CN109613239A - A rotavirus Test paper - Google Patents
A rotavirus Test paper Download PDFInfo
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- CN109613239A CN109613239A CN201811611785.0A CN201811611785A CN109613239A CN 109613239 A CN109613239 A CN 109613239A CN 201811611785 A CN201811611785 A CN 201811611785A CN 109613239 A CN109613239 A CN 109613239A
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/14—Reoviridae, e.g. rotavirus, bluetongue virus, Colorado tick fever virus
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Abstract
The present invention relates to a kind of colloidal gold immune chromatography tests of A rotavirus detection, and the colloidal gold immune chromatography test includes chromatographic film, and the chromatographic film is equipped with a detection line and a nature controlling line；Wherein, it is coated with the first anti-A rotavirus monoclonal antibody on the colloidal gold, the second anti-A rotavirus monoclonal antibody is coated in the detection line.The invention further relates to corresponding reagents.Anti-interference ability and sensitivity are improved when detecting A rotavirus.
Description
Technical field
The present invention relates to field of immunodetection, and in particular to the colloidal gold immune chromatography test of detection A rotavirus.
Background technique
Rotavirus (Rotavirus, RV) belongs to Reoviridae (Reoviridae) rotavirus
(Rotavirus) diplornavirus is the main pathogens for causing infant and the non-bacterial type diarrhea of various young animals.
Its main infection intestinal epithelial cell causes diarrhea to cause cellular damage.Rotavirus is popular in summer and autumn winter every year,
Route of infection is fecal oral route, and clinical manifestation is acute gastroenteritis, and in osmotic diarrhea disease, the course of disease is generally 7 days, and fever is held
It is 3 days continuous, it vomits 2~3 days, diarrhea 5 days, dewatering symptom seriously occurs.
There are three types of antigens, i.e. group antigen (VP6), neutralization antigen (VP7) and haemagglutinin antigen (VP4) for RV particle surface.Group
Antigen is related with various structures albumen, the inner capsid VP6 of mainly the 6th genetic fragment coding；Neutralizing antigen is by the 9th gene piece
The VP7 outer capsid glycoprotein of section coding；Haemagglutinin antigen is the outer capsid proteins VP4 encoded by genetic fragment 4, can be by albumen water
Solve enzyme hydrolysis.According to the antigenic specificity of VP6, it has been reported that the 7 groups of rotavirus serotypes (A~G).Wherein A groups are
Cause the main pathogens of infantile diarrhea, it is most commonly seen, and the case that human rotavirus's infection is more than 90% is also all this
Caused by group.
From 1973, when Bishop etc. studies infant's gastroenteritis, found from duodenal mucosa cell biopsy sample
After rotavirus, it has proved to be the main reason for causing infantile diarrhea disease, and the data in China showed in autumn and winter
The infantile diarrhea of 50%-60% is as caused by rotavirus.If being not treated in time after infant infection RV, will cause serious
Dehydration and acid poisoning, can threat to life when serious.Therefore there is pole to clinic to the diagnosis of RV infection in time, accurately and rapidly
Its important meaning.
Rotavirus detection at present mainly includes the following categories detection method: 1) using the inspection of rotavirus antibody sensitization blood cell
Survey method mainly detects rotavirus from the secretion such as saliva, excrement.The result of this method is not easy to repeat, and needs to have experience
Technical staff；2) it is solidifying to carry out polyacrylamide by extracting viral RNA in excrement for technique of polyacrylamide gel electrophoresis
Gel electrophoresis, electrophoresis form unique 11 zone, to determine whether infection rotavirus, but RNA extracts more difficult success and operation
Cumbersome time-consuming；3) electron microscopy directly detects the rotaviral particles of specific form in stool with Electronic Speculum, and sensibility is higher,
It is the classical way for identifying rotavirus.But correct diagnosis, and Electronic Speculum equipment are influenced since virion is easy to degrade usually
It is expensive, it is difficult to it is universal, therefore the application of electron microscopy is restricted significantly, is generally only used for studying；4) technique of gene detection,
High specificity, high sensitivity quantify accurately, have very high application value, but its complicated for operation, need in terms of etiological diagnosis
Want the technical staff of special training, special instrument and equipment；5) MBP enzyme linked immuno-adsorbent assay (ELISA) is most widely used at present
Immunologic detection method, sensitivity and specificity are high, but there is still a need for the machines of profession and personnel to operate；6) immune colloid gold
Technology, detection is quick and does not need any instrument and equipment, is suitble to on-site test.
Immune colloidal gold technique (Immune colloidal gold technique) is using colloidal gold as tracer label
Object is applied to a kind of novel immunolabelling technique of antigen-antibody, english abbreviation are as follows: GICT.Colloidal gold is by gold chloride
(HAuCl4) under the reducing agent such as white phosphorus, ascorbic acid, sodium citrate, tannic acid the effects of, polymerization becomes the gold of particular size
Grain, and since electrostatic interaction becomes a kind of stable colloidal state, referred to as colloidal gold.Colloid gold particle surface is negatively charged, with
It attracts each other between the positive group of protein by electrostatic force, reaches and form firm combination within the scope of Van der Waals force, together
When colloid gold particle rough surface be also the essential condition to form absorption.
However, colloid gold particle is connected to after antigen-antibody, it is very unstable, it is easy to because the variation of charge is so that antigen
Antibody is detached from, and causes the antigen-antibody amount of each colloid gold particle connection to greatly reduce, to cause the reduction of sensitivity.
Therefore, environment pH, ionic strength, the dosage of colloid gold particle, the molecular weight of antigen-antibody and antigen-antibody is dense
Degree etc. will affect the absorption of antigen-antibody, and then influence the sensitivity of testing result, so, although immune colloidal gold technique is
A kind of method of comparative maturity, but it is specific to every kind of specific detection substance, detection effect is still different.
On the other hand, it is based on Colloidal Gold, when detecting the wheel virus antigen in fecal sample, is also faced with excrement sample
The problem of interference of each substance such as swill, medicament residue, hemoglobin, leucocyte present in this.
Accordingly, in the colloidal gold immunochromatographimethod detection of A rotavirus, exist and improve sensitivity and the anti-interference energy of raising
The tight demand of power.
Summary of the invention
In view of this, the test paper is in A groups of wheels of detection the purpose of the present invention is to provide a kind of colloidal gold immune chromatography test
Have while very high anti-interference ability when shape virus and realizes higher sensitivity.
In this regard, the present invention provides following technical schemes.
In a first aspect, the present invention provides a kind of colloidal gold immune chromatography test, the colloidal gold immune chromatography test packet
Chromatographic film is included, the chromatographic film is equipped with a detection line and a nature controlling line；
Wherein, it is coated with the first anti-A rotavirus monoclonal antibody on the colloidal gold, forms colloid gold label albumen
Compound is coated with the second anti-A rotavirus monoclonal antibody in the detection line.
In one embodiment, the package amount of the described second anti-A rotavirus monoclonal antibody is 0.5~1mg/
mL。
In one embodiment, the amount of the colloid gold label albumen composition is 30~40OD.
In one embodiment, the package amount of the described first anti-A rotavirus monoclonal antibody is 15~30 μ g/
mL。
In one embodiment, anti-animal IgG antibody is coated on the nature controlling line.
In a preferred embodiment, the package amount of the anti-animal IgG antibody is 0.5~1mg/mL.
Second aspect, the present invention provides a kind of reagent, including sample pad pretreatment fluid, the sample pad pretreatment fluid packet
Include buffer, salt ion, closed protein, surfactant.
In a preferred embodiment, the buffer is Tris buffer, in phosphate buffer, borate buffer solution
One kind.
In a preferred embodiment, the salt ion is one of sodium chloride, potassium chloride, magnesium chloride.
In a preferred embodiment, the closed protein is one of bovine serum albumin(BSA), casein, ovalbumin
Or it is a variety of.
In a preferred embodiment, the surfactant is nonionic surfactant.
The third aspect, the present invention provides a kind of kits, including colloidal gold immune chromatography test and Ben Fa of the invention
Bright reagent.
Fourth aspect, provides colloidal gold immune chromatography test of the invention and/or reagent of the invention is used to prepare inspection
Survey the purposes of the kit of A rotavirus.
5th aspect, the present invention provides a kind of measuring methods of A rotavirus, and the method includes using the present invention
Colloidal gold immune chromatography test and/or reagent of the invention the step of.
The invention has the benefit that
By adjusting the package amount of colloidal gold, the package amount of the package amount of coated antibody and labelled antibody, meanwhile, cooperation is originally
The sample pad pretreatment fluid of invention improves the anti-interference ability and detection sensitivity of A rotavirus.
Specific embodiment
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Throughout the specification, unless otherwise specified, terms used herein are interpreted as usual in this field
Used meaning.Therefore, unless otherwise defined, all technical and scientific terms used herein has leads with belonging to the present invention
The identical meaning of the general understanding of field technique personnel.Contradiction if it exists, this specification are preferential.
In the present invention, statement " first " and " second " is only used for retouching when modifying anti-A rotavirus monoclonal antibody
Purpose is stated, for substance defined by differentiation；Without limiting order or primary and secondary in any way, also not specific kind of qualifier matter
Class.
In the present invention, colloidal gold immune chromatography test includes the sample that sequentially fixed and end interconnects on bottom plate
Pad, colloidal gold pad, chromatographic film, water absorption pad, it is suitable on membrane body along the direction from sample pad to water absorption pad in the chromatographic film
It is secondary that the detection line T and nature controlling line C being spaced from is distributed with.
Wherein,
The first anti-A rotavirus monoclonal antibody is coated in the colloidal gold pad,
The second anti-A rotavirus monoclonal antibody is coated in the detection line,
Anti- animal IgG antibody is coated on the nature controlling line,
Preferably,
The package amount of A rotavirus monoclonal antibody anti-for described first can be 15~30 μ g/mL.For example, institute
The package amount for stating the first anti-A rotavirus monoclonal antibody can be 15 μ g/mL, 16 μ g/mL, 17 μ g/mL, 18 μ g/mL, 19
μg/mL、20μg/mL、21μg/mL、22μg/mL、23μg/mL、24μg/mL、25μg/mL、26μg/mL、27μg/mL、28μg/
mL,29μg/mL,30μg/mL.The package amount of first anti-A rotavirus monoclonal antibody is controlled into the model in 15~30 μ g/mL
It can effectively guarantee the sensitivity and specificity of Test paper when enclosing interior.
The package amount of A rotavirus monoclonal antibody anti-for described second can be 0.5~1mg/mL.For example, the
The package amount of secondary antibody A rotavirus monoclonal antibody can for 0.5mg/mL, 0.55mg/mL, 0.6mg/mL, 0.65mg/mL,
0.7mg/mL,0.75mg/mL,0.8mg/mL,0.85mg/mL,0.9mg/mL,0.95mg/mL,1mg/mL.By the second anti-A groups of wheels
The package amount of shape viral monoclonal antibodies can effectively guarantee the spirit of Test paper when controlling in the range of 0.5~1mg/mL
Sensitivity and specificity.
It can be 30~40OD for the amount of the colloid gold label albumen composition.For example, colloid gold label albumen is multiple
The amount for closing object can be 30OD, 31OD, 32OD, 33OD, 34OD, 35OD, 36OD, 37OD, 38OD, 39OD, 40OD.Colloidal gold mark
Remember the sensitivity and specificity that can effectively guarantee Test paper when the amount of albumen composition is in the range of 30~40OD.
In the present invention, with after the anti-A rotavirus monoclonal antibody of colloid gold label first, centrifugal concentrating is needed to receive
Collection, it is preferred that centrifugal force is 7741~12096 × g.
It can be 0.5~1mg/mL for the package amount of the anti-animal IgG antibody.For example, anti-animal IgG antibody
Package amount can be 0.5mg/mL, 0.55mg/mL, 0.6mg/mL, 0.65mg/mL, 0.7mg/mL, 0.75mg/mL, 0.8mg/
mL,0.85mg/mL,0.9mg/mL,0.95mg/mL,1mg/mL.The package amount of anti-animal IgG antibody is controlled in 0.5~1mg/
It can guarantee the colored intensity of nature controlling line when in the range of mL, effectively convenient for the interpretation of result.
In the present invention, the animal IgG can be selected from rabbit igg, chicken IgG or mouse IgG, preferably mouse IgG.
In the present invention, the anti-animal IgG antibody can be monoclonal antibody or polyclonal antibody, it is preferably polyclonal
Antibody.
In the present invention, chromatographic film may be, for example: cellulose mixture film, nitrocellulose filter, Predator film, nitrification are fine
Tie up plain film or glass fibre element film, preferably nitrocellulose filter.
In the present invention, sample pad pretreatment fluid includes buffer, salt ion, closed protein, surfactant.
For the type of buffer, the present invention is one in Tris buffer, phosphate buffer and borate buffer solution
Kind.
For the dosage of buffer, those skilled in the art can select according to the type of buffer, for example, making
The content of 40~100mM can preferably be used using the concentration being greater than equal to 40mM with when Tris buffer.
For the type of salt ion, the present invention is selected from one of sodium chloride, potassium chloride and magnesium chloride or a variety of, preferably
Sodium chloride.
For the dosage of salt ion, those skilled in the art can select according to the type of salt ion, for example, making
With such as 0.4~2% (mass volume ratio) can be used when sodium chloride.
For the type of closed protein, the present invention be selected from one of bovine serum albumin(BSA), casein and ovalbumin or
A variety of, preferably in bovine serum albumin(BSA), casein and ovalbumin two kinds.
For the dosage of closed protein, such as 5%~20% (mass volume ratio) can be used, wherein bovine serum albumin
The content of white, casein or ovalbumin is respectively the 50% of closed protein content.
For the type of surfactant, the present invention is selected from nonionic surfactant, preferably triton x-100 (TX-
100)。
For the dosage of surfactant, those skilled in the art can select according to the type of surfactant,
Ratio can use such as 0.5~1% (percent by volume) when using triton x-100.
During lot of experiments, the present inventor is found surprisingly that, sample pad pretreatment fluid of the invention cooperates this
The package amount of the package amount of invention colloidal gold, the package amount of coated antibody and labelled antibody can improve the inspection of A rotavirus
Guarantee higher sensitivity while the anti-interference ability of survey.In this regard, it is presumed that this is because sample pad of the invention is located in advance
Reasonably combined use of the liquid to different closed proteins and surfactant, salt ion is managed, while eliminating nonspecific reaction
Also the specific reaction between detection antigen and antibody is enhanced.
In the present invention, kit may include as needed container, operation instructions, immune auxiliaries and/or for into
Other structures and/or reagent needed for the judgement of row result.
Include but is not limited to for other structures needed for carrying out result judgement in kit of the invention, for sampling knot
Structure, for carrying out contrast structure and/or for Observe and measure process or the structure of result.
Include but is not limited to for other reagents needed for carrying out result judgement in kit of the invention, it is detergent, dilute
Release liquid, color developing agent and/or terminate liquid, and other pretreatment fluids in addition to sample pad pretreatment of the invention.For example, of the invention
Before testing, diluted sample well known in the art can be used；Preferably, sodium chloride-containing dilution；It is highly preferred that
The dilution ratio of sample is 1:30~1:40.
Before testing, using diluted sample, in the detection process, colloidal gold pad is coated with the first anti-A groups of wheels
Shape viral monoclonal antibodies, detection line (T line) is coated with the second anti-A rotavirus Dan Ke in the chromatographic film of immune chromatography test paper
Grand antibody, nature controlling line (C line) are coated with anti-animal IgG antibody.First first with colloid gold label of A rotavirus in sample
Anti- A rotavirus monoclonal antibody reactive, is moved in chromatographic film with Sidestream chromatography effect, until detection line (T line) and second
Anti- A rotavirus monoclonal antibody reactive forms interlayer structure, and in neat red stripes, according to red stripes, whether there is or not sentence
Determine result.The anti-A rotavirus monoclonal antibody of the first of colloid gold label continues to move along, until nature controlling line (C line) and anti-
The reaction of animal IgG antibody indicates that detection reaction system is effective in neat red stripes.
The present invention is described in more detail by the following examples, but the present invention is not limited to these Examples.
The label of the anti-A rotavirus monoclonal antibody of embodiment 1 first
It by the label buffer of appropriate amount, is slowly added to colloidal gold solution and mixes, add the appropriate first anti-A groups of colyliforms
Viral monoclonal antibodies are eventually adding terminate liquid, form first anti-A rotavirus monoclonal antibody-colloidal gold composite, then
By obtaining colloid gold label albumen composition after purification after appropriateness centrifugation, sprayed in the colloidal gold pad.
The coating of the anti-A rotavirus monoclonal antibody of embodiment 2 second
Film coating buffer is put on station, first corresponding clean container is added in the accurate film coating buffer that measures, then measures second
Anti- A rotavirus monoclonal antibody is added in corresponding membrane coating buffer, is mixed, and keeps the anti-A groups of colyliforms of detection line (T line) second sick
The final concentration of 1.2mg/mL of malicious monoclonal antibody.
The coating of the anti-animal IgG antibody of embodiment 3
Film coating buffer is put on station, it is first accurate to measure the corresponding clean container of required film coating buffer addition, then measure
Required sheep anti-mouse igg polyclonal antibody is added in corresponding membrane coating buffer, is mixed, is kept nature controlling line (C line) sheep anti-mouse igg polyclonal
The final concentration of 1mg/mL of antibody.
The optimization of the anti-A rotavirus labeling of monoclonal antibody condition of embodiment 4 first
6 clean vials are taken, number is 1 to 6, and every bottle is separately added into 1000 μ l colloidal gold solutions；Then 1~6
The 0.1M borate buffer solution (BB) that 100 μ l pH value are 9.0 is separately added into number bottle；Be then respectively adding 5,10,15,20,
25,30 the first anti-A rotavirus monoclonal antibody of μ g；It is finally separately added into 100 μ, 10% sodium chloride solution, observation develops the color, simultaneously
Colloidal gold albumen composition purified water is diluted 4 times, is swept in 450nm~700nm wave-length coverage with spectrophotometer
It retouches, records λ max.
Using the anti-A rotavirus monoclonal antibody of colloid gold label first, optimum mark condition is filtered out, to guarantee to try
The overall performance of agent box is good, and the results are shown in Table 1.
Table 1
As shown in Table 1, about 15 μ g of the saturation capacity of labelled protein, to guarantee kit performance, the first anti-A rotavirus list
The best package amount of clonal antibody is 15~30 μ g/mL.
The optimization of the label centrifugal force of embodiment 5
It by the label buffer of appropriate amount, is slowly added to colloidal gold solution and mixes, add the appropriate first anti-A groups of colyliforms
Viral monoclonal antibodies are eventually adding terminate liquid, form first anti-A rotavirus monoclonal antibody-colloidal gold composite, then
It is centrifuged by different centrifugal force as shown in Table 2, obtains antibody-colloidal gold compound after purification.Phenomenon is observed, as a result
As shown in table 2.
Table 2
Precipitating is loose, and supernatant is colourless or pale red corresponds to centrifugal rotational speed and meets the requirements, as shown in Table 2, centrifugal force 7741
It meets the requirements when~12096 × g.
The optimization of the anti-A rotavirus monoclonal antibody package amount of embodiment 6 second
Using film coating buffer, the second anti-A rotavirus monoclonal antibody is diluted to 0.3mg/mL, 0.5mg/ respectively
Then mL, 0.7mg/mL, 0.9mg/mL, 1.0mg/mL, 1.2mg/mL spray to nitrocellulose filter and form detection line, preparation
Enterprise's reference material is detected at test paper, the results are shown in Table 3.
Table 3
As shown in Table 3, when the second anti-A rotavirus monoclonal antibody peridium concentration is 0.5mg/mL~1mg/mL, inspection
It is best to survey effect, meets standard requirements.
Embodiment 7 marks the optimization (optimization of colloid gold label albumen composition amount) of colloidal gold package amount used
The colloidal gold composite of label after purification is redissolved into liquid with colloidal gold and is diluted to OD=25, OD=30, OD=respectively
35, OD=40, OD=45；Then it sprays in colloidal gold pad, is prepared into test paper detection enterprise's reference material, the results are shown in Table 4.
Table 4
| Colloidal gold package amount | Limit of identification | Positive coincidence rate | Negative match-rate |
| OD=25 | 1 ︰ 32 is weakly positive | 7/10 | 10/10 |
| OD=30 | 1 ︰ 32 is the positive | 10/10 | 10/10 |
| OD=35 | 1 ︰ 32 is the positive | 10/10 | 10/10 |
| OD=40 | 1 ︰ 32 is the positive | 10/10 | 10/10 |
| OD=45 | 1 ︰ 32 is the positive | 10/10 | 9/10 |
As shown in Table 4, colloidal gold package amount it is too low or it is excessively high will affect testing result, will appear vacation as package amount is excessively high
The positive, and package amount is all satisfied standard requirements in 30~40OD.
The optimization of 8 sample dilution ratio of embodiment
By required detection sample/enterprise's limit of identification reference material, carried out with dilution according to extension rates different in table 5
After dilution, detected with the test paper prepared, the results are shown in Table 5.
Table 5
As shown in Table 5, detection limit reference material is the positive with testing result at 1:10~1:50 times of diluted；
And for the sample of cross jamming, if sample extension rate is too low, it will lead to the result of false positive.The above result shows that ideal
Sample loading alternative be by sample to be tested according to 1:30~1:40 dilution after detect.
The screening of 9 sample pad pretreatment fluid of embodiment
The optimization of 9.1 sample pad pretreatment fluid components
Sample pad is pre-processed using the sample pad pretreatment fluid of various combination shown in table 6, after oven drying
It is spare, be prepared into test paper, to detection kit (colloidal gold method) enterprise's reference material of A rotavirus and clinical interference sample into
Row detection, the results are shown in Table 7.
Table 6
Table 7
As shown in Table 7, the limit of identification of combination 8,10,12,20,22,24,32,34 and 36, positive coincidence rate, feminine gender
Coincidence rate and cross sample detection are able to satisfy requirement, and said combination is characterized in that, containing two different closed proteins, surface is living
Property agent be TX-100.
The optimization of each content of material in 9.2 sample pad pretreatment fluids
It will be shown below component and prepare sample pad pretreatment fluid: 40mM buffer, 0.6% chlorination according to content as follows
Sodium, 8% closed protein (wherein the content of bovine serum albumin(BSA) and casein be respectively closed protein total content 50%), 1%
TX-100 changes the content of a certain component when the content of components other in sample pad pretreatment fluid remains unchanged.As a result such as table
Shown in 8~11.
The optimization of buffer content
Table 8
As the result is shown: PB, Tris or BB buffer of 40~100mM content, limit of identification meet detection titre not
Lower than 32 times, positive coincidence rate, negative match-rate are also all satisfied condition, and also do not find vacation in the detection of cross jamming sample
Sun.
The optimization of sodium chloride content
Table 9
As the result is shown: when sodium chloride content is 0.4%~2%, limit of identification meets detection titre and is not less than 32 times,
Positive coincidence rate and negative match-rate are 100%, and do not find false sun in the detection of cross jamming sample.
(wherein the content of bovine serum albumin(BSA) and casein is respectively closed protein total content for the optimization of closed protein content
50%)
Table 10
As the result is shown: for closed protein content at 5%~20%, limit of identification meets detection titre not less than 32
Times, positive coincidence rate and negative match-rate are 100%, and do not find false sun in the detection of cross jamming sample.
The optimization of TX-100 content
Table 11
| TX-100 content | Limit of identification | Positive coincidence rate | Negative match-rate | Cross jamming sample |
| 0.25% | 1 ︰ 32 is weakly positive | 8/10 | 10/10 | False sun is not found |
| 0.5% | 1 ︰ 32 is the positive | 10/10 | 10/10 | False sun is not found |
| 1% | 1 ︰ 32 is the positive | 10/10 | 10/10 | False sun is not found |
| 1.5% | 1 ︰ 32 is the positive | 10/10 | 9/10 | False sun is not found |
| 2% | 1 ︰ 32 is weak property | 9/10 | 5/10 | False sun is not found |
As the result is shown: at 0.5%~1%, limit of identification meets detection titre and is not less than 32 times TX-100 content,
Positive coincidence rate and negative match-rate are 100%, and do not find false sun in the detection of cross jamming sample.
10 performance test of embodiment
According to the specification requirement of respective product, while fecal sample is detected, analyzes kit more of the present invention and market
Generally acknowledge the clinical correlation of the preferable kit of quality.
According to the specification requirement of respective product, 1 A rotavirus antigen positive fecal sample is diluted, after dilution
Sample is detected after carrying out gradient dilution according still further to multiple described in table 13, analyzes kit more of the present invention and matter is generally acknowledged in market
Measure the colour developing of positive gradient and sensitivity of preferable kit.
Optional 3 fecal samples will be diluted according to mode fecal sample shown in table 14, then with kit of the present invention and market
Generally acknowledge that the preferable kit of quality is detected, analyzes kit more of the present invention and the preferable kit inspection of quality is generally acknowledged in market
Survey the case where result is interfered by sample.
10.1 generally acknowledge the preferable A rotavirus antigen detection kit (prior art) of quality and of the invention with market
Kit is compared, as a result as shown in table 12.
Table 12
As shown in Table 12, the positive coincidence rate of kit of the present invention and commercial reagent box is 93.18%, and negative match-rate is
100%, total coincidence rate is 99.15%, meets the testing requirements of A rotavirus detection kit.
10.2 generally acknowledge the preferable A rotavirus antigen detection kit (prior art) of quality and of the invention with market
Kit carries out the detection of positive sample gradient, as a result as shown in table 13.
Table 13
As shown in Table 13, the present invention is suitable with the sensitivity of the prior art, but positive colour developing gradient of the invention is more bright
It is aobvious.
10.3 generally acknowledge the preferable A rotavirus antigen detection kit (prior art) of quality and of the invention with market
Kit carries out the detection of clinical sample difference sampling amount, as a result as shown in table 14.
Table 14
As shown in Table 14, in a certain range (1~4 drop, 1 about 35 μ l of drop), enhancing of the present invention with sampling amount, detection
As a result still guarantee that unanimously sampling amount result is identical to specifications with the prior art；And the prior art goes out with the increase of sampling amount
The phenomenon that existing negative findings become positive findings.To sum up, illustrate kit of the present invention under equal conditions, anti-interference ability is more
It is good.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in the attached claims.
Claims (9)
1. a kind of colloidal gold immune chromatography test, it is characterised in that: the colloidal gold immune chromatography test includes chromatographic film, described
Chromatographic film is equipped with a detection line and a nature controlling line；
Wherein, it is coated with the first anti-A rotavirus monoclonal antibody on the colloidal gold, is coated with second in the detection line
Anti- A rotavirus monoclonal antibody.
2. colloidal gold immune chromatography test according to claim 1, wherein the second anti-A rotavirus monoclonal
The package amount of antibody is 0.5~1mg/mL.
3. described in any item colloidal gold immune chromatography tests according to claim 1~2, wherein the first anti-A groups of colyliforms disease
The package amount of malicious monoclonal antibody is 15~30 μ g/mL.
4. described in any item colloidal gold immune chromatography tests according to claim 1~3, wherein be coated on the nature controlling line
Anti- animal IgG antibody；Preferably, the package amount of the anti-animal IgG antibody is 0.5~1mg/mL.
5. a kind of reagent, it is characterised in that: the reagent includes sample pad pretreatment fluid, wherein the sample pad pretreatment fluid
Including buffer, salt ion, closed protein, surfactant.
6. reagent according to claim 5, wherein the buffer is Tris buffer, phosphate buffer, BB buffering
One of liquid；Preferably, the salt ion is one of sodium chloride, potassium chloride, magnesium chloride；Preferably, the closing egg
White is one of bovine serum albumin(BSA), casein, ovalbumin or a variety of；Preferably, the surfactant is nonionic
Surfactant.
7. a kind of kit, wherein the kit includes the described in any item colloidal gold immunochromatographimethod examinations of Claims 1 to 4
Paper and the described in any item reagents of claim 5~6.
8. the described in any item colloidal gold immune chromatography tests of Claims 1 to 4 and/or claim 5~6 are described in any item
Reagent is used to prepare the purposes of the kit of detection A rotavirus.
9. a kind of measuring method of A rotavirus, wherein the method includes using Claims 1 to 44 described in any item
The step of colloidal gold immune chromatography test and/or claim 5~6 described in any item reagents.
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| CN201811611785.0A CN109613239B (en) | 2018-12-27 | 2018-12-27 | Group A rotavirus detection test paper |
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| CN201811611785.0A CN109613239B (en) | 2018-12-27 | 2018-12-27 | Group A rotavirus detection test paper |
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| CN107271660A (en) * | 2017-06-22 | 2017-10-20 | 苏州万木春生物技术有限公司 | A kind of preparation method of test paper for human chorionic gonadotrophin detection |
| CN107287167A (en) * | 2017-06-22 | 2017-10-24 | 四川迈克生物新材料技术有限公司 | The hybridoma of the anti-A rotavirus monoclonal antibody of mouse and its monoclonal antibody of secretion can be secreted |
| CN108169492A (en) * | 2017-12-15 | 2018-06-15 | 东北农业大学 | It is a kind of to be used to detect colloidal gold immuno-chromatography test paper strip of bovine rota and its preparation method and application |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
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| CN103901198A (en) * | 2012-12-26 | 2014-07-02 | 深圳先进技术研究院 | Immune test paper for detecting group A rotaviruses, and its making method |
| CN107271660A (en) * | 2017-06-22 | 2017-10-20 | 苏州万木春生物技术有限公司 | A kind of preparation method of test paper for human chorionic gonadotrophin detection |
| CN107287167A (en) * | 2017-06-22 | 2017-10-24 | 四川迈克生物新材料技术有限公司 | The hybridoma of the anti-A rotavirus monoclonal antibody of mouse and its monoclonal antibody of secretion can be secreted |
| CN108169492A (en) * | 2017-12-15 | 2018-06-15 | 东北农业大学 | It is a kind of to be used to detect colloidal gold immuno-chromatography test paper strip of bovine rota and its preparation method and application |
| CN109085353A (en) * | 2018-06-13 | 2018-12-25 | 比杭生物科技（杭州）有限公司 | A kind of Candida albicans colloidal-gold detecting-card and its application |
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