CN106814189A - A kind of kit for detecting clostridium difficile and application thereof - Google Patents

A kind of kit for detecting clostridium difficile and application thereof Download PDF

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Publication number
CN106814189A
CN106814189A CN201611187231.3A CN201611187231A CN106814189A CN 106814189 A CN106814189 A CN 106814189A CN 201611187231 A CN201611187231 A CN 201611187231A CN 106814189 A CN106814189 A CN 106814189A
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Prior art keywords
clostridium difficile
antibody
dilution
goat
kit
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CN201611187231.3A
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CN106814189B (en
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黄绣川
邱兵
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Jiaxing Practical Medical Technology Co.,Ltd.
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Chengdu Jin Si Wei Biotechnology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)

Abstract

The invention discloses a kind of enzyme-linked immunosorbent assay (ELISA) kit of clostridium difficile, it includes following component：Dilution, confining liquid, the ELISA Plate for being coated with clostridium difficile antibody, ELIAS secondary antibody, positive reference substance, cleaning solution, nitrite ion and terminate liquid, wherein, dilution is that every 100ml contains 1mlNP 40,0.5~2mlTritonX 100,1gBSA, the phosphate buffer of 0.05mlTween20.The invention also discloses purposes of the aforementioned agents box in clostridium difficile is detected, kit of the present invention can be good with efficient detection clostridium difficile, application prospect.

Description

A kind of kit for detecting clostridium difficile and application thereof
Technical field
The present invention relates to a kind of kit for detecting clostridium difficile and application thereof.
Background technology
Clostridium difficile (C.DIFF) is a kind of Gram-positive, has the bacillus fusiformis of gemma, obligate anaerobic, is unique The anaerobic bacteria of nosocomial infection can be caused, be also to cause the bacterium that gemma can be uniquely formed in nosocomial infection pathogen.Clostridium difficile The diarrhoea main cause that infection causes causes flora ecological disturbance because the use of antibiotic causes flora cell component to change, So that clostridium difficile flora is bred, diarrhoea is ultimately resulted in.Incidence higher has been considered to occur in those long-term exposures In antibiotic and in the low old and child patient of the immunocompetence with serious potential comorbidities.The symptom change of infection It is in extensive range, from asymptomatic state or slight C. difficile infection to serious and life-threatening state.It is difficult at the beginning of 21 century The incidence and seriousness of difficult clostridium infection steeply rise, and in developed country, clostridium difficile is the primary of nosocomial infection diarrhoea The cause of disease, the antibiotic-associated diarrhea and nearly all pseudomembranous colitis of up to 20% report are all by clostridium difficile sense What dye caused.Therefore, efficient detection clostridium difficile is extremely important.
Clinically various detection modes are continued to develop, including from initial product strain culture, cytotoxicity is detected And toxin neutralization test, the most frequently used till now enzyme linked immunological two-step method, three-step approach and pcr gene detection method.Pcr gene is detected Method detection sensitivity is too high, but caused false positive rate is also high.ELISA two step method there is also that Detection results are not good to ask Topic is, it is necessary to further improve.
The content of the invention
Object of the present invention is to provide a kind of new kit for detecting clostridium difficile.
The enzyme-linked immunosorbent assay (ELISA) kit of clostridium difficile of the present invention, it includes following component：Dilution, confining liquid, bag ELISA Plate, ELIAS secondary antibody, positive reference substance, cleaning solution, nitrite ion and the terminate liquid for being had clostridium difficile antibody, wherein, dilution Liquid is that every 100ml contains 1mlNP-40,0.5~2mlTritonX-100,1gBSA, the phosphate-buffered of 0.05mlTween20 Liquid.
Wherein, dilution is that every 100ml contains 1mlNP-40,0.5~2mlTritonX-100,1gBSA, The phosphate buffer of 0.05mlTween20.
The dilution is that every 100ml contains 1mlNP-40,0.5~1mlTritonX-100,1gBSA, The phosphate buffer of 0.05mlTween20；The further preferred dilution be every 100ml contain 1mlNP-40, 0.5mlTritonX-100,1gBSA, the phosphate buffer of 0.05mlTween20.
Preferably, the confining liquid contains 2gBSA, 1g-5g trehalose, 1ml-5mlFBS per 100ml.Further preferably Ground, the confining liquid is the phosphate buffer that every 100ml contains 2gBSA, 2g-3g trehalose, 2ml-3mlFBS；It is preferred that described Confining liquid is the phosphate buffer that every 100ml contains 2gBSA, 3g trehalose, 3mlFBS.
Preferably, coated clostridium difficile antibody is the anti-clostridium difficile glutamte dehydrogenase monoclonal of mouse on the ELISA Plate The mixture of antibody and goat-anti Clostridium difficile toxin A polyclonal antibody and goat-anti Clostridium difficile toxin B polyclonal antibody.
Preferably, the ELISA Plate for being coated with clostridium difficile antibody is prepared as follows：Antibody is taken, dilution is made Solution, the micro- g holes of the 0.2-0.5 in terms of antibody are added in the hole of ELISA Plate, and 4 DEG C are coated with 10-14 hours.
Preferably, the antibody in the ELIAS secondary antibody is respectively the goat-anti clostridium difficile paddy ammonia of horseradish peroxidase-labeled The goat-anti Clostridium difficile toxin A polyclonal antibody and horseradish mistake of acidohydrogenase polyclonal antibody and horseradish peroxidase-labeled The mixture of the goat-anti Clostridium difficile toxin B polyclonal antibody of oxide enzyme mark.
Preferably, the ELIAS secondary antibody includes ELIAS secondary antibody 1 and ELIAS secondary antibody 2, and ELIAS secondary antibody 1 is that every 100ml contains 1g- The goat-anti clostridium difficile glutamte dehydrogenase Anti-TNF-α of 2gBSA, 5ml-20mlFBS, 20-50 μ g horseradish peroxidase-labeleds The phosphate buffer of body, 50ml glycerine；ELIAS secondary antibody 2 is that every 100ml contains 1g-2gBSA, 5ml-20mlFBS, 20-50 μ g The goat-anti Clostridium difficile toxin A polyclonal antibody of horseradish peroxidase-labeled, the sheep of 20-50 μ g horseradish peroxidase-labeleds Anti- Clostridium difficile toxin B polyclonal antibody, the phosphate buffer of 50ml glycerine.
Preferably, the cleaning solution is the phosphate buffer containing 0.05%Tween20；
The nitrite ion includes the A liquid containing peroxide and the B liquid containing tetramethyl benzidine.
The terminate liquid is the sulfuric acid solution that concentration is 0.5M-2M.
Present invention also offers purposes of the aforementioned agents box in clostridium difficile is detected.
NP-40：Nonidet P40.
TritonX-100：Triton X-100.
BSA：Bovine serum albumin(BSA).
Tween20：Polysorbas20.
FBS：Hyclone.
The cooperation that dilution of the present invention passes through each component, can effectively help the antigen in antibody capture sample, improve The sensitiveness of product.Using kit of the present invention, using ELISA two step method can efficient quick detection clostridium difficile, should With having good prospects.
The present invention is described in further details below by specific embodiment, but is not to limit of the invention System, the above of the invention, according to the ordinary technical knowledge and customary means of this area, not departing from, the present invention is above-mentioned Under the premise of basic fundamental thought, the modification of other diversified forms can also be made, is replaced or is changed.
Specific embodiment
The present invention of embodiment 1 is used to detect the kit of clostridium difficile
1st, the composition of kit of the present invention
(1) dilution of the present invention：
Formula 1：PBS (PH7.4), 1%NP-40,0.5%TritonX-100,1%BSA, 0.05%Tween20
Formula 2：PBS (PH7.4), 1%NP-40,1%TritonX-100,1%BSA, 0.05%Tween20
Formula 3：PBS (PH7.4), 1%NP-40,2%TritonX-100,1%BSA, 0.05%Tween20
PBS：Phosphate-buffered salt is molten
(2) inventive closure liquid
Formula 1：PBS, 2%BSA, 1% trehalose, 1%FBS
Formula 2：PBS, 2%BSA, 2% trehalose, 2%FBS
Formula 3：PBS, 2%BSA, 3% trehalose, 3%FBS
Formula 4：PBS, 2%BSA, 5% trehalose, 5%FBS
(3) remaining component：It is coated with ELISA Plate, HRP ELIAS secondary antibodies, positive reference substance (the GLDH restructuring of specific antibody Antigen, ToxinA/B recombinant antigens), cleaning solution, nitrite ion, terminate liquid.
The ELISA Plate of specific antibody：Specific antibody is the anti-clostridium difficile glutamte dehydrogenase monoclonal antibody of mouse, sheep Anti- Clostridium difficile toxin A polyclonal antibody and goat-anti Clostridium difficile toxin B polyclonal antibody, are purchased from Nanjing bronze object biotechnology Co., Ltd；After each antibody is diluted with carbonate, added with the micro- g holes of 0.2-0.5,4 DEG C are coated with 10-14 hours, wherein, mouse resists The independent hole of clostridium difficile glutamte dehydrogenase monoclonal antibody, goat-anti Clostridium difficile toxin A polyclonal antibody and goat-anti are difficult Difficult clostridial toxin B polyclonal antibodies mix.
HRP ELIAS secondary antibodies：The goat-anti clostridium difficile glutamte dehydrogenase polyclonal antibody of horseradish peroxidase-labeled, it is peppery The goat-anti Clostridium difficile toxin A polyclonal antibody of root peroxidase labelling, the difficult shuttle of goat-anti of horseradish peroxidase-labeled Verticillium toxin B polyclonal antibodies, purchased from Wuhan Bo Oute bio tech ltd, are made every 100ml and contain 1g-2gBSA, 5ml- 20mlFBS, antibody 20-50 micrograms (every kind of antibody 20-50 micrograms), the phosphate buffer of 50ml glycerine, pH5.0-7.0.
Positive reference substance：Contain glutamte dehydrogenase recombinant antigen, the phosphate buffer of ToxinA/B recombinant antigens.Paddy Propylhomoserin dehydrogenase recombinant antigen, ToxinA/B recombinant antigens are purchased from Nanjing Zhong Ding Bioisystech Co., Ltd.
Cleaning solution：The phosphate buffer containing 0.05mlTween20 per 100ml.
Nitrite ion：A liquid containing peroxide and the B liquid containing TMB (tetramethyl benzidine).Have purchased from Beijing Suo Laibao science and technology Limit company, 100 microlitres/hole adds colour developing after the mixing of the ratio such as A liquid and B liquid when using.
Terminate liquid：Contain the sulfuric acid solution of 0.5M-2M per 1L.
Hereinafter beneficial effects of the present invention are illustrated with the mode of experimental example：
The comparing of experimental example 1 and existing universal diluent
1st, experiment material
Kit of the present invention：Using the kit of the embodiment of the present invention 1, wherein confining liquid is formula 2, and dilution is adopted respectively With the dilution of formula 1~formula 10.
Formula 1：PBS (PH7.4), 1%NP-40,0.5%TritonX-100,1%BSA, 0.05%Tween20
Formula 2：PBS (PH7.4), 1%NP-40,1%TritonX-100,1%BSA, 0.05%Tween20
Formula 3：PBS (PH7.4), 1%NP-40,2%TritonX-100,1%BSA, 0.05%Tween20
Formula 4：PBS (PH7.4), 0.1%NP-40,1%BSA, 0.05%Tween20
Formula 5：PBS (PH7.4), 1%NP-40,1%BSA, 0.05%Tween20
Formula 6：PBS (PH7.4), 5%NP-40,1%SDS, 1%BSA, 0.05%Tween20
Formula 7：PBS (PH7.4), 1%NP-40,1%SDS, 1%BSA, 0.05%Tween20
Formula 8：PBS (PH7.4), 1%NP-40,2%SDS, 1%BSA, 0.05%Tween20
Formula 9：PBS (PH7.4), 1%NP-40,1%SDS, 0.5%TritonX-100,1%BSA, 0.05% Tween20
Formula 10：PBS (PH7.4), 1%NP-40,2%SDS, 0.5%TritonX-100,1%BSA, 0.05% Tween20
Contrast agents box：In addition to dilution is universal diluent, remaining component is with kit of the present invention, general dilution Liquid is：PBS (PH7.4), 1%BSA, 0.05%Tween20.
2nd, experimental technique
Take GLDH recombinant antigens, positive sample 1,2 (producing malicious clostridium difficile culture supernatant), negative sample (common large intestine bar Bacterium culture supernatant) and blank (blank is dilution), detected respectively using kit of the present invention and contrast agents box.
Detection method is as follows：
Respectively with different diluted recombinant antigen, negative sample, positive sample, and using dilution as blank pair According to being separately added into 50 microlitres of antigen/negative sample/positive sample/dilutions being coated with inside the ELISA Plate hole of specific antibody Liquid is compareed and 50 microlitres of HRP ELIAS secondary antibodies, and after 37 DEG C are incubated 1 hour, TMB colour developings, colour developing terminates rear terminate liquid and terminates, and 450nm wavelength readings.
3rd, experimental result
Experimental result is as shown in table 1 below：
The result that table 1 is detected using different diluent
Antigen/sample Universal diluent The formula of formula 12 Formula 3 Formula 4 Formula 5 Formula 6 Formula 7 Formula 8 Formula 9 Formula 10
GLDH recombinant antigens 1.550 1.659 1.067 0.998 1.086 1.894 0.252 0.452 0.332 0.898 0.776
Negative sample 0.119 0.140 0.100 0.230 0.056 0.117 0.408 0.034 0.068 0.200 0.170
Positive sample 1 0.299 1.999 1.565 0.798 0.071 0.430 0.211 0.078 0.089 0.689 0.430
Positive sample 2 0.278 1.966 1.047 0.490 0.075 0.405 0.243 0.069 0.035 0.442 0.331
Blank 0.062 0.055 0.056 0.088 0.100 0.058 0.062 0.055 0.047 0.068 0.098
As upper table 1 understands, positive sample 1 and positive sample 2 are detected using universal diluent, its OD value is low, as a result in weak The positive, close to the cutoff values of this kit, easily causes erroneous judgement.
And inventive formulation (formula 1,2,3) is used, the OD values of positive sample are significantly larger than universal diluent, also above it His formula, for it is actually detected when, the sensitiveness of product is high, optimization formula 1 and 2, is most preferably formulated 1, positive sample during detection Sheet 1 and positive sample 2 are strong positive, while the detected value of positive control (recombinant antigen) is not less than the detection of universal diluent Value.
Confirmatory experiments of the ToxinA and ToxinB to dilution：
ToxinA the and ToxinB the results of table 2
Because this kit further comprises two detections of antigen of ToxinA/ToxinB, therefore utilize ToxinA/ToxinB Two antigens are verified to dilution of the present invention.The result detects positive sample as shown in upper table 2 using universal diluent 1 and positive sample 2, its OD value is low, as a result in weakly positive, close to the cutoff values of this kit, easily causes erroneous judgement.And use Dilution (formula 1) of the present invention can detect that positive sample 1 and positive sample 2 are strong positive, while positive control (is recombinated anti- It is former) detected value and universal diluent detected value no significant difference.Therefore, dilution (formula 1) of the present invention can effectively improve The sensitiveness of this reagent kit product.
The comparing of experimental example 2 and existing general confining liquid
1st, experiment material
Kit of the present invention：Using the kit of the embodiment of the present invention 1, wherein confining liquid is formula 1~formula 4, dilution Liquid is respectively adopted formula 1.
Contrast agents box：In addition to dilution is general confining liquid, remaining component is with kit of the present invention, general closing Liquid is：PBS (PH7.4), 1%BSA, 0.05%Tween20.
2nd, experimental technique
Take GLDH recombinant antigens, positive sample 1,2 (producing malicious clostridium difficile culture supernatant), negative sample (common large intestine bar Bacterium culture supernatant) and blank (blank is dilution), detected respectively using kit of the present invention and contrast agents box.
Detection method is as follows：
The ELISA Plate for being coated with specific antibody is closed with different confining liquids respectively, the closing fluid-tight of each formula After having closed, a part is in 4 DEG C of preservations, while another part carries out accelerated test in 37 DEG C of constant incubators, it is right after 10 days The ELISA Plate of each condition carries out ELISA detections.
Detecting step：Be coated with inside the ELISA Plate hole of specific antibody be separately added into 50 microlitres of antigen/negative samples/ Positive sample/dilution control and 50 microlitres of HRP ELIAS secondary antibodies, after 37 DEG C are incubated 1 hour, TMB colour developings, colour developing terminates after terminating Liquid terminates, and in 450nm wavelength readings.
3rd, experimental result
Experimental result is as shown in table 3 below：
The result that table 3 is detected using different confining liquids
As upper table 3 understands, closed protective is carried out to ELISA Plate using general confining liquid, ELISA Plate accelerates 10 days at 37 DEG C Afterwards, the OD values of its detection recombinant antigen and positive sample are decreased obviously, it was demonstrated that ELISA Plate interior antibody activity decrease, ELISA Plate is steady It is qualitative poor.And closed protective is carried out to ELISA Plate using inventive closure liquid (formula 1- is formulated 4), ELISA Plate is in 37 DEG C of acceleration After 10 days, the OD values of its detection recombinant antigen and positive sample with accelerate before (4 DEG C preserve) be almost as good as, it was demonstrated that it is anti-in ELISA Plate Body is active and is held essentially constant, and ELISA Plate stability is preferable, wherein, optimization formula 2 and 3 is most preferably formulated 3.
Confirmatory experiments of the ToxinA and ToxinB to confining liquid：
ToxinA the and ToxinB the results of table 4
Because this kit further comprises two detections of antigen of ToxinA/ToxinB, therefore utilize ToxinA/ToxinB Two antigens are verified to inventive closure liquid.The result is carried out using general confining liquid as shown in upper table 4 to ELISA Plate Closed protective, after ELISA Plate accelerates 10 days at 37 DEG C, the OD values of its detection recombinant antigen and positive sample are decreased obviously, it was demonstrated that ELISA Plate interior antibody activity decrease, ELISA Plate less stable.And inventive closure liquid (formula 1- is formulated 4) is used to ELISA Plate Closed protective is carried out, after ELISA Plate accelerates 10 days at 37 DEG C, (4 before the OD values and acceleration of its detection recombinant antigen and positive sample DEG C preserve) be almost as good as, it was demonstrated that ELISA Plate interior antibody activity and be held essentially constant, ELISA Plate stability is preferable.
To sum up, kit of the present invention can be with efficient detection clostridium difficile, and product stability is good, long shelf-life, using preceding Scape is good.

Claims (10)

1. a kind of enzyme-linked immunosorbent assay (ELISA) kit of clostridium difficile, it is characterised in that：It includes following component：
Dilution, confining liquid, the ELISA Plate for being coated with clostridium difficile antibody, ELIAS secondary antibody, positive reference substance, cleaning solution, colour developing Liquid and terminate liquid, wherein, dilution is that every 100ml contains 1mlNP-40,0.5~2mlTritonX-100,1gBSA, The phosphate buffer of 0.05mlTween20.
2. kit according to claim 1, it is characterised in that：The dilution be every 100ml contain 1mlNP-40, 0.5~1mlTritonX-100,1gBSA, the phosphate buffer of 0.05mlTween20；It is preferred that the dilution is every 100ml Contain 1mlNP-40,0.5mlTritonX-100,1gBSA, the phosphate buffer of 0.05mlTween20.
3. kit according to claim 1, it is characterised in that：The confining liquid contains 2gBSA, 1g-5g sea per 100ml Algae sugar, 1ml-5mlFBS.
4. kit according to claim 1, it is characterised in that：The confining liquid is that every 100ml contains 2gBSA, 2g-3g The phosphate buffer of trehalose, 2ml-3mlFBS；It is preferred that the confining liquid is every 100ml contain 2gBSA, 3g trehalose, The phosphate buffer of 3mlFBS.
5. kit according to claim 1, it is characterised in that：Coated clostridium difficile antibody is mouse on the ELISA Plate Anti- clostridium difficile glutamte dehydrogenase monoclonal antibody and goat-anti Clostridium difficile toxin A polyclonal antibody and goat-anti clostridium difficile The mixture of toxin B polyclonal antibodies.
6. kit according to claim 5, it is characterised in that：The ELISA Plate for being coated with clostridium difficile antibody according to It is prepared by following method：Antibody is taken, dilution is made solution, the micro- g holes of the 0.2-0.5 in terms of antibody are added in the hole of ELISA Plate, 4 DEG C of bags By 10-14 hours.
7. kit according to claim 1, it is characterised in that：Antibody in the ELIAS secondary antibody is respectively horseradish peroxide The goat-anti clostridium difficile glutamte dehydrogenase polyclonal antibody of compound enzyme mark and the goat-anti of horseradish peroxidase-labeled are difficult The mixing of the goat-anti Clostridium difficile toxin B polyclonal antibody of difficult clostridial toxin A polyclonal antibodies and horseradish peroxidase-labeled Thing.
8. kit according to claim 7, it is characterised in that：The ELIAS secondary antibody includes ELIAS secondary antibody 1 and enzyme mark two Anti- 2, ELIAS secondary antibody 1 is the sheep that every 100ml contains 1g-2gBSA, 5ml-20mlFBS, 20-50 μ g horseradish peroxidase-labeleds Anti- clostridium difficile glutamte dehydrogenase polyclonal antibody, the phosphate buffer of 50ml glycerine；ELIAS secondary antibody 2 is that every 100ml contains There is the goat-anti Clostridium difficile toxin A Anti-TNF-α of 1g-2gBSA, 5ml-20mlFBS, 20-50 μ g horseradish peroxidase-labeleds Body, the goat-anti Clostridium difficile toxin B polyclonal antibody of 20-50 μ g horseradish peroxidase-labeleds, the phosphate of 50ml glycerine delay Fliud flushing.
9. kit according to claim 1, it is characterised in that：The cleaning solution is the phosphorus containing 0.05%Tween20 Phthalate buffer；
The nitrite ion includes the A liquid containing peroxide and the B liquid containing tetramethyl benzidine；
The terminate liquid is the sulfuric acid solution that concentration is 0.5M-2M.
10. kit described in claim 1~9 any one detect clostridium difficile in purposes.
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CN110806478A (en) * 2019-11-24 2020-02-18 天津市宝坻区人民医院 Clostridium difficile detection kit
CN111763263A (en) * 2020-07-09 2020-10-13 宁夏医科大学 Preparation method and detection kit of clostridium difficile antigen and polyclonal antibody
CN113267621A (en) * 2021-05-14 2021-08-17 北京金诺百泰生物技术有限公司 Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit
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