CN101776690A

CN101776690A - Immunoassays kit of microcystin-LR and detection method thereof

Info

Publication number: CN101776690A
Application number: CN201010105246A
Authority: CN
Inventors: 何恩奇; 钮伟民; 吴庆刚; 刘文卫; 周磊; 黄飚; 张艺
Original assignee: Wuxi Center for Disease Control and Prevention
Current assignee: Wuxi Center for Disease Control and Prevention
Priority date: 2010-01-28
Filing date: 2010-01-28
Publication date: 2010-07-14

Abstract

The invention relates to an immunoassays kit of microcystin-LR and a detection method thereof, which belong to the technical field of the photo stimulation chemiluminescence immunoassay. The kit consists of a white non-transparent micro-porous plate (1), an MC-LR standard product (2), (3) luminous micro particles which are connected with MC-LR-BSA (3), monoclonal antibody solution of biotinylation antiMC-LR (4) and light-sensing micro particles being enveloped with Streptavidin (5). The detection method comprises the following steps: luminous micro particles being enveloped with MC-LR-BSA, MC-LR standard product or sample and monoclonal antibody solution of biotinylation antiMC-LR are sequentially added into the non-transparent white micro-porous plate to be reacted in dark place, and then the light-sensing micro particles being enveloped with Streptavidin are added to be detected after incubation. A photo stimulation chemical luminous detection instrument is used for the detection, and the strength of the light signal is proportional to the concentration of MC-LR in the sample, so the content of MC-LR is measured referring to the standard curve. The method is used for the detection of the MC-LR content in the water. The kit has simple components, the detection time is short, the sensibility is high, and the operation is simple and convenient.

Description

A kind of immunoassay kits of microcapsule algae toxin and detection method thereof

Technical field

The light-induced chemiluminescent immunoassay kit and the detection method thereof of a kind of microcapsule algae toxin (MC-LR) are used for the detection of water body MC-LR content, belong to light-induced chemiluminescent immunoassay (LICA) technical field.

Background technology

Microcystin (Microcystin) is the hepatotoxin that a class that blue-green alga bloom discharges has strong toxic and side effect, and wherein microcapsule algae toxin (MC-LR) is the strongest a kind of of this toxin poisoning.Studies show that MC-LR can cause poultry, domestic animal and wild animal death.And the MC-LR of low dosage just can cause people's hepar damnification even liver cancer in the potable water.Algae toxin in the fresh water water body has become global environment problem at present, and the Microcystin poisoning often takes place all over the world.By the potable water of algae endotoxin contamination and aquatic products, bring huge threat to human health, China has listed Microcystin in the water quality detection index in the new edition " drinking water standard " of enforcement on the 1st July in 2007, and standard is 1 μ g/mL.

MC-LR is a kind of ring-type seven peptides, and molecular weight 995.2 is soluble in water, non-volatility, and chemical property and stable all can not be destroyed it as high temperature, illumination, proteinase etc.The coagulating sedimentation of water technology, filtration can not under the effect of some bacterium can be degraded its effective removal under ultraviolet irradiation and in the water.

Because the damage that causes of MC-LR is quick and irreversible, thereby sets up the content of toxins that quick sensitive detecting method monitors in the water body and will play good forewarning function.Utilize plysiochemical characteristic such as molecular weight, chromophore and the reactivity of MC-LR, the assay method of having set up at present MC-LR has bioassay method, chemical assay and immunoassay, and biological toxicity test wherein, phosphoprotein phosphatase inhibition method (PPI), HPLC detection method and ELISA detection method are methods comparatively commonly used both at home and abroad at present.When traditional detection method was used widely, people also constantly introduced new technology the detection of MC-LR, in the hope of setting up more responsive, more stable, easier MC-LR detection technique.

Light-induced chemiluminescent immunoassay (LICA) is the chemiluminescence immunoassay technology of new generation based on the nanoscale high molecular particle, and this technology will be widely used in the interaction of research biomolecule.Be called AlphaLISA analytic approach (PE company) again, be described as disposable ELISA.Its cardinal principle is the homogeneous chemistry luminescence technology that is produced by optical excitation, and it has fast, homogeneous phase (disposable), highly sensitive, broad quantum and characteristics easy and simple to handle.LICA reagent is formed the about 188nm of mean particle dia, surface coverage polysaccharide hydrogel by photosensitive particulate that contains Photoactive compounds and the luminous particle that contains luminophor.Hydrogel can reduce non-specific binding, simultaneously, increases the suspension of particulate.Particulate is covalently bound by the functional group and the biomolecule of hydrogel surface.It is long-pending that nano_scale particle has increased reacted surface greatly, and each microparticle surfaces is covered with hundreds and thousands of biomolecule, can catch target molecule.

The central principle of LICA technology is the generation and the transmission of singlet oxygen.After being subjected to red laser (680nm) irradiation, photosensitive particulate can make the oxygen in the surrounding environment be converted into singlet oxygen, and the life span of singlet oxygen only is 4 microseconds.Of short duration life span has determined the propagation diameter of singlet oxygen very little (being about 200nm).If luminous particle just can be accepted singlet oxygen within the 200nm scope, and send the light (520nm-620nm) of high level.On the contrary, if do not have luminous particle in the 200nm diameter range, singlet oxygen will fall back to ground state oxygen and not have signal to produce.This depend on two kinds of particulates mutually approaching chemical energy transmission be the basis of LICA homogeneous reaction.Usually in this reaction system, the concentration of particulate is very low.The probability of two kinds of mutual random collisions of particulate is very low, and therefore, the background of reaction system is very faint.If be coated on the bio-molecular interaction of microparticle surfaces, the distance of two particulates that furthered for example forms the sandwich or receptor-ligand compound of immunity, so just the energy-producing effective transmission of energy and send light signal.

Summary of the invention

The object of the present invention is to provide kit and the detection method thereof of a kind of MC-LR of detection, be used for detection water body MC-LR content.

Technical scheme of the present invention: the light-induced chemiluminescent immunoassay kit of a kind of microcapsule algae toxin (MC-LR), by White-opalescent microwell plate (1), MC-LR standard items (2), be coated with the luminous particle (3) of MC-LR-BSA, the anti-MC-LR monoclonal anti of biotinylation liquid solution (4) and the photosensitive particulate (5) that is coated with Streptavidin are formed.

Described kit detects the method for MC-LR, and the basis of mensuration is the labelled immune reaction.In the White-opalescent microwell plate, add luminous particle, MC-LR standard items or the sample of handling well, the anti-MC-LR monoclonal anti of the biotinylation liquid solution that are coated with MC-LR-BSA successively and carry out immune response; Then add the photosensitive particulate that is coated with Streptavidin and react, reaction back sensed light signal is to add the detected value drawing standard curve of MC-LR standard items, to add the MC-LR content of detected value from the typical curve calculation sample of sample.

Concrete operations are: get luminous particle 20 μ L, MC-LR standard items that are coated with MC-LR-BSA or the sample 20 μ L that handle well and the anti-MC-LR monoclonal anti of biotinylation liquid solution 20 μ L and be added to successively in the White-opalescent microwell plate, hatched 30 minutes for 37 ℃; Add the photosensitive particulate 175 μ L that are coated with Streptavidin, hatched 10 minutes for 37 ℃; Sensed light signal on the light-induced chemiluminescent detector calculates MC-LR content the sample from typical curve.

Beneficial effect of the present invention: MC-LR-BSA on the luminous particle and free MC-LR competition are connected on the anti-MC-LR monoclonal antibody of biotinylation, formed compound with bag by the photosensitive particulate of Streptavidin again, excite down at ruddiness, generation and transmission by the singlet ion-oxygen, NE BY ENERGY TRANSFER is produced fluorescence to luminous particle, detect with light activation luminous detection instrument, MC-LR concentration is inversely proportional in light signal strength and the sample, and the reference standard curve records MC-LR content in the sample.The present invention is used for the detection of water body MC-LR content, and this detection kit is simple in structure, and easy and simple to handle, detection time is short, highly sensitive.

Description of drawings

Fig. 1: the kit synoptic diagram that detects MC-LR.1, White-opalescent microwell plate, 2, the MC-LR standard items, 3, be coated with the luminous particle of MC-LR-BSA, 4, the anti-MC-LR monoclonal anti of biotinylation liquid solution, 5, be coated with the photosensitive particulate of Streptavidin.

Fig. 2: MC-LR-LICA reacts synoptic diagram.

Fig. 3: MC-LR-LICA canonical plotting.

Embodiment

Embodiment 1: preparation kit and detection water sample

Be coated with the luminous particle preparation of MC-LR-BSA: in centrifuge tube, add the 1mg luminous particle, add 12.5 μ L 1%Tween-20,0.05mg MC-LR-BSA artificial antigen, 10 μ L hydroboration cyanogen sodium, 2-(N-morpholine) ethyl sulfonic acid (MES) damping fluid with 0.1M, pH 6.0 adds to 200 μ L with volume, 37 ℃ of lucifuge oscillating reactionss 48 hours.Carboxymethoxylamine half hydrochloride (CMO) solution that adds 10 μ L 0.3M pH 5.0 seals not binding site, and it is centrifugal that 37 ℃ of lucifuges were hatched after 1 hour, separates the luminous particle that has been connected MC-LR-BSA, and the dilution back is standby.

The preparation of standard items MC-LR reagent: (0ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL 5ng/mL), dilutes from the pure product of MC-LR and obtains, and dilution is PBS (0.01mmol/L, pH 7.4).

The composition of kit:

(1), White-opalescent microwell plate (12 * 8 hole can be split as single hole).

1 (2), * be coated with the luminous particle of MC-LR-BSA: 2mL.

(3), 6 * MC-LR standard items, the 1.0mL/ bottle, concentration of standard solution is: 0,0.1,0.2,0.5,1,5ng/mL.

(4), the anti-MC-LR monoclonal anti of 1 * biotinylation liquid solution 2mL.

1 (5), * be coated with the photosensitive particulate of Streptavidin: 20mL.

Points for attention during mensuration

1, uses before all reagent to be gone up to room temperature (18-30 ℃).

2, immediately all reagent are put back to 2-8 ℃ after the use.

3, hatch in the process at all constant temperature, avoid irradiate light.

Detect step

Sample preparation:

The clarification water sample can be directly used in detection.If the water sample muddiness, get water sample 2mL behind the mixing in the 3mL test tube, the centrifugal 20min of 3000rpm shifts clear layer to another test tube, and is to be detected.

Get and be coated with each 20 μ L of MC-LR-BSA luminous particle, MC-LR standard items or the sample of handling well, three kinds of reagent of the anti-MC-LR antibody-solutions of biotinylation and be added in the White-opalescent microwell plate, hatched 30 minutes for 37 ℃; Add 175 μ L and be coated with the photosensitive particulate of Streptavidin, hatched 10 minutes for 37 ℃; Sensed light signal on the light-induced chemiluminescent detector calculates MC-LR content the sample from typical curve.The results are shown in Table 1, try to achieve by typical curve that the contained MC-LR concentration of water sample is respectively 0.342,1.21ng/mL.

Table 1

Claims

1. microcapsule algae toxin, be called for short MC-LR, light-induced chemiluminescent immunoassay kit, it is characterized in that by (1) White-opalescent microwell plate, (2) MC-LR standard items, (3) be coated with the luminous particle of MC-LR-BSA, the anti-MC-LR monoclonal anti of (4) biotinylation liquid solution and (5) are coated with the photosensitive particulate of Streptavidin and form.

2. the method with the described kit detection of claim 1 MC-LR is characterized in that adding successively luminous particle, MC-LR standard items or the sample of handling well, the anti-MC-LR monoclonal anti of the biotinylation liquid solution that are coated with MC-LR-BSA and carries out immune response in the White-opalescent microwell plate; Then add the photosensitive particulate that is coated with Streptavidin and react, reaction back sensed light signal is to add the detected value drawing standard curve of MC-LR standard items, to add the MC-LR content of detected value from the typical curve calculation sample of sample.

3. the method for detection MC-LR according to claim 2, it is characterized in that being operating as: get luminous particle 20 μ L, MC-LR standard items that are coated with MC-LR-BSA or the sample 20 μ L that handle well and the anti-MC-LR monoclonal anti of biotinylation liquid solution 20 μ L and be added to successively in the White-opalescent microwell plate, hatched 30 minutes for 37 ℃; Add the photosensitive particulate 175 μ L that are coated with Streptavidin, hatched 10 minutes for 37 ℃; Sensed light signal on the light-induced chemiluminescent detector calculates MC-LR content the sample from typical curve.