotin-NHS (Roche) was 18-25 DEG C of coupling 2 hours, and ratio is: and the F of 1mol (ab ') 2: 10mol biotin. Use lysineProcess 20 minutes with blocking-up coupling reaction, PBS+ azide dialysis for product at 18-25 DEG C.
Embodiment 2: diagnosis of PVL-producing staphylococcus aureus bacterial strain
1. prepare biological sample
At GP agar (Difco:10g/l peptone, 5g/l yeast extract, 17g/l agar; Sigma:5g/lNaCl,1g/l glucose) go up preculture from clinical staphylococcus aureus strains. Cultivate after 18 hours for 37 DEG C and verify purity, thenBy some colonies (about 107CFU/ml) be seeded to the 5mlCCY culture medium (Difco:30g/ in 25ml glass taper blake bottleL yeast extract, 20g/l casamino acid; Sigma:3.11g/lNa2HPO42H2O，0.41g/lKH2PO4, 23g/l thirdKetone acid) in. 37 DEG C of shakes were cultivated after 18 hours, and centrifugal (8000g, 4 DEG C, 10 minutes) collect culture supernatants, then short-termBe kept at 4 DEG C or-20 DEG C for subsequent use.
Each bacterial strain is equipped with identifier (A or LY add 6 bit digital), and if bacterial strain PVL-producing is labeled as "+",If not PVL-producing, is labeled as "-".
2.ELISA measures
With the coated elisa plate hole (Greiner, 100 μ l/ holes) of anti-LukS antibody 18A4E10, for this reason, with PBS (SigmaPBS, pH7.4; 0.1% sodium azide) dilute this antibody to 10 μ g/ml, little at environment temperature (about 22 DEG C) incubation 18 with plateTime. With after PBS polysorbas20 (BioradImmunoWash1575machine) washing 5 times, by PBS tween (0.05%) for plate/10% milk powder/0.5%BSA (Sigma) is in saturated 2 hours of environment temperature (150 μ l/ hole).
After PBS tween (0.05%) washing 5 times, 37 DEG C of incubation culture supernatants 75 minutes.
After PBS tween (0.05%) washing 5 times, by the 1/100 rabbit antibody 176-89 that is diluted in PBS-T5% milk powder, mistake37 DEG C of incubations of F (ab ' 2) fragment solution of oxide enzyme labeling 75 minutes. PBS-tween adds the TMB of 75 μ l molten after washing 5 timesLiquid (TMB substrate, Sigma), plate lucifuge incubation 30 minutes. By adding the 1NH of 75 μ l2SO4EventuallyOnly reaction. Use the reading (OD) of MicroPlateMPReader680 (Biorad) in 450nm assay plate.
3. result
The results are shown in Fig. 1, this figure has provided the OD of each bacterial strain.
This result shows, has 12 strains clearly to be detected, and in 14 parts of PVL-bacterial strains, have in 15 parts of PVL+ bacterial strains3 parts obtain false positive results.
These results clearly illustrate that, adopt conventional Radioimmunoassay of vascular endothelial growth can distinguish PVL+ bacterial strain and PVL-bacterial strain,But for the latter's shortage specificity.
Embodiment 3: diagnosis of PVL-producing staphylococcus aureus bacterial strain after heating pretreatment biological sample
1. sample treatment
The biological sample of preparing as embodiment 2 is carried out to heat treated at 95 DEG C, and the time is 10 to 30 minutes, according to realityThe scheme of executing described in example 2 is determined OD.
Table 1 has provided OD result, and its Graphics Processing is more than 10 minutes, to make PVL sex change.
Table 1
| Strain number | PVL | Do not heat | Heat 10 minutes | Heat 20 minutes | Heat 30 minutes |
| LY990179 | + | 0.839 | 2.126 | 2.76 | ＞3 |
| A960420 | + | 1.73 | 2.844 | ＞3 | ＞3 |
| A990438 | - | 1.545 | 0.852 | 0.25 | 0 |
| A980642 | + | 0.458 | 1.364 | 2.325 | ＞3 |
| LY990301 | - | 2.123 | 0.105 | 0 | 0 |
| HT20020275 | + | 0.729 | 2.269 | ＞3 | ＞3 |
| LY990333 | - | 0.615 | 0.202 | 0 | 0 |
| HT20040540 | + | 1.178 | ＞3 | ＞3 | ＞3 |
2.ELISA measures
Repeat the process described in the 2nd of above-described embodiment 2, it is pre-95 DEG C of heating 1 hour that what difference was to use isThe culture supernatants of 29 bacterial strains (15 parts of PVL+, 14 parts of PVL-) of processing.
3. result
The results are shown in Fig. 2, wherein provided the OD of each bacterial strain.
These results show, method of the present invention has identified all PVL+ bacterial strains, your pupil of its OD level withPVL-bacterial strain is differentiated mutually.
Therefore, pretreatment can improve the specificity of method of the present invention.
Embodiment 4: adopt diagnosis of PVL-producing staphylococcus Portugal after polytype modification treatment pretreatment biological sampleGrape coccus bacterial strain
1. sample treatment
Use from clinical 3 parts of PVL+ bacterial strains (LY990084, A92007, LUG855) and 3 parts of PVL-bacterial strains(LY990333, LY991321, RN6911), process as follows:
-non-processor (0),
-95 DEG C of heat denatured 1 hour (1),
-sour sex change, with the KH of final concentration 0.1M2PO4Oscillation treatment 30 minutes, then adds NaOH neutralization (2),
-combined degeneration, with the KH of final concentration 0.1M2PO4Oscillation treatment is carried out sour sex change for 10 minutes, and then 95 DEG C are boiled 10 pointsClock, then adds NaOH neutralization (3).
2.ELISA measures
Repeat process as described in Example 2, what difference was use is according to above-mentioned the 1st pretreated bacterial strainWith with the 1/1000 rabbit antibody 176-89 that is diluted in PBS-tween 5% milk powder, F (ab ' 2) fragment of peroxidase labelling is moltenLiquid.
3. result
The results are shown in Fig. 3, wherein provided the OD of each bacterial strain.
These results demonstrations, no matter how sample is processed, and the OD value of all PVL-bacterial strains is between 0.09 and 0.181.
For PVL+ bacterial strain, OD value after treatment is higher than PVL-bacterial strain. By chemical treatment, heat treatment and both combinationsThe increase of processing the OD obtaining has significance,statistical (being respectively p=0.024, p < 0.001, p=0.018), and heat placeManage seemingly effective under these conditions.
Therefore, pretreatment can improve the specificity of conventional Radioimmunoassay of vascular endothelial growth.
Embodiment 5: method of the present invention can earlier detection PVL-producing staphylococcus aureus
1. prepare biological sample
According to the method preculture described in embodiment 2 from clinical 4 parts of staphylococcus aureus strains (2 parts of PVL+ bacteriumStrain and 2 parts of PVL-bacterial strains). Then by colony for carry out richness in the CCY culture medium of the 5ml of 25ml glass taper blake bottleCollection inoculation. After inoculation, immediately culture medium is sampled, 37 DEG C of shaken cultivation, and at postvaccinal 1h, 2h, 3h and 18h are continuousSampling. Centrifugal (8000g, 4 DEG C, 10 minutes) collect culture supernatants, short-term preservation in 4 DEG C or-20 DEG C for subsequent use. As embodimentHeat treated sample described in 3.
2.ELISA measures
Repeat the process described in the 2nd of above-described embodiment 2, what difference was to use is above-mentioned the 1st pretreatedThe culture supernatants of bacterial strain.
3. result
Table 2 has provided OD result, and it shows that method of the present invention can detect that PVL (opens from cultivating 2 hours very rapidlyBeginning can clearly detect).
Table 2
Embodiment 6: the PVL of the method for the present invention of sampling in can earlier detection biological samples
1. prepare biological sample
Clinical samples is that the broncho-pulmonary with the patient of staphylococcus aureus PVL+ bacterial strain or PVL-strain infection is inhaledGo out thing, bronchovesicular liquid and from the fester of dermapostasis or from the fester of deep suppuration focus. Of the present invention in useMethod is analyzed these samples before and is stored in-20 DEG C. Room temperature makes it homogeneous for 15 minutes by sample vortex oscillation after meltingChange. Through centrifugal 10 minutes (4000rpm, 15 DEG C), supernatant is transferred to Eppendorf pipe, then 95 DEG C of sex change 1 hour.
For the unusual sample of thickness, first sample 50/50 is diluted in to PBS, pH7.4, then vortex oscillation 15 minutes.Through centrifugal 10 minutes (4000rpm, 15 DEG C), supernatant is transferred to Eppendorf pipe, then 95 DEG C of sex change 1 hour.
For the supernatant of thickness, it 50/50 is diluted in to normal heptane (Merck), and then vortex oscillation 10 minutes.Through centrifugal 10 minutes (4000rpm, 15 DEG C), thoroughly remove the upper strata phase that is equivalent to normal heptane, and by lower floor's phase transfer to anotherEppendorf pipe, then 95 DEG C of sex change 1 hour.
2.ELISA measures
Repeat the process described in the 2nd of above-described embodiment 2, what difference was to use is above-mentioned the 1st pretreatedBiological samples.
3. result
Table 3 and 4 has provided PVL measurement result, and these results show, regardless of the methicillin-sensitivity of bacterial strain, allCan direct-detection to the PVL in biological samples, and do not have false positive (be PVL-bacterial strain sample none be detected as and containPVL). In addition do not affect detecting PVL with normal heptane pretreatment sample.
Table 3
| Sample | Sample type | Pretreated | Bacterial strain* | The amount (μ g/ml) of PVL |
| 17 | BAL fluid | Nothing | PVL+ | ＜0.05 |
| 13 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 18 | The little lavation of bronchovesicular | Nothing | PVL- | ＜0.05 |
| 22 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 11 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 8 | Bronchus aspirate | PBS+ normal heptane | PVL- | ＜0.05 |
| 16 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 10 | Bronchus aspirate | Nothing | PVL- | ＜0.05 9 --> |
| 19 | Phlegm | Nothing | PVL- | ＜0.05 |
| 14 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 15 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 12 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 7 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 5 | Bronchus aspirate | Nothing | PVL- | ＜0.05 |
| 23 | Joint fluid | Nothing | PVL- | ＜0.05 |
| 20 | Peritoneal fluid | Nothing | PVL- | ＜0.05 |
| 4 | Drainage-fluid | Nothing | PVL+ | ＜0.05 |
| 2 | Abscess | Nothing | PVL+ | 1 |
| 3 | Abscess | Nothing | PVL- | ＜0.05 |
| 1 | Drainage-fluid | Nothing | PVL- | ＜0.05 |
| 5 | Bronchus aspirate | PBS | PVL+ | 1.2 |
| 5 | Bronchus aspirate | PBS+ normal heptane | PVL+ | 1.2 |
| 6 | Bronchus aspirate | PBS+ normal heptane | PVL+ | 1.2 |
*According to Vandeneschetal.2Whether described method detects in staphylococcus aureus separator by PCRThere is the coding LukS-PV of PVL and LukF-PV gene and determine that described bacterial strain is PVL-or PVL+.
Table 4
*According to Vandeneschetal.2Whether described method detects in staphylococcus aureus separator by PCRThere is the coding LukS-PV of PVL and LukF-PV gene and determine that described bacterial strain is PVL-or PVL+.
**According to Vandeneschetal.2Described method detects the methoxy west of encoding in staphylococcus aureus separatorThe mecA gene of woods resistance.
Document
1：WardPD，TurnerWD，1980，Infect.Immun.，28(2)：393-397
2：Vandeneschetal.，2003，EmergInfectDis，9(8)：978-984
3：DufourPetal.，2002，ClinInfectDis，35：819-824
4：CribierBetal，1992，Dermatology，185：175-185
5：FreneyJetal.，2000，PrécisdeBactériologieClinique[Handbookofclinicalbacteriology]，40：298and793-794
6：J.Histochem.Cytochem.45：481-491，1997
7：Labandeira-ReyMetal，Science2007，inpress
<110>Biomerieux SA
National Health and Medicine Inst.
Clo Derby nail (unit of length)-University Lyon 1-Claude Bernard
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Claims (7)
1. in the biological sample that may contain staphylococcus aureus, determine the gold that produces P-VLThe method of staphylococcus aureus, wherein said definite by adopting conventional Radioimmunoassay of vascular endothelial growth detection Pan Dun-Valentine to kill whiteCytokine and carrying out, is characterized in that described biological sample to carry out pretreatment to make P-VLSex change.
2. the method for claim 1, the Radioimmunoassay of vascular endothelial growth that it is characterized in that described routine is sandwich method.
3. the method for claim 2, is characterized in that described Radioimmunoassay of vascular endothelial growth uses at least one anti-Pan Dun-Valentine to kill whiteCytokine monoclonal antibody.
4. the method for claim 1, is characterized in that described Radioimmunoassay of vascular endothelial growth uses at least one anti-Pan Dun-Valentine to kill whiteCytokine monoclonal antibody.
5. the method for any one in claim 1 to 4, is characterized in that described pretreatment is the temperature between 60 to 100 DEG CHeat at least 10 minutes.
6. the method for any one in claim 1 to 4, is characterized in that it comprises definite being present in described biological sampleStaphylococcus aureus is the additional step of methicillin-resistant bacterium or methicillin-sensitivity bacterium.
7. the method for claim 5, is characterized in that it comprises definite golden yellow grape being present in described biological sampleCoccus is the additional step of methicillin-resistant bacterium or methicillin-sensitivity bacterium.
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| FR0654336 | 2006-10-18 | ||
| FR0654336 | 2006-10-18 | ||
| FR0753081 | 2007-02-06 | ||
| FR0753081A FR2912219A1 (en) | 2007-02-06 | 2007-02-06 | In vitro diagnostic process of Staphylococcus aureus producers of panton velentine leukocidin by biological sample, comprises detecting panton velentine leukocidin by routine immunological test using anti-PVL monoclonal antibody |
| PCT/FR2007/052165 WO2008050041A1 (en) | 2006-10-18 | 2007-10-16 | Method for in vitro diagnosis of pvl-producing staphylococcus aureus |
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| WO2013071175A1 (en) * | 2011-11-11 | 2013-05-16 | Alere San Diego, Inc. | Devices and methods for detection of panton-valentine leukocidin (pvl) |
| US20160108106A1 (en) * | 2013-05-21 | 2016-04-21 | Arsanis Biosciences Gmbh | Generation of highly potent antibodies neutralizing the lukgh (lukab) toxin of staphylococcus aureus |
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| CN1425919A (en) * | 2003-01-30 | 2003-06-25 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method |
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| CN1425919A (en) * | 2003-01-30 | 2003-06-25 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method |
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| Title |
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| IMMUNOSSAY OF THE MRSA-RELATED TOXIC PROTEIN, LEUKOCIDIN, WITH SCANNING ELECTROCHEMICAL MICROSCOPY.;KASAI S ET AL;《ANALYTICAL CHEMISTRY》;20001201;第72卷(第23期);5761-5765 * |
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