CN105785015A

CN105785015A - Kit and method for rapidly and highly sensitively detecting OA

Info

Publication number: CN105785015A
Application number: CN201610268286.0A
Authority: CN
Inventors: 王平; 邱先鑫; 苏凯麒; 钟隆洁; 方佳如; 田玉兰; 邹瞿超
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2016-04-25
Filing date: 2016-04-25
Publication date: 2016-07-20

Abstract

The invention discloses a kit and method for rapidly and highly sensitively detecting OA, belongs to the field of food detection, and particularly relates to synthesis of a conjugate OA-BSA with BSA and OA and labeling of a monoclonal antibody of OA with HRP for formation of HRP-MAb. The kit comprises a detachable 8*12 ELISA plate labeled by OA-BSA, a standard at an OA series concentration, an OA monoclonal antibody HRP-MAb labeled by an HRP labeling kit, 10* washing concentrate, a chromogenic substrate TMB and a stopping solution HCl. A detection limit of the method is 0.19 ng/ml, and is converted into a detection limit 9.5 [mu]g/kg of the OA in shellfish meat, a detection range is 0.2 to 5 ng/ml, and is converted into a detection range 10 to 250 [mu]g/kg of the OA in the shellfish meat, the total OA content of shellfish is specified by the import and export standards not to exceed 160 [mu]g/kg, so that the kit well meets a detection requirement, and meanwhile, the detection limit is far lower than those of a PP2A-based kit (50 [mu]g/kg) and mouse bioassay (200 [mu]g/kg). In addition, the kit is low in cost, easy to operate, short in reaction time and applicable to field rapid detection, and 84 samples can be detected at the same time.

Description

The test kit of a kind of fast high-sensitive detection okadaic acid toxin and method

Technical field

The present invention relates to field of food detection, particularly relate to a kind of fast high-sensitive detection land for growing field crops soft The test kit of sponge acid toxin and method.

Background technology

Okadaic acid (OA) is the fat-soluble sea of one produced by fin algae and Prorocentrum micans Ocean biotoxin.Variety classes shellfish achieves biotoxin shellfish by edible marine algae In enrichment, but shellfish itself is not affected by this toxin, and the mankind are edible contaminated shellfish After class, diarrhoeal mussel poisoning (DSP) and gastrointestinal disturbance can be caused.By zoopery, OA has been proved to have carcinogenecity, mutagenesis and immunotoxicity.EU Committee (EC) Cmax in regulation shellfish meat is less than 160ug/kg.Mouse bioassay is once by as mensuration The standard method of OA.But its selectivity and precision are relatively low and relate to ethical issues, in 2011 January in year is prohibited.Efficiently liquid phase and the method sensitivity of HPLC-MS Height, result are accurate, but equipment is much more expensive, time-consuming, be also required to skilled operator, and Sample pre-treatments is had higher requirements.Other technologies such as cell sensor etc. is also used for shellfish poison The detection of element, but be also unfavorable for realizing field quick detection.Therefore in the urgent need to extremely sensitive Method realizes the OA toxin quickly detecting in shellfish meat.

Summary of the invention

Present invention aims to the deficiencies in the prior art, the method utilizing enzyme linked immunological, Proposing the test kit of a kind of field quick detection okadaic acid, this test kit has high pass Amount, low detection limit, easy and simple to handle, the shortest, the advantage of low cost, fill up this skill domestic The blank of art.

It is an object of the invention to be achieved through the following technical solutions:

The test kit of a kind of fast high-sensitive detection okadaic acid toxin, including OA-BSA The ELISA Plate of detachable 8*12 of labelling, the standard substance of OA series concentration, HRP labelling try OA monoclonal antibody HRP-MAb of agent box labelling, 10 × concentrated cleaning solution, chromogenic substrate TMB, stop buffer HCl.

Further, the preparation process of OA-BSA is: the OA powder of (1) 50 μ g is dissolved in In 50 μ l methanol solutions, obtain OA solution.(2) by the EDC of 10ul 20mg/ml and The NHS of 10ul 10mg/ml is added in OA solution, and slowly concussion 2 hours, are activated OA solution, wherein, the solvent of EDC and NHS is 0.1M MES, pH=5.5.(3) The BSA of 400 μ l 1mg/ml is dissolved in the PBS of 0.01M pH=7.2, by It is added dropwise in the OA solution of activation.(4) sustained response under the conditions of liquid earthquake mixed above 4 hours, obtain OA-BSA solution, bag filter MW=10000 is placed on volume integral simultaneously Cool down after several 20% ethanol boil.(5) above-mentioned OA-BSA solution bag filter is dialysed, Dialysis solution is the PBS of 0.01MpH=7.2, and dialysis solution is changed once for every eight hours, Change four times altogether, obtain OA-BSA stock solution.

Further, the preparation process of the ELISA Plate of the detachable 8*12 of OA-BSA labelling is: (1) Coated elisa plate, hatches, and washes plate；It is coated the carbonate buffer that buffer is 0.05M pH9.6 Liquid, containing 1.59gNaCO in 1L carbonate buffer solution₃、2.93gNaHCO₃With 0.1M NaCl； (2) sealase target, hatches, and washes plate；Confining liquid is the BSA solution of mass fraction 1%.

Further, the incubation temperature of ELISA Plate is 35 DEG C, and incubation time is 1h.

Further, 10 × concentrated cleaning solution is the PBS+1%Tween20 of 0.1M, during use Dilute 10 times.

Further, standard concentration be 0,0.2,0.5,1,2,5ng/ml.

A kind of method detecting okadaic acid toxin, comprises the following steps:

(1) respectively standard substance and sample are added the detachable 8*12's of OA-BSA labelling In ELISA Plate hole, in each hole, add HRP-MAb, hatch, wash plate；

(2) add chromogenic substrate TMB, hatch；

(3) add stop buffer HCl, at 450nm, measure absorbance；

(4) absorbance according to standard substance draws the standard curve of detection OA toxin, according to Standard curve calculates the concentration of toxin in sample.

Further, the pretreatment process adding the sample in ELISA Plate hole is: takes 0.2g and blends shellfish Meat adds 1ml volume fraction 90% methanol, and vortex concussion 5min, 7000rpm are centrifuged 3min, Taking supernatant liquid filtering, filter is 0.25um organic system, dilute 10 times to be detected.

Further, OA-BSA 100ul, confining liquid 200ul, standard substance and sample 100ul respectively, HRP-MAb 50ul, TMB 100ul, HCl 100ul.

Further, the incubation time 30min in step (1), in step (2) when hatching Between 15min.

The present invention compared with prior art provides the benefit that: technical solution of the present invention is relative to mice Bioanalysis, efficient liquid phase or the method for HPLC-MS, cell sensor Method, operate the simplest, it is not necessary to professional operator and operating environment, cost is more For cheap, react the rapidest.The detection of this test kit is limited to 0.19ng/ml, less than based on The test kit of other principles detection OA.Detection can be completed in 90 minutes, have simultaneously High-throughout feature, once can detect 84 samples, is highly suitable for the quick detection at scene, And filled up the blank in this field domestic.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of described test kit；

Fig. 2 is the S type curve chart of described test kit detection okadaic acid concentration range；

Fig. 3 is the canonical plotting of described test kit detection okadaic acid；

Fig. 4 is the specificity curve chart of described test kit.

Detailed description of the invention

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but not It is to limit the present invention.

Embodiment 1: the test kit of a kind of fast high-sensitive detection okadaic acid toxin, tool Body includes the ELISA Plate of detachable 8*12 of OA-BSA labelling, the standard of OA series concentration Product, OA monoclonal antibody HRP-MAb of HRP labelling kit labelling, 10 × concentration are washed Wash liquid, chromogenic substrate TMB, stop buffer HCl.

Embodiment 2: the preparation of antigen coated microplate

(1) antigen coated ELISA Plate

Using the carbonate buffer solution of 0.05M pH 9.6 as being coated liquid, in ELISA Plate, every hole adds Enter 100ul, hatch 1h for 35 DEG C.

(2) plate is washed

Outwelling the liquid in ELISA Plate, the concentration adding 200ul dilution 10 times in every hole is washed Wash liquid, shake 2min, repeat above step 3 times.

(3) close

In ELISA Plate, every hole adds the BSA solution of 1%, hatches 1h for 35 DEG C.

(4) plate is washed, encapsulation

Wash plate by the step in (2), then antigen coated ELISA Plate loaded in aluminium foil bag, Putting into desiccant, vacuum sealer carries out vacuum-pumping and sealing, 4 DEG C of storages.

Embodiment 3: the determination of detection range

In test kit add 100ul OA series concentration, 0,0.005,0.01,0.02,0.05, 0.1,0.2,0.5,1,2,5,10,20,50,100,200ng/ml, every kind of concentration 3 Individual repeating hole, adds HRP-MAb, washes plate, be subsequently adding TMB, add after hatching after hatching Enter HCl, survey absorbance at 450nm, be the hole of 0 by every hole absorbance A and concentration Absorbance average A₀Compare, draw A/A₀.Fig. 2 is along with OA concentration change A/A₀Value, as seen from the figure, in the range of OA concentration range is 0.2-5ng/ml, A/A₀With OA concentration presents good linear relation, and Fig. 3 is to elect 5 points in the range of this to mark Directrix curve, obtains r²It is 0.9839, presents good linear relationship, the therefore line of this test kit Property scope is defined as 0.2-5ng/ml.

Embodiment 4: the specificity of test kit

Different types of toxin such as dinophysistoxin (DTX), Saxidomus poison is added in test kit Element (STX), gonyatoxin (GTX), scallop toxin (PTX), every kind of toxin is every 3 repeating holes of individual concentration, subsequent process reference example 3.Specificity cross reacting rate comes Represent.Fig. 3 is the not isotoxin cross reaction to this test kit, the IC of DTX₅₀For The IC of 6.382ng/ml, OA₅₀For 1.504ng/ml, therefore the intersecting of DTX and this test kit Response rate is 23.6%, and on the one hand this cross reacting rate is less, still further aspect, China shellfish The content of apoplexy due to endogenous wind DTX is almost 0.And the IC of other several toxin₅₀All tend to very big, hand over Fork response rate < 0.2%.

Embodiment 5: detection limit, recovery of standard addition, the mensuration of the coefficient of variation

(1) sample mark-on: take nontoxic shellfish meat, shellfish meat kind is Mytilus edulis, scallop, Concha Ostreae, 10g Blend 20s, in shellfish meat, inject corresponding toxin amount, spiked levels is 20,40,80,160, 200ug/kg, is subsequently adding the methanol 50ml of 90%, stirs 2min, weighs shellfish meat 2g, and 3 Pipe, 7000rpm is centrifuged 3min, takes supernatant 0.25um organic system filter and filters, dilute Release 10 times to be detected.

(2) adding serial standards in test kit, 2 repeating holes, other holes add respectively Entering blank value, 7 repeating holes, the mark-on sample in (1), 9 repeating holes are 9, follow-up Experimentation reference example 3.Detection limit computational methods by surveys blank well concentration put down The standard deviation of average+3 × blank well concentration；Recovery of standard addition is by every three holes in 9 holes Averaging, 3 then obtained take average again, then divided by spiked levels, calculate change simultaneously Different coefficient.Calculate the spacing 0.19ng/ml of detection of this test kit；Table 1 reclaims for mark-on Rate and the coefficient of variation, it can be seen that the response rate of this test kit, between 75-105%, makes a variation Coefficient is at 2-16%.

Table 1 recovery of standard addition and the coefficient of variation

Concentration (ug/kg)	Mussel	Concha Ostreae	Scallop
				20	84.47% ± 2.75%	82.35% ± 1.79%	75.78% ± 8.11%
40	94.01% ± 6.43%	89.91% ± 9.49%	95.26% ± 7.16%
				80	80.29% ± 2.82%	102.56% ± 11.33%	87.74% ± 15.62%
160	88.96% ± 15.93%	99.92% ± 11.15%	102.34% ± 9.68%
				200	83.23% ± 8.43%	78.96% ± 8.27%	81.89% ± 15.46%

Embodiment 6: test kit detection actual sample (should compared with efficient liquid phase (HPLC) With example)

(1) take the poisonous shellfish meat that 0.2g blends and add the methanol that 1ml volume fraction is 90%, whirlpool Rotation concussion 5min, 7000rpm are centrifuged 3min, take supernatant 0.25um organic system filter Filter, dilute 10 times to be detected.

(2) adding serial standards, 2 repeating holes to shown test kit, other holes are respectively Add poisonous shellfish meat sample in (1), 3 repeating holes, 22 samples, subsequent experimental process Reference example 3.And detect same shellfish meat sample with HPLC.Table 2 is described test kit Result with Liquid Detection actual sample.The detection of this test kit is limited to 0.19ng/ml, front place The extension rate of reason process is 50 times, so being converted in shellfish meat as 9.5ug/kg.Can see Going out in the sample of detected 20, described test kit detects that 3 sample datas are higher than 9.5ug/kg, and the testing result of test kit is close with the result of HPLC, and this reagent is described The effectiveness of box.

Table 2 test kit detects actual sample data with HPLC

Claims

1. the test kit of a fast high-sensitive detection okadaic acid toxin, it is characterised in that Including the ELISA Plate of detachable 8*12 of OA-BSA labelling, the standard substance of OA series concentration, OA monoclonal antibody HRP-MAb of HRP labelling kit labelling, 10 × concentrated cleaning solution, Chromogenic substrate TMB, stop buffer HCl.

Test kit the most according to claim 1, it is characterised in that the preparation of OA-BSA Cheng Wei:

The OA powder of (1) 50 μ g is dissolved in 50 μ l methanol solutions, obtains OA solution.

(2) NHS of EDC and the 10ul 10mg/ml of 10ul 20mg/ml is added to OA solution In, slowly concussion 2 hours, obtain the OA solution of activation, wherein, EDC's and NHS Solvent is 0.1M MES, pH=5.5.

(3) BSA of 400 μ l 1mg/ml is dissolved in the PBS of 0.01M pH=7.2 In, it is added dropwise in the OA solution of activation.

(4) sustained response 4 hours under the conditions of liquid earthquake mixed above, obtain OA-BSA solution, Bag filter MW=10000 is placed on after volume fraction 20% ethanol boils simultaneously and cools down.

(5) being dialysed by above-mentioned OA-BSA solution bag filter, dialysis solution is 0.01MpH=7.2's PBS, dialysis solution is changed once for every eight hours, changes four times altogether, obtains OA-BSA Stock solution.

Test kit the most according to claim 1, it is characterised in that OA-BSA labelling can The preparation process of the ELISA Plate of dismounting 8*12 is:

(1) coated elisa plate, hatches, and washes plate；It is coated the carbonic acid that buffer is 0.05M pH9.6 Salt buffer, containing 1.59gNaCO in 1L carbonate buffer solution₃、2.93gNaHCO₃With 0.1M NaCl；

(2) sealase target, hatches, and washes plate；Confining liquid is the BSA solution of mass fraction 1%.

Test kit the most according to claim 3, it is characterised in that the incubation temperature of ELISA Plate Being 35 DEG C, incubation time is 1h.

Test kit the most according to claim 1, it is characterised in that 10 × concentrated cleaning solution is The PBS+1%Tween20 of 0.1M, dilutes 10 times during use.

Test kit the most according to claim 1, it is characterised in that standard concentration is 0, 0.2、0.5、1、2、5ng/ml。

7. utilize a method for test kit detection okadaic acid toxin described in claim 1, It is characterized in that, comprise the following steps:

(1) respectively standard substance and sample are added the enzyme mark of the detachable 8*12 of OA-BSA labelling In plate hole, in each hole, add HRP-MAb, hatch, wash plate；

(2) add chromogenic substrate TMB, hatch；

(3) add stop buffer HCl, at 450nm, measure absorbance；

Method the most according to claim 7, it is characterised in that add the sample in ELISA Plate hole Pretreatment process be: take 0.2g and blend shellfish meat and add 1ml volume fraction 90% methanol, vortex Concussion 5min, 7000rpm are centrifuged 3min, take supernatant liquid filtering, and filter is that 0.25um has Machine system, dilute 10 times to be detected.

Method the most according to claim 7, it is characterised in that OA-BSA100ul, closes Liquid 200ul, standard substance and sample 100ul, HRP-MAb 50ul, TMB 100ul respectively, HCl 100ul。

Method the most according to claim 7, it is characterised in that hatching in step (1) Time 30min, the incubation time 15min in step (2).