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.
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.