A kind of reagent composition for immunochromatography for canine coronavirus and detection method
Technical field
The present invention relates to one and carry highly sensitive, it is possible to suppression nonspecific reaction for detecting the reagent composition for immunochromatography of canine coronavirus or thing treatment fluid to be checked.The invention further relates to suppress nonspecific reaction, the easiest and measure the immunochromatography detection method of thing to be checked accurately.
Background technology
The pathogen of canine coronavirus disease is canine coronavirus (Canine Corona Virus, CCV).The gastroenteritis differed with weight as Clinical symptoms, the watery diarrhea in lethal clinically.Sudden onset, psychotic depression, appetite are useless absolutely, vomit, discharge that stench is dilute soft and the feces of band mucus.Vomiting often can last for days, until having alleviated before there is diarrhoea.After, feces is by essence shape, half pasty state to water sample, in orange or green, and the blood that interior mucous sum amount does not waits.Rapid dehydration, loses weight.The most sick dog of the source of infection and band poison dog, the sick dog toxin expelling time is 14 days, and the ability keeping contact to infect is the most longer.Sick dog is blurted out saliva, nose liquid and the outside toxin expelling of feces through respiratory tract, digestive tract, pollutes feedstuff, drinking-water, cage tool and surrounding, has passed to susceptible animal directly or indirectly.CCV can be survived 69 days in feces, also can keep the infectiousness of a few days, the most once fall ill in water, then be difficult to prevent spread and epidemic.Canine coronavirus is distributed widely in the whole world, is one of epidemic disease that China is supported dog industry, Fur Animal Feeding industry harm maximum.
In recent years, people are wide to the external diagnosis reagent case of detection antigen or the research ratio of portable diagnostic device.Dog class disease detection aspect, if Canine Parvovirus, canine distemper virus detection kit are a kind of common device for immunochromatography.In this case, antigen is present in the secretions such as tear of dog, saliva, feces, utilizes the monoclonal antibody that can identify virus to further investigate the device for immunochromatography of this Viral diagnosis.But, in the case of the canine coronavirus that detection sample is feces, owing to containing Multiple components in feces, and composition is the most variant with proportion, affects the accuracy of immunochemistry binding assay.In order to make the impact of sample to be checked minimize, it has been proposed that use various compensatory device that it is processed.
EDTA does not possess sufficient effect for stabilized hemoglobin contained in the samples such as feces at present, and the stabilization effect that the water soluble metal complexes of use transition metal ions the obtains effect than only use EDTA is high.In traditional device for immunochromatography, the background coloration of chromatographic film, hydrophobic binding and the material of electric interactions effect can be eliminated by described compositions, such as surfactant, macromolecule stabilizer, reach to weaken or eliminate the effect of non-specific binding.In order to suppress nonspecific reaction, in buffer agent, generally use various additive, such as bovine serum albumin, casein, Polyethylene Glycol, Tween20 etc..
Nonionic surfactant (such as Tween20, Triton X-100 etc.) is tested by the present invention.During it was found that utilize the thing to be checked in immunochromatographyassay assay sample, nonspecific reaction still exists.On this basis, the present invention proposes one and carries highly sensitive, it is possible to reagent composition for immunochromatography and the detection method for detecting canine coronavirus of suppression nonspecific reaction.
Summary of the invention
The technical problem to be solved in the present invention
In traditional immuno-chromatography detection device, EDTA does not possess sufficiently effect for stabilized hemoglobin contained in the samples such as feces at present, when utilizing the thing to be checked in immunochromatographyassay assay sample, nonspecific reaction still exists, and the stabilization effect using the water soluble metal complexes of transition metal ions to obtain is higher than an effect of use EDTA.
Compared with prior art, the invention provides one and can suppress nonspecific reaction and highly sensitive reagent composition for immunochromatography or thing treatment fluid to be checked.On this basis, the invention provides a kind of sample need not be carried out pretreatment, by sample is directly appended to detect device sample absorption site, can rapidly simplicity and carry out the device for immunochromatography detected accurately.Such as, it is provided that a kind of by by feces diluted, being directly added drop-wise to detect the sample absorption site of device, can simplicity and carry out the immuno-chromatography detection device of dog medical diagnosis on disease accurately rapidly.
It addition, the invention still further relates to, by by detectable substances such as feces, tear, salivas, after processing with thing treatment fluid mixing to be checked, directly be added drop-wise to detect the sample absorption site of device, can simplicity and carry out the detection technique detected accurately rapidly.
The means of solution problem
In order to solve problems of the prior art, it is an object of the invention to provide a kind of detectable device compositions being applicable to dog viroid infection early stage and corresponding immunochromatographydetecting detecting test strip, canine coronavirus method is simple, the detection time is short, low cost, specificity are higher to utilize the reagent strip of the present composition to detect.
The immunochromatography compositions of the present invention is very effective to secretions detections such as excrement and urine of dogs, tear, salivas, the thing to be checked after dilution is added drop-wise to sample absorption site, can suppress the nonspecific reaction of impurity in sample, thus reach sensitivity, detect thing to be checked.The detection object of the present invention is canine coronavirus, and organism sample is feces.
In the present invention, the Buffer types in reagent composition for immunochromatography has: acetate buffer, phosphate buffer, borate buffer, Tris hydrochloride buffer etc., preferably Tris hydrochloride buffer.Concentration is the scope of 0.01-250mM, if concentration is less than 0.01mM, cushioning effect is insufficient.When concentration is more than 250mM, higher than necessary concentration, cause waste.
The chelating agen of one of the composition of reagent composition for immunochromatography of the present invention, containing amino and carboxylic acid functional and with heavy metal ion and alkaline-earth metal ions, the compound of complexation can be formed, preferably aminophosphonic acid quasi-chelate compound, the concentration of chelating agen is 0.0001-0.005mM
Scope, concentration less than 0.0001mM time, it is impossible to suppression nonspecific reaction.
The example of the nonionic surfactant of one of the reagent composition for immunochromatography composition of the present invention has: polyoxypropylene alkyl ether, and polyoxyethylene sorbitan fatty acid ester (trade name " Tween " series), polyoxyethylene are to t-octyl phenyl ether (trade name " Triton " series).The content of nonionic surfactant is the scope of the 0.01-10% of immunochromatography reagent compositions, the scope of preferably 0.5-1%.When content is more than 1%, the impact preferably suppressing nonspecific reaction will not be brought.
In order to realize the object of the invention, the present invention provides a kind of colloidal gold immunochromatographydetection detection test paper bar for detecting canine coronavirus according to above-mentioned reagent composition for immunochromatography, and described colloidal gold immunochromatographydetection detection test paper bar comprises:
1) monoclonal antibody and the nitrocellulose membrane of two two bands of anti-igg of canine coronavirus antigen it are coated;
2) glass fibre membrane of the monoclonal antibody containing colloid gold label canine coronavirus antigen.
Wherein, two described anti-igg are sheep anti-mouse igg antibody.
Further, in described nitrocellulose membrane, the concentration of the monoclonal antibody of canine coronavirus antigen is 1mg/mL, and the concentration of described sheep anti-mouse igg antibody is 1mg/mL.
As preferably, in described glass fibre membrane, the labelling pH value of the monoclonal antibody of the canine coronavirus antigen of colloid gold label is 8.5, and it is 40 μ g/mL that monoclonal antibody is combined concentration with gold colloidal.
As preferably, described colloid gold particle average diameter is 25nm.
Foregoing immune chromatography detecting test paper strip, also includes base plate, sample pad and absorbent paper, described base plate is pasted nitrocellulose membrane, pastes absorbent paper above nitrocellulose membrane, the most mutually overlaps sticking glass fibrous membrane and sample pad below nitrocellulose membrane.
Present invention also offers a kind of method preparing aforementioned colloidal gold immunochromatographydetection detection test paper bar, comprise the steps:
1) monoclonal antibody of canine coronavirus antigen is prepared;
2) preparation of nitrocellulose membrane: the monoclonal antibody of dilution canine coronavirus antigen and sheep anti-mouse igg antibody;After nitrocellulose membrane is affixed on base plate, drawing monoclonal antibody and the sheep anti-mouse igg antibody of above-mentioned canine coronavirus antigen, form detection line and nature controlling line respectively on nitrocellulose membrane, room temperature is coated overnight, standby;
3) preparation of glass fibre membrane: it is 8.5 that gold colloidal adjusts antibody pH value, it is 40 μ g/mL that monoclonal antibody is combined concentration with gold colloidal, after stabilizer treatment, by the colloidal gold solution even spread of good for labelling monoclonal antibody to the glass fibre membrane handled well, lyophilization, standby;
4) last in step 2) top of nitrocellulose membrane for preparing pastes absorbent paper, the most mutually overlaps sticking glass fibrous membrane and sample pad in the lower section of nitrocellulose membrane.
Effect of the invention is that
The reagent composition for immunochromatography of the present invention contains buffer, chelating agen and nonionic surfactant, after processing the detection object in detection sample, it is possible to suppression nonspecific reaction, makes sensitivity strengthen, it is judged that result is accurate.
Another important function of the detection technique of the present invention is: if without the reagent composition for immunochromatography special handling sample pad of the present invention and label pad, then with thing treatment fluid to be checked, thing to be checked can be carried out mixing in advance and processes by direct immunochromatography, and in the way of developping solution, it is added drop-wise to the sample absorption site of device for immunochromatography, directly detecting antigen, the easiest diagnoses dog class disease.
The immunochromatographydetecting detecting test strip device of the present invention, using double-antibody sandwich method and immunochromatography technique detection canine coronavirus antigen, compared with other detection techniques, its detection sample need not special handling, reagent and sample consumption are minimum, and sample size can as little as 1 μ L~2 μ L;Can be not only used for Detection of antigen it can also be used to antibody test.And the detection time can be greatly shortened, be not required to use the expensive instrument such as fluorescence microscope, enzyme mark detector, it is possible to qualitatively detection canine coronavirus antigen, testing result understand be prone to judge, result of the test can preserve for a long time.Colloidal gold immunochromatographydetection detection test paper bar the most of the present invention have high specificity, highly sensitive, simple to operate, detection quick and precisely, need not the advantage such as equipment of complexity, the requirement of Clinical Laboratory can be met, be also very suitable for the clinical sample detections such as the place that field condition, outpatient service, household person and experiment condition do not possess and use.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the test film of device for immunochromatography;Wherein, 1 is sample pad, and 2 is label pad, and 3 is chromatographic film, and 4 is detection line, and 5 is nature controlling line, and 6 is adsorptive pads, and 7 is backboard.
Fig. 2 is that reagent paper detector of the present invention detects canine coronavirus result;Wherein, from left to right be respectively do not detect, negative findings, positive findings.
Detailed description of the invention
Only the present invention is described in detail for following example, but is not limited to the scope of the present invention.
Embodiment 1
Reagent composition for immunochromatography:
Buffer agent in 1.1 reagent composition for immunochromatography does not specially require, and has as Buffer types: acetate buffer, phosphate buffer, borate buffer, Tris hydrochloride buffer etc., preferably Tris hydrochloride buffer.Concentration is the scope of the scope of the scope of 0.01-250mM, preferably 10-200mM, more preferably 30-180mM.If concentration is less than 0.01mM, cushioning effect is insufficient.When concentration is more than 250mM, higher than necessary concentration, cause waste.
1.2, as the chelating agen of one of the composition of reagent composition for immunochromatography of the present invention, are not particularly limited, as long as it can be used as the ligand with multiple dentate.As chelating agen in the present invention, containing amino and carboxylic acid functional and with heavy metal ion and alkaline-earth metal ions, the compound of complexation can be formed, preferably aminophosphonic acid quasi-chelate compound, the concentration of chelating agen is 0.0001-0.005mM
Scope, the scope of preferably 0.0005-0.002mM, concentration less than 0.0001mM time, it is impossible to suppression nonspecific reaction.
1.3 have as the example of the nonionic surfactant of one of the reagent composition for immunochromatography composition of the present invention: polyoxypropylene alkyl ether, polyoxyethylene sorbitan fatty acid ester (trade name " Tween " series), polyoxyethylene are to t-octyl phenyl ether (trade name " Triton " series).The content of nonionic surfactant is the scope of the 0.01-10% of immunochromatography reagent compositions, the scope of preferably 0.5-1%.When content is more than 1%, the impact preferably suppressing nonspecific reaction will not be brought.
Embodiment 2
Immunoassay test strip device:
2.1 label pad, sample pad generally selects glass fibre membrane.Containing colloid gold particle and monoclonal antibody or the conjugate of its fragment of polyclonal antibody in label pad.
2.2 chromatography medias mainly use nitrocellulose membrane.Chromatography media is drawn on it monoclonal antibody or the multi-resistance identifying antigen, and the two of identification tag resist.
2.3
Adsorptive pads selects filter paper.
Coating binder or Continuous pressing device for stereo-pattern on one face of 2.4 backboards so that it is have adhesivity, on this gluing surface, laminating arranges sample pad part, label part, chromatography media part, water absorbent portion.
2.5 resolution principle brief descriptions
After immersing sample with sample pad (through immunoreagent compositions-treated) end, sample solution passes through capillarity swimming from the bottom up along test strips, dissolve the label being dried in label pad, if with the presence of thing to be checked in testing sample, then label can direct swimming to detection line (T) and nitrocellulose membrane on line material generation immunoreation, thus colloid gold particle is assembled, form red lines, then other golden labeling antibodies not being combined with line material continue through capillarity swimming forward, sheep anti mouse two anti-generation association reaction with control line (C), also red lines are formed, two red line are i.e. formed on coated film, represent that sample is positive;If without thing to be checked in testing sample, be then combined with T line line material without antibody, thus T line redness lines disappear, represent that sample is negative.Whether C line effectively sets for inspection gold-marking immunity chromatography method itself, so no matter whether there is thing to be checked in sample, control line C should develop the color.If control line C does not develops the color, then explanation test strips lost efficacy.It is possible with being pre-mixed sample with thing treatment fluid to be checked with immunochromatography, in the way of developping solution, is then added drop-wise in sample pad (without any process), the inspection with as above-mentioned carrying out, result similar to the above can be reached.
Embodiment 3
Canine coronavirus colloidal gold immunochromatographydetection detection test paper bar preparation method is as follows:
The preparation of 3.1 labels
The monoclonal antibody [phosphate buffer (pH7.4 is diluted to 1mg/ml)] of 0.1ml canine coronavirus antigen is added in the colloidal gold solution liquid of 1ml, left at room temperature 1 minute, add the bovine serum albumin (BSA) of 100 μ l 10%, after being sufficiently stirred for, it is centrifuged 15 minutes with 8000rpm.After removing supernatant, add above-mentioned phosphate buffer, make labelled reagent solution.
The preparation of the detection unit in 3.2 chromatographic film
With phosphate buffer (pH7.4), the monoclonal antibody of canine coronavirus antigen being diluted to 1.0mg/ml, two anti-are diluted to 1.0mg/ml, draw film instrument through metal spraying, both are drawn in chromatographic film simultaneously, the most standby.
The assembling of 3.3 test strips
Test strips consists of a liner plate, is stained with sample pad, labeling pad, chromatographic film and adsorptive pads the most in order.The plate posted is cut into strip wide for 3mm and is canine coronavirus colloidal gold immuno-chromatography test paper strip, seal in then test strips is loaded together with desiccant aluminium foil bag and preserve.
3.4
Measure
During detection, the fecal specimens of animal to be checked is picked with aseptic cotton carrier, at 1mL PBS (pH7.2,0.01mol/L) in mixing, at the bottom of bulky grain is sunken to pipe after, take supernatant and drop in this test strips, about 5-10min reads ELISA test strip result, during reading, test strips horizontal positioned, front is observed.
3.5 results judge
If only having one the red line colour developing of C line on coated film, representing that testing result is negative, illustrating not exist canine coronavirus in testing sample;If C line and two red lines of T line all develop the color on coated film, represent that testing result is positive, illustrate to exist canine coronavirus in testing sample;If do not developed the color at C line, then show that test strips lost efficacy.
3.6 present invention application effect citings:
(1) specific test: with normal saline to canine coronavirus, hepatitis infectiosa canis virus II type, Canine Parvovirus, canine adenovirus Ⅰ, canine distemper virus, canine parainfluenza virus culture fluid doubling dilution to 1:130, take 120 μ L respectively to drip in sample pad, additionally set normal saline and do negative control.In addition to canine coronavirus is positive, other sample is all negative reaction, is repeated 3 times rear result identical, illustrates that the method has higher specificity.
(2) sensitivity tests: canine coronavirus making doubling dilution to 1:130 with normal saline, take 120 μ L respectively and drip in sample pad, reagent paper is still positive, and is repeated 3 times rear result identical, and reagent paper sensitivity of the present invention is strong.
(3) stability test: the 3 of the present invention batches of test strips are individually positioned in 37 DEG C of constant incubators, room temperature, 4 DEG C, 10 parts of canine coronavirus samples, 10 parts of negative canine coronavirus negative sample of detection simultaneously are taken out every 1 month, result shows: 6 months test strips to the present invention of 37 DEG C of effects are without destruction, it was demonstrated that this test strips is the most relatively stable;The test strips room temperature of the present invention place 12 months the most relatively stable.
Embodiment 4
With Korea S's import canine coronavirus test strip (gloomy Pood company of Korea S), Shanghai biotech firm canine coronavirus test strip (Shanghai Quicking Biotech Co., Ltd.), clinical 80 parts of samples are detected respectively with the canine coronavirus colloidal gold immunochromatographydetection detection test paper bar of embodiment 3 preparation respectively, contrast clinical symptoms simultaneously.Analyze testing result.
Result shows (table 1), the same two kinds of test strips of the test strip of the present invention are 100% to the positive coincidence rate of detection sample, the present invention is 98% with Bionote Inc.'s ELISA test strip negative match-rate, is 97.5% with Shanghai spirit detection negative match-rate soon, and total coincidence rate is 98.5%.Therefore, from experimental data it can be shown that the colloidal gold immunochromatographydetection detection test paper bar of the reagent composition for immunochromatography of present invention employing and preparation is better than most of commodity test strips.
Table 1 is with two kinds of ELISA test strip sample comparing results
Embodiment 5
The colloidal gold immunochromatographydetection detection test paper bar prepared by commercial ELISA kit (Shanghai haze derivatives) and embodiment 3 detects 80 parts of CCV fecal specimens simultaneously and calculates both total coincidence rates, analyzes testing result.As shown in table 2, the immunochromatographydetecting detecting test strip of the present invention is 97.5% with total coincidence rate of ELISA.
Table 2 test strips compares with ELISA kit testing result
Above-described embodiment is preferably a kind of embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any substitute mode that all should be equivalence without departing from the core essence of the present invention and the change of principle, belongs to the scope of protection of present invention.