CN101603961A - A kind of light-induced chemiluminescent immunoassay kit of chloromycetin and detection method thereof - Google Patents
A kind of light-induced chemiluminescent immunoassay kit of chloromycetin and detection method thereof Download PDFInfo
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Abstract
The light-induced chemiluminescent immunoassay kit and the detection method thereof of a kind of chloromycetin (CAP) belong to the light-induced chemiluminescent immunoassay technical field.Add in proper order to the opaque white color microwell plate: the anti-CAP antibody of luminous particle, CAP standard items or testing sample, rabbit and the biotinylation goat anti-rabbit antibody that are coated with CAP-OVA, the lucifuge reaction, add the photosensitive particulate that is coated with Streptavidin again, hatch the back and detect.CAP-OVA on the luminous particle and free CAP competition are connected to CAP antibody, formed complex with biotinylation goat anti-rabbit antibody and 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, CAP concentration is inversely proportional in light signal strength and the sample, and the reference standard curve records CAP content in the sample.The present invention is used for the detection of food CAP content such as honey, milk, egg, and kit is simple in structure, and detection time is short, highly sensitive, easy and simple to handle.
Description
Technical field
A kind of light-induced chemiluminescent immunoassay kit of chloromycetin and detection method thereof belong to light-induced chemiluminescent immunoassay (LICLIA) technical field, are used for the detection of food CAP content.
Background technology
(Chloramphenicol CAP) is a kind of broad-spectrum antibiotic of Cheap highly effective to chloromycetin, and Gram-positive and negative bacteria are all had the good restraining effect, therefore once is widely used in the farming and animal husbandry.But animal derived food can cause multiple disease along with food chain is taken in for a long time by human body.The lighter destroys the equilibrium state of normal flora in the human body, and flora imbalance makes human body produce the drug-fast bacteria pearl, and giving from now on, ill use antibiotic therapy brings harmful effect; Allergic reaction can appear in the people of microbiotic allergic constitution, jeopardizes health.Can disturb the synthetic of bone marrow cell protein when serious, and it is synthetic to suppress juvenile cell DNA, causes granulocytopenia, causes malignant diseases such as alpastic anemia, haemolysis, purpura.
Toxic and side effect in view of CAP, international food educational circles classifies them as banning drugs, European Union, the U.S. etc. all in rules regulation CAP residual limit standard be " zero tolerance " (Zerotolerance), promptly must not detect, according to European Union " 2002/657/EC " standard code, to require detection limit be 0.3 μ g/kg to the minimum of CAP in the animal derived food.Soon U.S. FDA is also made corresponding regulation.China Ministry of Agriculture has stipulated that CAP must not detect in the edible tissues of all food animals, and it is deleted from " Chinese veterinary drug allusion quotation ", classifies banning drugs as.And corresponding SN0219-93 and the SCT3018-2004 industry standard formulated, in line with international standards.
Be to adapt to higher examination criteria, the detection technique level must improve accordingly, one of popular project that nearly ten years CAP detection techniques are international experts and scholars' research always.The detection method of CAP is divided into physical-chemical process and immuno-chemical method, the former has liquid phase chromatography (LC), high performance liquid chromatography (HPLC), mass spectroscopy (MS) etc., the latter mainly is that enzyme is exempted from method (ELISA), and the sensitivity of detection all reaches ppb (μ g/kg) rank.The detection method of current main-stream is LC---the employed method of China's industry standard, and ELISA.In recent years, Chinese scholars was developed physical detection new methods such as liquid chromatography tandem mass spectrometry (LC-MS/MS), liquid chromatography electrospray ionization mass spectrometry (LC-EITMS), microbiological analysis again, and the application of ELISA method has also been had further research.
Light-induced chemiluminescent immunoassay (LICLIA) is the chemiluminescence immunoassay technology of new generation based on the nanoscale high molecular particle, is called the AlphaLISA analytic approach again, and this technology will be widely used in the interaction of research biomolecule.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.LICLIA 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, increases the suspension of particulate simultaneously.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 covers hundreds and thousands of biomolecule, can catch target molecule.
The central principle of LICLIA 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).When 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 LICLIA 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 CAP of detection, be used for detection product CAP content such as honey.
The present invention mainly adopts light induced chemiluminescence immunoassay method (LICLIA) to detect CAP.Its technical scheme: the light-induced chemiluminescent immunoassay kit of a kind of chloromycetin CAP, by (1) White-opalescent microwell plate, (2) CAP standard items, (3) be coated with the luminous particle of CAP-OVA, (4) the antibody dried frozen aquatic products of the anti-CAP of rabbit, (5) biotinylation goat anti-rabbit antibody dried frozen aquatic products and (6) are coated with six article of photosensitive particulate of Streptavidin and form.
Be coated with the preparation of the luminous particle (3) of CAP-OVA: in centrifuge tube, add the 1mg luminous particle, add 12.5 μ L 1%Tween-20,0.05mg CAP-OVA artificial antigen, 10 μ L hydroboration cyanogen sodium, use 0.1M, the 2-of pH 6.0 (N-morpholine) ethyl sulfonic acid damping fluid adds to 200 μ L with volume, 37 ℃ of lucifuge oscillating reactionss 48 hours, add 10 μ L 0.3M, the carboxymethoxylamine half hydrochloride solution of pH 5.0 seals not binding site, 37 ℃ of lucifuges were hatched 1 hour, centrifugal purification, behind the unreacted reagent of flush away, separate the luminous particle that obtains being coated with CAP-OVA, the dilution back is standby.
CAP standard items (2) dilute from the pure product of CAP and obtain, and dilution is the 0.05mmol/L that contains mass concentration 10% methyl alcohol, the PBS of pH 7.4, the CAP concentration of CAP standard items is respectively: 0ng/mL, 0.02ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 25ng/mL.
With the method for described kit detection CAP, the basis of mensuration is the labelled immune reaction.In the White-opalescent microwell plate, add the luminous particle that is coated with CAP-OVA in turn, CAP standard items or the sample of handling well, anti-CAP antibody of rabbit and biotinylation goat anti-rabbit antibody carry out immune response; Then add the photosensitive particulate that is coated with Streptavidin and react the back sensed light signal,, calculate the CAP content of sample from typical curve with the detected value that adds sample to add the detected value drawing standard curve of CAP standard items.
The method of described detection CAP, it is operating as: get the luminous particle that is coated with CAP-OVA, CAP standard items or the sample of handling well, the anti-CAP antibody of certain density rabbit and biotinylation goat anti-rabbit antibody totally four kinds of each 20 μ L of reagent are added to the White-opalescent microwell plate, hatch 20 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 CAP content the sample from typical curve.
The method of described detection CAP, its sample preparation:
Honey sample is handled: the 2g sample fully mixes with 4mL distilled water, adds 4mL ethyl acetate, the concussion 10min that jumps a queue, the centrifugal 10min of 3000g; Get the 2mL supernatant to clean glass tube, it is to be measured that 50 ℃ of gentle nitrogen stream evaporates to dryness, residue fully are dissolved in the dilution of 1mL dilution standard product usefulness;
Egg sample is handled: behind the 3g sample homogeneous, add 6mL ethyl acetate, concussion 10min, the centrifugal 10min of 3000g gets the 2mL supernatant to clean glass tube, 50 ℃ of gentle nitrogen stream evaporates to dryness, with the residue of 1mL n-hexane dissolution drying, the dilution that adds 1mL dilution standard product usefulness fully mixes the centrifugal 10min of 3000g; Drawing lower floor's water is used for detecting;
Milk sample is handled: the 5mL milk sample, and the centrifugal 15min of 2000rpm gets 2.5mL lower floor skimmed milk and fully mixes with 5mL ethyl acetate, and concussion 10min leaves standstill 5～10min again; Shift the 4mL upper solution in clean glass tube, gentle nitrogen stream evaporate to dryness; Residue is dissolved in the dilution of 200 μ L dilution standard product usefulness, and is to be measured.
Beneficial effect of the present invention: this detection kit is simple in structure, and easy and simple to handle, detection time is short, highly sensitive.
Description of drawings
Fig. 1 detects the kit synoptic diagram of CAP.1, White-opalescent microwell plate, 2, the CAP standard items, 3, be coated with the luminous particle of CAP-OVA, 4, the antibody dried frozen aquatic products of the anti-CAP of rabbit, 5, biotinylation goat anti-rabbit antibody dried frozen aquatic products, 6, be coated with the photosensitive particulate of Streptavidin.
Fig. 2 CAP-LICLIA reacts synoptic diagram.
Fig. 3 CAP-LICLIA canonical plotting.
Embodiment
Embodiment 1 preparation kit
Be coated with the luminous particle preparation of CAP-OVA:
In centrifuge tube, add the 1mg luminous particle, add 12.5 μ L 1%Tween-20,0.05mgCAP-OVA 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 coated with CAP-OVA, and the dilution back is standby.
The preparation of CAP standard items reagent: (0ng/mL, 0.02ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL 25ng/mL), dilutes from the pure product of CAP and obtains, and dilution is the PBS (0.05mmol/L, pH 7.4) that contains 10% methyl alcohol.
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 CAP-OVA: 2mL.
(3), 6 * CAP standard items, the 1.0mL/ bottle, standard items CAP concentration is: 0,0.02,0.1,1,10,25ng/mL.
(4), the anti-CAP antibody of 1 * rabbit dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
(5), 1 * biotinylation goat anti-rabbit antibody dried frozen aquatic products, time spent 0.5mL dissolved in distilled water.
1 (6), * be coated with the photosensitive particulate of Streptavidin: 20mL.
Points for attention during mensuration
1. before using all reagent are gone up to room temperature (18-30 ℃).
2. immediately all reagent are put back to 2-8 ℃ after using.
3. hatch at all constant temperature and avoid irradiate light in the process.
Embodiment 2: detect honey, egg, milk sample
Sample preparation:
Honey sample is handled: the 2g sample fully mixes with 4mL distilled water, adds 4mL ethyl acetate, the concussion 10min that jumps a queue, the centrifugal 10min of 3000g.Get the 2mL supernatant to clean glass tube, 50 ℃ of gentle nitrogen stream evaporates to dryness, residue fully is dissolved in 1mL standard items dilution, and is standby.
Egg sample is handled: behind the 3g sample homogeneous, add 6mL ethyl acetate, concussion 10min, the centrifugal 10min of 3000g.Get the 2mL supernatant to clean glass tube, 50 ℃ of gentle nitrogen stream evaporates to dryness with the residue of 1mL n-hexane dissolution drying, add 1mL standard items dilution and fully mix.The centrifugal 10min of 3000g.Drawing lower floor's water is used for detecting.
Milk sample is handled: the 5mL sample, and the centrifugal 15min of 2000rpm gets 2.5mL lower floor skimmed milk and fully mixes with 5mL ethyl acetate, and concussion 10min leaves standstill 5～10min again.Shift the 4mL upper solution in clean glass tube, gentle nitrogen stream evaporate to dryness.Residue is dissolved in 200 μ L standard items dilutions, and is to be measured.
Get be coated with CAP-OVA luminous particle, CAP standard items or the sample of handling well, the anti-CAP antibody of certain density rabbit and biotinylation goat anti-rabbit antibody each 20 μ L of totally four kinds of reagent be added to the White-opalescent microwell plate, hatched 20 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 CAP content the sample from typical curve.The results are shown in Table 1, try to achieve by typical curve that this example honey, egg, the contained CAP concentration of milk sample are respectively 0.748,0.334,0.017ng/mL.
Table 1
Claims (6)
1, the light-induced chemiluminescent immunoassay kit of a kind of chloromycetin CAP, it is characterized in that by (1) White-opalescent microwell plate, (2) CAP standard items, (3) be coated with the luminous particle of CAP-OVA, (4) the antibody dried frozen aquatic products of the anti-CAP of rabbit, (5) biotinylation goat anti-rabbit antibody dried frozen aquatic products and (6) are coated with six article of photosensitive particulate of Streptavidin and form.
2, kit according to claim 1, it is characterized in that being coated with the preparation of the luminous particle (3) of CAP-OVA: in centrifuge tube, add the 1mg luminous particle, add 12.5 μ L 1%Tween-20,0.05mgCAP-OVA artificial antigen, 10 μ L hydroboration cyanogen sodium, use 0.1M, the 2-of pH 6.0 (N-morpholine) ethyl sulfonic acid damping fluid adds to 200 μ L with volume, 37 ℃ of lucifuge oscillating reactionss 48 hours, add 10 μ L 0.3M, the carboxymethoxylamine half hydrochloride solution of pH5.0 seals not binding site, 37 ℃ of lucifuges were hatched 1 hour, and centrifugal purification is behind the unreacted reagent of flush away, separate the luminous particle that obtains being coated with CAP-OVA, the dilution back is standby.
3, kit according to claim 1, it is characterized in that CAP standard items (2), from the pure product of CAP, dilute and obtain, dilution is the 0.05mmol/L that contains mass concentration 10% methyl alcohol, the PBS of pH 7.4, and the CAP concentration of CAP standard items is respectively: 0ng/mL, 0.02ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL, 25ng/mL.
4, a kind of method that detects CAP with the described kit of claim 1, it is characterized in that in the White-opalescent microwell plate, adding in turn the luminous particle that is coated with CAP-OVA, CAP standard items or the sample of handling well, anti-CAP antibody of rabbit and biotinylation goat anti-rabbit antibody carry out immune response; Then add the photosensitive particulate that is coated with Streptavidin and react the back sensed light signal, adding the detected value drawing standard curve of CAP standard items, calculate CAP content the sample from typical curve with the detected value that adds sample.
5, the method for detection CAP according to claim 4, it is characterized in that being operating as: get the luminous particle that is coated with CAP-OVA, CAP standard items or the sample of handling well, the anti-CAP antibody of certain density rabbit and biotinylation goat anti-rabbit antibody totally four kinds of each 20 μ L of reagent are added to the White-opalescent microwell plate, hatch 20 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 CAP content the sample from typical curve.
6, according to the method for claim 4 or 5 described detection CAP, it is characterized in that sample preparation:
Honey sample is handled: the 2g sample fully mixes with 4mL distilled water, adds 4mL ethyl acetate, the concussion 10min that jumps a queue, the centrifugal 10min of 3000g; Get the 2mL supernatant to clean glass tube, it is to be measured that 50 ℃ of gentle nitrogen stream evaporates to dryness, residue fully are dissolved in the dilution of 1mL dilution standard product usefulness;
Egg sample is handled: behind the 3g sample homogeneous, add 6mL ethyl acetate, concussion 10min, the centrifugal 10min of 3000g; Get the 2mL supernatant to clean glass tube, 50 ℃ of gentle nitrogen stream evaporates to dryness, with the residue of 1mL n-hexane dissolution drying, the dilution that adds 1mL dilution standard product usefulness fully mixes the centrifugal 10min of 3000g; Drawing lower floor's water is used for detecting;
Milk sample is handled: the 5mL milk sample, and the centrifugal 15min of 2000rpm gets 2.5mL lower floor skimmed milk and fully mixes with 5mL ethyl acetate, and concussion 10min leaves standstill 5～10min again and shifts the 4mL upper solution in clean glass tube, gentle nitrogen stream evaporate to dryness; Residue is dissolved in the dilution of 200 μ L dilution standard product usefulness, and is to be measured.
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