CN106093403A - A kind of method with double antibody sandwich ELISA detection beta lactamase of optimization - Google Patents

A kind of method with double antibody sandwich ELISA detection beta lactamase of optimization Download PDF

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Publication number
CN106093403A
CN106093403A CN201610356629.9A CN201610356629A CN106093403A CN 106093403 A CN106093403 A CN 106093403A CN 201610356629 A CN201610356629 A CN 201610356629A CN 106093403 A CN106093403 A CN 106093403A
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China
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hole
lactamase
antibody
plate
beta
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Inventor
于国萍
邵美丽
岳崇慧
吴昊
藏小丹
范美婧
陈媛
陈超
王艳菲
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Northeast Agricultural University
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Northeast Agricultural University
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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Abstract

The present invention provides a kind of method with double antibody sandwich ELISA detection beta lactamase of optimization, beta lactamase antigen 40U/mL coated elisa plate, the Rabbit Anti LACTB 1:800 by volume dilution of 0.8mg/ml, it is coated, volume fraction 5%BSA sealase target, adds testing sample, if negative control, the β lactamase Antibody 1:3000 by volume dilution of 100ug/ml, hatches；The dilution of 1mg/ml sheep anti mouse lgG HRP volume ratio 1:5000 adds, and hatches；TMB one-component nitrite ion develops the color；Terminate reaction, survey OD450Nm, by testing sample mean OD value with negative control group OD value than judging that in sample, beta-lactam enzyme concentration is negative or positive.The method has detection, and speed is fast, highly sensitive, simple operation and other advantages.

Description

A kind of method with double antibody sandwich ELISA detection beta-lactamase of optimization
Technical field
The present invention relates to the detection method of beta-lactamase, examining with double antibody sandwich ELISA of a kind of optimization The method surveying beta-lactamase.
Background technology
Beta-lactamase can crack Penicillin in Milk, cephalosporins, makes these class antibiotic at milk Middle minimizing or removal, so be otherwise known as " solution anti-agent ".Penicillin, cephalosporins etc. are by beta-lactam enzymatic lysis Rear generation penicilloic acids etc., the acid of penicillin thiazole becomes Penicilloyl protein with protein bound, and it is to cause penicillin anaphylaxis One of main matter so that there being the people of allergic constitution that the symptom such as skin pruritus, dyspnea occurs.2009, China defended Life portion discloses " may the non-food stuff material of illegal interpolation and the food additive kind list (second of easy abuse in food Batch) ", the most just contain beta-lactamase.
At present, both at home and abroad to the detection method research of beta-lactamase also in the starting stage, beta-lactamase in Lac Bovis seu Bubali Detection method have: cylinder plate method, iodimetric titration, acidity quantitative method, high performance liquid chromatography etc., but these methods exist time-consuming long, False negative and false positive phenomenon, high in cost of production shortcoming, and DASELISA immunization can effectively detect antigen, has Detection speed is fast, highly sensitive, simple operation and other advantages.Therefore, this experiment utilize Rabbit Anti-LACTB and β- Lactamase-Antibody (8A5.A10) sets up double-antibody sandwich elisa detection method, for the detection of beta-lactamase and have Pipe department provides method fast and effectively.
Summary of the invention
It is an object of the invention to provide a kind of method with double antibody sandwich ELISA detection beta-lactamase of optimization.
It is an object of the invention to be achieved through the following technical solutions:
Double antibody sandwich method concrete operations are a kind of technology based on antigen antibody reaction detection antigen, by specific antibody It is attached on solid phase carrier form insolubilized antibody, is subsequently adding determined antigen and combines formation immune complex, the most enzyme-added after washing Traget antibody, antigen is combined formation enzyme labelled antibody-antigen-insolubilized antibody complex in immune complex, adds substrate colour developing, logical Cross microplate reader mensuration OD value thus judge the content of antigen.Concretely comprise the following steps:
1) beta-lactamase antigen it is coated: beta-lactamase is antigen coated in ELISA Plate, every hole 100 μ L, 4 DEG C are overnight, Next day washes plate；
2) it is coated polyclonal antibody: by Rabbit Anti-LACTB that concentration is 0.8mg/ml by Rabbit Anti- LACTB: the ratio of carbonate buffer solution volume ratio 1:800 is diluted, the solution 100 μ L/ hole coated elisa plate after dilution, 4 DEG C overnight incubation, washes plate next day；
3) close: with confining liquid sealase target, every hole 200 μ L, hatch 2h, wash plate for 37 DEG C；
4) sample-adding: add testing sample, set negative control, every hole 100 μ L simultaneously, every part of sample at least adds diplopore, 37 DEG C Hatch 1h, wash plate；
5) monoclonal antibody is added: concentration is that the β-lactamase-Antibody of 100ug/ml is by β-lactamase- The ratio of Antibody:PBS buffer volume ratio 1:3000 is diluted, and the solution after dilution adds enzyme mark version, every hole 100 μ L, hatches 1h, washes plate for 37 DEG C；
6) ELIAS secondary antibody is added: add sheep anti mouse lgG-HRP, every hole 100 μ L, hatch 1h, wash plate for 37 DEG C；
7) colour developing: adding TMB one-component nitrite ion, every hole 100 μ L in ELISA Plate, under room temperature, lucifuge develops the color about 15min；
8) reaction is terminated: often hole adds stop buffer 50 μ L termination reaction, determination experiment knot in 20min after terminating reacting Really.
9) OD is measured450Nm: when measuring worthwhile P/N >=2.1 of OD of 450nm by microplate reader, it is determined that sample is positive, works as P/N During ＜ 2.1, it is determined that sample is negative, and wherein P is testing sample mean OD value, and N is negative control group OD value, β during P/N=2.1- Lactamase concentrations is 25U/ml.
Above-mentioned plate of washing is: discard remaining liq in ELISA Plate, with cleaning mixture 250 μ L/ hole detersive enzyme target, every hole washing 3 Secondary, each 3-5min, filter paper pats dry；
Described confining liquid is volume fraction 5%BSA, volume fraction 7.5%BSA, volume fraction 2.5%BSA, mass fraction One or two or more kinds in 5% defatted milk powder, volume fraction 1%BSA, mass fraction 1% defatted milk powder, described cleaning mixture is PH7.4, the PBS of 0.15M；Described stop buffer is 2M H2SO4。
The antigen coated concentration of described beta-lactamase is 40U/mL, for 0.01M pH9.6 carbonate buffer solution by β-interior Amidase is diluted to 40U/mL gained from 200U/mL.
Described ELIAS secondary antibody is by sheep anti mouse lgG-HRP:PBS by the sheep anti mouse lgG-HRP PBS of 1mg/ml Buffer volume ratio 1:5000 dilution gained.
Described confining liquid is preferably volume fraction 5%BSA.
Described TMB one-component nitrite ion main component is 3,3 ', 5,5 '-tetramethyl benzidine.
Double antibody sandwich ELISA is optimized by the present invention, and polyclonal antibody is coated time and dilution times respectively The determination of number, monoclonal antibody extension rate, enzyme labelled antibody incubation time and diluted concentration, confining liquid condition, during substrate-function Between etc. select, according to choice of experimental conditions optimum condition.
Experiment show that the antibody time of being coated can not be the shortest, and it can not be the most firm that time the shortest antibody is combined with ELISA Plate, shadow Ringing experimental result, so this experiment employing 4 DEG C is overnight, pH9.6,0.01M carbonate buffer solution, as being coated liquid, is strengthened combining energy Power.And monoclonal antibody and ELIAS secondary antibody are diluted multiple optimization, select optimal extension rate for testing, get rid of by In the uncertain factor that monoclonal antibody and ELIAS secondary antibody concentration discomfort cause.Also need to off-period and substrate-function time It is measured, so that it is determined that optimum reaction condition, the evaluation of repeated property, it was demonstrated that the method repeatability that the present invention provides is good, this Method detection least concentration is 25U/mL, and therefore the present invention establishes stable double-antibody sandwich elisa detection method.
Accompanying drawing explanation
The curve of Fig. 1 antigen optium concentration；
Fig. 2 polyclonal antibody and the selection of monoclonal antibody working concentration；
The selection of Fig. 3 enzyme labelled antibody working condition；
The selection of Fig. 4 confining liquid；
The selection of Fig. 5 off-period；
The selection of Fig. 6 substrate-function time；
Detailed description of the invention
Below in conjunction with the accompanying drawings technical scheme is further described, but is not limited thereto, every to this Inventive technique scheme is modified or equivalent, without deviating from the spirit and scope of technical solution of the present invention, all should contain In protection scope of the present invention.
The determination of embodiment 1 best operating condition:
(1) beta-lactamase antigen is most preferably coated the determination of concentration:
The method using chessboard square formation, combination is tested as shown in Figure 1:
1) beta-lactamase antigen it is coated: with 0.01M pH9.6 carbonate buffer solution, beta-lactamase is dilute from 200U/mL Release and be coated in ELISA Plate (diluted concentration gradient is 30U/mL, 40U/mL, 50U/mL, 60U/mL), every hole 100 μ L, 4 DEG C of mistakes At night, next day washes plate；
2) it is coated polyclonal antibody: by Rabbit Anti-LACTB that concentration is 0.8mg/ml by Rabbit Anti- LACTB: the ratio of carbonate buffer solution volume ratio 1:800 is diluted, the solution 100 μ L/ hole coated elisa plate after dilution, 4 DEG C overnight incubation, washes plate next day；
3) close: with 5%BSA confining liquid sealase target, every hole 200 μ L, hatch 2h, wash plate for 37 DEG C；
4) sample-adding: beta-lactamase is diluted to 200U/mL alternate embodiment 1 with 0.01M pH9.6 carbonate buffer solution In testing sample, set negative control (phosphate buffer), every hole 100 μ L simultaneously, every part of sample adds 5 holes, hatches 1h for 37 DEG C, Wash plate；
5) monoclonal antibody is added: concentration is that the β-lactamase-Antibody of 100ug/ml is by β-lactamase- Antibody:PBS buffer volume ratio 1:500, the ratio of 1:1000,1:1500,1:2000,1:2500,1:3000,1:3500 Being diluted, the solution after dilution adds enzyme mark version, every hole 100 μ L, hatches 1h, washes plate for 37 DEG C；
6) ELIAS secondary antibody is added: delayed by sheep anti mouse lgG-HRP:PBS by the sheep anti mouse lgG-HRP PBS of 1mg/ml Rush the dilution of liquid volume ratio 1:5000, every hole 100 μ L, hatch 1h, wash plate for 37 DEG C；
7) colour developing: adding TMB one-component nitrite ion, every hole 100 μ L in ELISA Plate, under room temperature, lucifuge develops the color about 15min；
8) reaction is terminated: every hole adds 2M H2SO4Stop buffer 50 μ L terminates reaction, measures after terminating reacting in 20min Experimental result.
9) OD is measured450Nm: measure the OD value of 450nm by microplate reader, selects close to 1, and comparison feminine gender value is less than 0.2, P/N Antigen when value is maximum is optium concentration.Wherein P is testing sample OD value, and N is negative control group OD value.
Monoclonal antibody be respectively 1:500,1:1000,1:1500,1:2000,1:2500,1:3000,1:3500 dilution, 1:5000 dilutes ELIAS secondary antibody, then measures OD450nm value and P/N value by microplate reader.Selecting close to 1, comparison feminine gender value is little In 0.2, antigen when P/N value is maximum is optium concentration.
As it is shown in figure 1, antigen coated concentration most preferably 40U/mL.
(2) monoclonal antibody and the determination of polyclonal antibody best effort concentration:
The method using chessboard square formation, tests according to combination shown in Fig. 2:
1) beta-lactamase antigen it is coated: with 0.01M pH9.6 carbonate buffer solution, beta-lactamase is dilute from 200U/mL Releasing 40U/mL to be coated in ELISA Plate, every hole 100 μ L, 4 DEG C overnight, and next day washes plate；
2) it is coated polyclonal antibody: by Rabbit Anti-LACTB that concentration is 0.8mg/ml by Rabbit Anti- LACTB: carbonate buffer solution volume ratio 1:500, the ratio of 1:600,1:700,1:800,1:900 are diluted, after dilution Solution 100 μ L/ hole coated elisa plate, 4 DEG C of overnight incubation, wash plate next day；
3) close: with 5%BSA confining liquid sealase target, every hole 200 μ L, hatch 2h, wash plate for 37 DEG C；
4) sample-adding: beta-lactamase is diluted to 200U/mL alternate embodiment 1 with 0.01M pH9.6 carbonate buffer solution In testing sample, set negative control (phosphate buffer), every hole 100 μ L simultaneously, every part of sample adds 5 holes, hatches 1h for 37 DEG C, Wash plate；
5) monoclonal antibody is added: concentration is that the β-lactamase-Antibody of 100ug/ml is by β-lactamase- Antibody:PBS buffer volume ratio 1:500, the ratio of 1:1000,1:1500,1:2000,1:2500,1:3000,1:3500 Being diluted, the solution after dilution adds enzyme mark version, every hole 100 μ L, hatches 1h, washes plate for 37 DEG C；
6) ELIAS secondary antibody is added: delayed by sheep anti mouse lgG-HRP:PBS by the sheep anti mouse lgG-HRP PBS of 1mg/ml Rush the dilution of liquid volume ratio 1:5000, every hole 100 μ L, hatch 1h, wash plate for 37 DEG C；
7) colour developing: adding TMB one-component nitrite ion, every hole 100 μ L in ELISA Plate, under room temperature, lucifuge develops the color about 15min；
8) reaction is terminated: every hole adds 2M H2SO4Stop buffer 50 μ L terminates reaction, measures after terminating reacting in 20min Experimental result.
9) OD is measured450Nm: measure the OD value of 450nm by microplate reader, selects close to 1, and comparison feminine gender value is less than 0.2, P/N Antigen when value is maximum is optium concentration.Wherein P is testing sample OD value, and N is negative control group OD value.
Above-mentioned plate of washing is: discard remaining liq in ELISA Plate, with pH7.4, and, 0.15MPBS cleaning mixture 250 μ L/ hole detersive enzyme Target, every hole washing 3 times, each 3min, filter paper pats dry；
From figure 2 it can be seen that when polyclonal antibody is diluted to 1:800, when monoclonal antibody is diluted to 1:3000, P/N Being worth the highest, sensitivity is the highest.Therefore, choosing polyclonal antibody 1:800, monoclonal antibody 1:3000 is as working concentration.
(3) enzyme labelled antibody incubation time and the determination of diluted concentration:
Step is the method according to chessboard square formation, tests according to combination shown in Fig. 3, determines enzyme labelled antibody incubation time With diluted concentration:
1) beta-lactamase antigen it is coated: with 0.01M pH9.6 carbonate buffer solution, beta-lactamase is dilute from 200U/mL Releasing 40U/mL to be coated in ELISA Plate, every hole 100 μ L, 4 DEG C overnight, and next day washes plate；
2) it is coated polyclonal antibody: by Rabbit Anti-LACTB that concentration is 0.8mg/ml by Rabbit Anti- LACTB: the ratio of carbonate buffer solution volume ratio 1:800 is diluted, the solution 100 μ L/ hole coated elisa plate after dilution, 4 DEG C overnight incubation, washes plate next day；
3) close: with 5%BSA confining liquid sealase target, every hole 200 μ L, hatch 2h, wash plate for 37 DEG C；
4) sample-adding: adding testing sample, set negative control (phosphate buffer) simultaneously, every hole 100 μ L, every part of sample adds 5 holes, hatch 1h, wash plate for 37 DEG C；
5) monoclonal antibody is added: concentration is that the β-lactamase-Antibody of 100ug/ml is by β-lactamase- The ratio of Antibody:PBS buffer volume ratio 1:3000 is diluted, and the solution after dilution adds enzyme mark version, every hole 100 μ L, hatches 1h, washes plate for 37 DEG C；
6) ELIAS secondary antibody is added: the sheep anti mouse lgG-HRP PBS of 1mg/ml is buffered by sheep anti mouse lgG-HRP:PBS Liquid volume ratio 1:3000,1:4000,1:5000,1:6000,1:7000 dilution, every hole 100 μ L, incubation time is respectively 37 DEG C 30min, 37 DEG C of 1h, 37 DEG C of 2h, wash plate；
7) colour developing: adding TMB one-component nitrite ion, every hole 100 μ L in ELISA Plate, under room temperature, lucifuge develops the color about 15min；
8) reaction is terminated: every hole adds 2M H2SO4Stop buffer 50 μ L terminates reaction, measures after terminating reacting in 20min Experimental result.
9) OD is measured450Nm: measure the OD value of 450nm by microplate reader.
Above-mentioned plate of washing is: discard remaining liq in ELISA Plate, with pH7.4, and, 0.15MPBS cleaning mixture 250 μ L/ hole detersive enzyme Target, every hole washing 3 times, each 3min, filter paper pats dry；
From figure 3, it can be seen that when polyclonal antibody is diluted to 1:800, and monoclonal antibody is diluted to 1:3000, other In the case of part is constant, enzyme labelled antibody is with 1:5000, and during 37 DEG C of 1h, P/N value is the highest, and estimating this condition of selection is the suitableeest diluted concentration And the response time.
(4) determination of confining liquid kind:
Step is the method according to chessboard square formation, as shown in Figure 4 compound mode, determines confining liquid kind.Antigen, polyclone Antibody, monoclonal antibody, ELIAS secondary antibody all select the conditions such as optium concentration and the response time that above-mentioned experiment determines, by confining liquid Select mass fraction 1% defatted milk powder, mass fraction 5% defatted milk powder, volume fraction volume fraction 1%BSA, volume integral respectively Number 2.5%BSA, volume fraction 5%BSA, volume fraction 7.5%BSA are closed, and every hole 200 μ L hatches 2h for 37 DEG C, other Step and condition, with (3), measure OD by microplate reader450Value, selects best effort concentration according to P/N value analysis.
Figure 4, it is seen that when polyclonal antibody is diluted to 1:800, and monoclonal antibody is diluted to 1:3000, and enzyme mark resists Body is with 1:5000, in the case of other conditions are constant, and result display 5%BSA (P/N) > 7.5%BSA (P/N) > 2.5%BSA (P/ N) > 5% defatted milk powder (P/N) > 1%BSA (P/N) > 1% defatted milk powder (P/N), therefore, selection 5%BSA is confining liquid.
(5) determination of off-period:
Step is the method according to chessboard square formation, as shown in Figure 5 compound mode, determines the confining liquid time.At above-mentioned (4) base On plinth, close with optimal confining liquid, and it is same to hatch 1h, 1.5h, 2h, 2.5h, 3h, other steps and condition at 37 DEG C respectively (4), OD is measured by microplate reader450Value, selects best effort concentration according to P/N value analysis.
From figure 5 it can be seen that when polyclonal antibody is diluted to 1:800, and monoclonal antibody is diluted to 1:3000, confining liquid For 5%BSA, enzyme labelled antibody is with 1:5000, in the case of other conditions are constant, off-period 37 DEG C of 2h (P/N) > 37 DEG C of 3h (P/ N)>37℃2.5h(P/N)>37℃1.5h(P/N)>37℃1h(P/N).Estimate selection 37 DEG C closing 2h is optimal off-period.
(6) determination of substrate-function time:
Step is the method according to chessboard square formation, as shown in Figure 6 compound mode, determines the substrate-function time.Use above-mentioned (1) optimum reaction condition that-(5) are selected, in the case of lucifuge substrate developing time choose respectively 10min, 15min, 20min, 25min, 30min, at the 2M H adding 50 μ L in every hole after substrate colour developing2SO4Stop buffer, then measures OD by microplate reader450 Value, other steps and condition, with (5), determine the suitableeest substrate-function time.
From fig. 6 it can be seen that when polyclonal antibody is diluted to 1:800, and monoclonal antibody is diluted to 1:3000, confining liquid For 5%BSA, closing 2h, enzyme labelled antibody is with 1:5000, in the case of other conditions are constant, and the P/N value as substrate-function 15min Maximum, therefore, room temperature 15min is the suitableeest developing time.
Embodiment 2
1) beta-lactamase antigen it is coated: beta-lactamase is antigen coated in ELISA Plate, every hole 100 μ L, 4 DEG C are overnight, Next day washes plate；
2) it is coated polyclonal antibody: by Rabbit Anti-LACTB that concentration is 0.8mg/ml by Rabbit Anti- LACTB: the ratio of carbonate buffer solution volume ratio 1:800 is diluted, the solution 100 μ L/ hole coated elisa plate after dilution, 4 DEG C overnight incubation, washes plate next day；
3) close: with 5%BSA confining liquid sealase target, every hole 200 μ L, hatch 2h, wash plate for 37 DEG C；
4) sample-adding: adding testing sample, set negative control (phosphate buffer) simultaneously, every hole 100 μ L, every part of sample adds 5 holes, hatch 1h, wash plate for 37 DEG C；
5) monoclonal antibody is added: concentration is that the β-lactamase-Antibody of 100ug/ml is by β-lactamase- The ratio of Antibody:PBS buffer volume ratio 1:3000 is diluted, and the solution after dilution adds enzyme mark version, every hole 100 μ L, hatches 1h, washes plate for 37 DEG C；
6) ELIAS secondary antibody is added: add sheep anti mouse lgG-HRP, every hole 100 μ L, hatch 1h, wash plate for 37 DEG C；
7) colour developing: adding TMB one-component nitrite ion, every hole 100 μ L in ELISA Plate, under room temperature, lucifuge develops the color about 15min；
8) reaction is terminated: every hole adds 2M H2SO4Stop buffer 50 μ L terminates reaction, measures after terminating reacting in 20min Experimental result.
9) OD is measured450Nm: when measuring worthwhile P/N >=2.1 of OD of 450nm by microplate reader, it is determined that sample is positive, works as P/N During ＜ 2.1, it is determined that sample is negative, and wherein P is testing sample mean OD value, and N is negative control group OD value, β during P/N=2.1- Lactamase concentrations is 25U/ml.
Above-mentioned plate of washing is: discard remaining liq in ELISA Plate, with pH7.4, and, 0.15MPBS cleaning mixture 250 μ L/ hole detersive enzyme Target, every hole washing 3 times, each 3min, filter paper pats dry；Described cleaning mixture is pH7.4, the PBS of 0.15M；Described termination Liquid is 2M H2SO4。
Embodiment 3
Step 1 on the basis of embodiment 2) in the antigen coated concentration of beta-lactamase be 40U/mL, for 0.01M Beta-lactamase is diluted to 40U/mL gained from 200U/mL by pH9.6 carbonate buffer solution.
Embodiment 4
On the basis of embodiment 3, ELIAS secondary antibody is by goat-anti by the sheep anti mouse lgG-HRP PBS of 1mg/ml Mus lgG-HRP:PBS buffer volume ratio 1:5000 dilution gained.
Embodiment 5
On the basis of embodiment 4, confining liquid is preferably volume fraction 5%BSA.
Embodiment 6 reproducibility:
(1) test in repeatability batch
Choosing with batch ELISA Plate assembled, 4 parts of different testing samples (positive and negative sample) are by implementing Example 5 step detects, and in same ELISA Plate, each sample carries out 5 parallel tests, operates journey according to well-established ELISA Sequence, carries out ELISA mensuration, surveys OD450Value, carries out statistic analysis result, and its coefficient of variation, 2.22%～7.14%, is less than 10%, degree of variation is the least, reproducible, table 1.
Replica test in 1 group of table
(2) test between repeatability batch
That selects 5 different times formulations is coated flat board, (positive to 4 parts of different testing samples at identical conditions Sample and negative sample) detect by embodiment 1 step, according to well-established ELISA operation sequence, carry out ELISA survey Fixed, calculate the coefficient of variation with a detection sample OD450 value, statistic analysis result, the coefficient of variation 2.01%～8.03%, Less than 10%.Illustrate reproducible, can test, table 2.
Replica test between 2 groups of table
Four, sensitivity assessment
Be diluted from beta-lactamase standard solution 200U/mL, be diluted to different concentration (150,100,75,50, 25,20U/ml), measure OD450 value with the double antibody sandwich ELISA set up, during P/N=2.1, beta-lactam enzyme concentration is 25U/ml, detection least concentration is 25U/mL, when P/N >=2.1, it is determined that sample is positive, as P/N ＜ 2.1, it is determined that sample For the positive.

Claims (5)

1. the method with double antibody sandwich ELISA detection beta-lactamase optimized, its step is as follows:
1) being coated beta-lactamase antigen: beta-lactamase is antigen coated in ELISA Plate, every hole 100 μ L, 4 DEG C overnight, next day Wash plate；
2) it is coated polyclonal antibody: by Rabbit Anti-LACTB that concentration is 0.8mg/ml by Rabbit Anti-LACTB: The ratio of carbonate buffer solution volume ratio 1:800 is diluted, the solution 100 μ L/ hole coated elisa plate after dilution, hatches for 4 DEG C Overnight, plate is washed next day；
3) close: with confining liquid sealase target, every hole 200 μ L, hatch 2h, wash plate for 37 DEG C；
4) sample-adding: adding testing sample, set negative control, every hole 100 μ L simultaneously, every part of sample at least adds diplopore, hatches for 37 DEG C 1h, washes plate；
5) monoclonal antibody is added: concentration is that the β-lactamase-Antibody of 100ug/ml is by β-lactamase- The ratio of Antibody:PBS buffer volume ratio 1:3000 is diluted, and the solution after dilution adds enzyme mark version, every hole 100 μ L, hatches 1h, washes plate for 37 DEG C；
6) ELIAS secondary antibody is added: add sheep anti mouse lgG-HRP, every hole 100 μ L, hatch 1h, wash plate for 37 DEG C；
7) colour developing: adding TMB one-component nitrite ion, every hole 100 μ L in ELISA Plate, under room temperature, lucifuge develops the color about 15min；
8) reaction is terminated: often hole adds stop buffer 50 μ L termination reaction, determination experiment result in 20min after terminating reacting；
9) OD is measured450Nm: measure the OD value of 450nm by microplate reader, when P/N >=2.1, it is determined that sample is positive, as P/N ＜ When 2.1, it is determined that sample is negative, and wherein P is testing sample mean OD value, and N is negative control group OD value, β during P/N=2.1-interior Amidase concentration is 25U/ml, for this method minimal detectable concentration；
Above-mentioned plate of washing is: discard remaining liq in ELISA Plate, and with cleaning mixture 250 μ L/ hole detersive enzyme target, every hole washs 3 times, often Secondary 3-5min, pats dry on filter paper.
The most in accordance with the method for claim 1, it is characterised in that: described confining liquid is volume fraction 5%BSA, volume fraction 7.5%BSA, volume fraction 2.5%BSA, mass fraction 5% defatted milk powder, volume fraction 1%BSA, mass fraction 1% defat One or two or more kinds in milk powder, described cleaning mixture is pH7.4, the PBS of 0.15M；Described stop buffer is 2M H2SO4。
The most in accordance with the method for claim 1, it is characterised in that: the antigen coated concentration of described beta-lactamase is 40U/mL.
The most in accordance with the method for claim 1, it is characterised in that: described ELIAS secondary antibody i.e. sheep anti mouse lgG-HRP is will The sheep anti mouse lgG-HRP of 1mg/ml presses sheep anti mouse lgG-HRP:PBS buffer volume ratio 1:5000 with PBS and dilutes institute ?.
The most in accordance with the method for claim 1, it is characterised in that: described confining liquid is volume fraction 5%BSA.
CN201610356629.9A 2016-05-26 2016-05-26 A kind of method with double antibody sandwich ELISA detection beta lactamase of optimization Pending CN106093403A (en)

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CN108445217A (en) * 2018-03-08 2018-08-24 山东绿都生物科技有限公司 A kind of fluorescent microsphere detection card quantitatively detecting beta-lactamase residues in milk
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CN112858694B (en) * 2021-02-05 2024-03-12 湖南泰新医药科技有限公司 Method for simultaneously measuring anti-PD-1 antibody and anti-PD-L1 antibody concentration in human serum

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