CN106290849A - A kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box - Google Patents
A kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box Download PDFInfo
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- CN106290849A CN106290849A CN201510243094.XA CN201510243094A CN106290849A CN 106290849 A CN106290849 A CN 106290849A CN 201510243094 A CN201510243094 A CN 201510243094A CN 106290849 A CN106290849 A CN 106290849A
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- Prior art keywords
- gold
- meningitis
- buffer
- immunochromatographyreagent reagent
- assay box
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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
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to field of biological detection, particularly relate to a kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box and its production and use.The present invention provides a kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box, including test card, test card includes base plate and is positioned at the sample pad, gold mark pad, nitrocellulose filter and the adsorptive pads that start to be arranged in order from sample-adding end of backplate surface, meningitis recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition is comprised on described gold mark pad, being coated with detection line and nature controlling line on described nitrocellulose filter, meningitis recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition on described gold mark pad use colloid gold label.Meningitis bacterium gold-immunochromatographyreagent reagent for assay box provided by the present invention has susceptiveness and specificity concurrently, has the advantages such as operation is fast and convenient, result is accurate, economic and practical.
Description
Technical field
The present invention relates to field of biological detection, particularly relate to a kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box and preparation method thereof and
Purposes.
Background technology
Neisseria meningitidis is called for short meningococcus, is the pathogen causing epidemic cerebrospinal meningitis.The mankind are its unique places
Main, can field planting on the nasopharynx part mucosa of the mankind, main by the spittle directly from air by caused by respiratory infectious, in the winter
At the beginning of last month of spring, season is the most popular.Epidemic cerebrospinal meningitis main clinical manifestation for generating heat, have a headache, vomit, mucocutaneous petechia,
Ecchymosis and meningeal irritation sign.Severe one can have septic shock and meningoencephalitis.Cerebrospinal fluid can be in suppurative change.Either exist
Developing country or developed country all can cause higher epidemic encephalitis M & M.
Epidemic cerebrospinal meningitis, especially fulminant type epidemic encephalitis disease progression are rapid, underlying cause of death be septicemia cause shock,
DIC and cerebral edema cerebral hernia.Therefore, diagnosis, tight disease observation early is the basis that primary disease is treated.
Summary of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box
And its production and use, it is used for solving the problems of the prior art.
For achieving the above object and other relevant purposes, the present invention is by the following technical solutions:
A first aspect of the present invention, it is provided that a kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box, including test card, described test card bag
Include base plate and be positioned at the sample pad, gold mark pad, nitrocellulose filter and the water suction that start to be arranged in order from sample-adding end of backplate surface
Pad, described gold mark pad comprises meningitis recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition, described nitrocellulose filter wraps
Being had detection line and nature controlling line, meningitis recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition on described gold mark pad use colloid
Gold labelling.
Preferably, described base plate is PVC base plate.
Preferably, on described nitrocellulose filter, detection line is positioned at side close to sample-adding end, and nature controlling line is positioned at from sample-adding end relatively
Remote side.
Preferably, described detection line is coated with meningitis recombinant antigen.
Preferably, nature controlling line is coated goat anti-rabbit igg polyclonal antibody.
Preferably, described sample pad use buffer process, described buffer selected from PBS, Tris-HCl buffer,
The combination of one or more in glycine buffer, borate buffer solution and citrate-phosphate salt buffer, the concentration of buffer
For 50-100mM.
Preferably, gold mark pad of the present invention is also through pretreatment, and the pre-treatment buffer used during pretreatment is selected from sweet ammonia
Acid buffer, Tris-HCl buffer, the combination of one or more of borate buffer solution, the concentration of buffer is 10-40mM.
Preferably, described buffer also includes increased response agent, described increased response agent selected from PEG4000, PEG6000,
Any one in PEG8000 and PEG20000, the concentration of described increased response agent is 10～50g/L.
Preferably, described buffer solution also includes surfactant, and described surfactant is selected from S-19TWEEN 20, S-20
In TWEEN 80, S-13TRITON X-45, S-14TRITON X-100, S-15TRITON X305 any one or
Multiple combination, the concentration of described surfactant is 20～40g/L.
Preferably, so that test kit has more preferably sensitivity and color developing effect, gold mark pad of the present invention is in pretreatment
Time the pre-treatment buffer that used include following component: fructose, aluminium hydroxide, glucose, sodium chloride and glycine, and fruit
The total concentration of sugar, aluminium hydroxide, glucose, potassium chloride and glycine is 4.5-6.5g/L, and the pH value of buffer is 7.3-7.5.
Preferably, each component concentration in buffer is:
Fructose 1.0-2.0g/L；Aluminium hydroxide 0.25-0.75g/L；Glucose 1.0-1.5g/L；Sodium chloride 0.25-0.5g/L；Sweet
Propylhomoserin 2.0-2.25g/L.
The solvent of described pre-treatment buffer is water.
Concretely comprising the following steps of described pretreatment: gold mark pad soaks in pretreatment fluid 1.5～2h, takes out and is put in 36～38 DEG C of drying.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the regulation of pH value.
Preferably, described test kit also includes getting stuck, described in get stuck and include that back card and upper cover, described back card are provided with test card draw-in groove,
Described test card is embedded in described test card draw-in groove, described on be covered with testing window and well, the position of described testing window and institute
Matching in the position stating detection line and nature controlling line, matches with the position of described sample pad in the position of described well.
Get stuck described in it is furthermore preferred that and get stuck for plastics.
Preferably, described detection kit meningitis bacterium in qualitative detection human serum/blood plasma.
Second aspect present invention provides the preparation method of described meningitis bacterium gold-immunochromatographyreagent reagent for assay box, comprises the steps:
1) with the meningitis recombinant antigen of colloid gold label, rabbit igg polyclonal antibody complex solution spraying gold mark pad, bag is prepared
Containing meningitis recombinant antigen, the gold mark pad of rabbit igg Anti-TNF-α nanocrystal composition；
2) on the detection line and nature controlling line of nitrocellulose filter, meningitis recombinant antigen and goat anti-rabbit igg Anti-TNF-α are sprayed respectively
Body, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On base plate, cutting prepares Test paper card；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
Preferably, gold mark pad of the present invention is also through pretreatment, and the pre-treatment buffer used during pretreatment is selected from sweet ammonia
Acid buffer, Tris-HCl buffer, the combination of one or more of borate buffer solution, the concentration of buffer is 10-40mM.
Preferably, described buffer also includes increased response agent, described increased response agent selected from PEG4000, PEG6000,
Any one in PEG8000 and PEG20000, the concentration of described increased response agent is 10～50g/L.
Preferably, described buffer solution also includes surfactant, and described surfactant is selected from S-19TWEEN 20, S-20
In TWEEN 80, S-13TRITON X-45, S-14TRITON X-100, S-15TRITON X305 any one or
Multiple combination, the concentration of described surfactant is 20～40g/L.
Preferably, so that test kit has more preferably sensitivity and color developing effect, gold mark pad of the present invention is in pretreatment
Time the pre-treatment buffer that used include following component: fructose, aluminium hydroxide, glucose, sodium chloride and glycine, and fruit
The total concentration of sugar, aluminium hydroxide, glucose, potassium chloride and glycine is 4.5-6.5g/L, and the pH value of buffer is 7.3-7.5.
Preferably, each component concentration in buffer is:
Fructose 1.0-2.0g/L；Aluminium hydroxide 0.25-0.75g/L；Glucose 1.0-1.5g/L；Sodium chloride 0.25-0.5g/L；Sweet
Propylhomoserin 2.0-2.25g/L.
The solvent of described pre-treatment buffer is water.
Concretely comprising the following steps of described pretreatment: gold mark pad soaks in pretreatment fluid 1.5～2h, takes out and is put in 36～38 DEG C of drying.
Described pre-treatment buffer can use the various conventional pH adjusting agent in this area to carry out the regulation of pH value.
Third aspect present invention provides described meningitis bacterium gold-immunochromatographyreagent reagent for assay box in the purposes of meningitis bacterium detection field.
The invention have the benefit that
Meningitis bacterium gold-immunochromatographyreagent reagent for assay box provided by the present invention has high sensitivity and high specific concurrently, it is possible to quickly detect brain
Film inflammation bacterium.Additionally, described detection kit has the advantages such as operation is fast and convenient, result is accurate, economic and practical.
Detailed description of the invention
Below by way of specific instantiation, embodiments of the present invention being described, those skilled in the art can be by disclosed by this specification
Content understand other advantages and effect of the present invention easily.The present invention can also be added by the most different detailed description of the invention
To implement or application, the every details in this specification can also be based on different viewpoints and application, in the essence without departing from the present invention
Various modification or change is carried out under god.
Before further describing the specific embodiment of the invention, it should be appreciated that protection scope of the present invention is not limited to following specific
Specific embodiments；It is also understood that the term used in the embodiment of the present invention is to describe specific specific embodiments,
Rather than in order to limit the scope of the invention；In description of the invention and claims, unless literary composition additionally clearly refers to
Going out, singulative " ", " one " and " this " include plural form.
When embodiment provides numerical range, it should be appreciated that unless the present invention is otherwise noted, two end points of each numerical range with
And any one numerical value all can be selected between two end points.Unless otherwise defined, all technology used in the present invention and section are academic
The same meaning that language and those skilled in the art of the present technique are generally understood that.In addition to the concrete grammar used in embodiment, equipment, material,
According to those skilled in the art's grasp to prior art and the record of the present invention, it is also possible to use and the embodiment of the present invention
Described in method, any method, equipment and the material of the similar or equivalent prior art of equipment, material realize the present invention.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use the art normal
Rule molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell cultivate, recombinant DNA technology and
The routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic updates；the series
METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS
IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The preparation of embodiment 1 test card of the present invention
1) using pre-treatment buffer that gold mark pad is carried out pretreatment, pre-treatment buffer is: fructose 1.5g/L, aluminium hydroxide
0.5g/L, glucose 1.25g/L, sodium chloride 0.3g/L, the aqueous solution of glycine 2.0g/L, pH=7.4, the concrete step of pretreatment
Suddenly it is: gold mark pad is soaked in pretreatment fluid 2h, takes out and be put in 37 DEG C of drying；Then with the meningitis weight of colloid gold label
Group antigen, rabbit igg polyclonal antibody complex solution spraying pretreated gold mark pad, prepare be coated meningitis recombinant antigen,
The gold mark pad of rabbit igg Anti-TNF-α nanocrystal composition, in solution, gold colloidal is 5:1 with the mass ratio of complex, and the concentration of solution is
10mg/ml, quantity for spray is 4ul/cm；
2) on the detection line and nature controlling line of nitrocellulose filter, spray meningitis recombinant antigen solution and the goat-anti of 1mg/ml respectively
Rabbit igg Anti-TNF-α liquid solution, quantity for spray is 1ul/cm, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On PVC base plate, cutting prepares the Test paper card of wide 3-5mm；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
The preparation of embodiment 2 contrast agent box
The preparation of comparative example test card: using 25mM glycine buffer pretreatment gold mark pad, other reagent and experimental technique are equal
With embodiment 1.
1) 25mM glycine buffer pretreatment gold mark pad is used, then with the meningitis recombinant antigen of colloid gold label, rabbit
IgG polyclonal antibody complex solution spraying pretreated gold mark pad, prepares and is coated many grams of meningitis recombinant antigen, rabbit igg
The gold mark pad of grand antibody complex, in solution, gold colloidal is 5:1 with the mass ratio of complex, and the concentration of solution is 10mg/ml, spray
Painting amount is 4ul/cm；
2) on the detection line and nature controlling line of nitrocellulose filter, spray meningitis recombinant antigen solution and the goat-anti of 1mg/ml respectively
Rabbit igg Anti-TNF-α liquid solution, quantity for spray is 1ul/cm, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On PVC base plate, cutting prepares the Test paper card of wide 3-5mm；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
The specificity experiments of embodiment 3 detection kit
Detection method:
1. Example 1 preparation test kit of the present invention and the contrast agent box of embodiment 2, test kit is placed on level table.
2. the preparation of testing sample: by four class experimental subjecies of table 1, samples from tester's serum, uses the PBS of 0.01M pH7.2
Buffer dilutes, and dilution ratio is 1:1..
3. every class experimental subject is 100 people, and the sample used by contrast experiment is identical, testing result such as table 1:
Table 1 detection kit specific test result
As seen from the above table, the detection kit of present invention no cross reaction equal to escherichia coli and dysentery bacterium, specificity is good.
In sum, detection kit provided by the present invention has good anti-interference and specificity, and has good spirit
Quick property, negative background is lower, effectively overcomes various shortcoming of the prior art and has high industrial utilization.
The principle of above-described embodiment only illustrative present invention and effect thereof, not for limiting the present invention.Any it is familiar with this skill
Above-described embodiment all can be modified under the spirit and the scope of the present invention or change by the personage of art.Therefore, such as
All that in art, tool usually intellectual is completed under without departing from disclosed spirit and technological thought etc.
Effect is modified or changes, and must be contained by the claim of the present invention.
Claims (10)
1. a meningitis bacterium gold-immunochromatographyreagent reagent for assay box, including test card, test card include base plate and be positioned at backplate surface from adding
Sample end starts sample pad, gold mark pad, nitrocellulose filter and the adsorptive pads being arranged in order, and described gold mark pad comprises meningitis
Recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition, described nitrocellulose filter is coated with detection line and nature controlling line, described
Meningitis recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition on gold mark pad use colloid gold label.
2. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that on described nitrocellulose filter, detection line is positioned at
Side close to sample-adding end, nature controlling line is positioned at from sample-adding end side farther out.
3. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that be coated with meningitis restructuring on described detection line anti-
Former.
4. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that be coated goat anti-rabbit igg Anti-TNF-α on nature controlling line
Body.
5. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that described sample pad uses buffer to process, described
Buffer is selected from PBS, Tris-HCl buffer, glycine buffer, borate buffer solution and citrate-phosphate
The combination of one or more in salt buffer, the concentration of buffer is 50～100mM.
6. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that described gold mark pad uses buffer to process, described
Buffer is selected from glycine buffer, Tris-HCl buffer, the combination of one or more of borate buffer solution, buffer
Concentration be 10～40mM.
Gold-immunochromatographyreagent reagent for assay box the most according to claim 6, it is characterised in that also include increased response agent in described buffer,
Any one in PEG4000, PEG6000, PEG8000 and PEG20000 of described increased response agent, described instead
The concentration answering reinforcing agent is 20～40g/L.
8. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that also include getting stuck, described in get stuck include back card and
Upper cover, described back card is provided with test card draw-in groove, and described test card is embedded in described test card draw-in groove, described on be covered with test
Window and well, the position of described testing window matches with the position of described detection line and nature controlling line, the position of described well
Match with the position of described sample pad.
9. gold-immunochromatographyreagent reagent for assay box as claimed in claim 1, it is characterised in that described detection kit is used for qualitative detection serum
Meningitis bacterium in/blood plasma.
10., according to the preparation method of the gold-immunochromatographyreagent reagent for assay box described in claim 1～9 any claim, specifically include as follows
Step:
1) with the meningitis recombinant antigen of colloid gold label, rabbit igg polyclonal antibody complex solution spraying pretreated gold mark
Pad, prepares the gold mark pad comprising meningitis recombinant antigen, rabbit igg Anti-TNF-α nanocrystal composition；
2) on the detection line and nature controlling line of nitrocellulose filter, meningitis recombinant antigen and goat anti-rabbit igg Anti-TNF-α are sprayed respectively
Body, prepares the nitrocellulose filter after being coated；
3) by sample pad, step 1) preparation gold mark pad, step 2) nitrocellulose filter prepared, adsorptive pads be pasted onto successively
On base plate, cutting prepares Test paper card；Finally Test paper is snapped fits into the prepared detection kit that gets stuck.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510243094.XA CN106290849A (en) | 2015-05-13 | 2015-05-13 | A kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510243094.XA CN106290849A (en) | 2015-05-13 | 2015-05-13 | A kind of meningitis bacterium gold-immunochromatographyreagent reagent for assay box |
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| CN106290849A true CN106290849A (en) | 2017-01-04 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN1298446A (en) * | 1998-04-29 | 2001-06-06 | 美国氰胺公司 | Vaccine containing recombinant pilin and used for resisting neisseria gonorrhoeae or neisseria meningitidis |
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2015
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|---|---|---|---|---|
| CN1292820A (en) * | 1998-01-14 | 2001-04-25 | 启龙股份公司 | i (Neisseria meningitidis) antigens |
| CN1298446A (en) * | 1998-04-29 | 2001-06-06 | 美国氰胺公司 | Vaccine containing recombinant pilin and used for resisting neisseria gonorrhoeae or neisseria meningitidis |
| CN101076731A (en) * | 2004-12-15 | 2007-11-21 | 金伯利-克拉克环球有限公司 | High efficient current immunity detecting for sample |
| US20090269733A1 (en) * | 2008-04-23 | 2009-10-29 | Azko, Inc. | Devices and Methods for the Rapid Analysis of Pathogens in Biological Fluids |
| CN102854318A (en) * | 2012-07-31 | 2013-01-02 | 吴克 | A and C type neisseria meningitides enzyme-linked diagnostic kit and preparation method thereof |
| CN103336113A (en) * | 2013-06-24 | 2013-10-02 | 苏州万木春生物技术有限公司 | Preparation method of human immunodeficiency virus (HIV) antibody detection test paper |
| CN104360085A (en) * | 2014-12-05 | 2015-02-18 | 重庆乾德生物技术有限公司 | AFP (alpha fetal protein) detection kit |
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| 王欣茹 等: "人血清中A、C、Y、W135群脑膜炎球菌荚膜多糖IgG抗体含量ELISA检测方法的建立", 《中国生物制品学杂志》 * |
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