CN103852580B - Staphylococcus aureus identification kit and preparation method thereof - Google Patents
Staphylococcus aureus identification kit and preparation method thereof Download PDFInfo
- Publication number
- CN103852580B CN103852580B CN201410057836.5A CN201410057836A CN103852580B CN 103852580 B CN103852580 B CN 103852580B CN 201410057836 A CN201410057836 A CN 201410057836A CN 103852580 B CN103852580 B CN 103852580B
- Authority
- CN
- China
- Prior art keywords
- polystyrene microsphere
- solution
- preparation
- concentration
- damping fluid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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
- G01N33/531—Production of immunochemical test materials
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
- G01N33/545—Synthetic resin
-
- 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
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention relates to a kind of staphylococcus aureus identification kit and preparation method thereof, comprise and detect reagent R1 and negative control reagent R2, negative control reagent R2 is the potpourri of polystyrene microsphere and microballoon damping fluid, it is characterized in that detecting the potpourri that reagent R1 adopts immune polystyrene microsphere and microballoon damping fluid, immune polystyrene microsphere adopts hydroxy functionalized polystyrene microsphere and at least two kinds of differential protein couplings to form.Hydroxy functionalized polystyrene microsphere preparation method, alkylated chitosan and polystyrene microsphere is adopted to react, utilize the high density hydroxyl that hydroxy functionalized Surfaces of Polystyrene Microparticles distributes, improve the coupling efficiency of hydroxy functionalized polystyrene microsphere and differential protein, differential protein in immune Surfaces of Polystyrene Microparticles coupling or antibody materials are improved greatly, there is the advantages such as detection sensitivity is high, accuracy is high, and loss is few.
Description
Technical field
The invention belongs to biotechnology and microorganism detection field, relate to a kind of staphylococcus aureus Rapid identification kit based on immune polystyrene microsphere agglutinating reaction.The staphylococcus aureus that the present invention is applicable in Clinical microorganism or food microorganisms sample detects fast.
Technical background
Staphylococcus is raw common bacterial strain of dwelling on human skin and mucous membrane.Wherein staphylococcus aureus can cause the pyogenic infection on a large scale comprising epidermis and suppurate, food poisoning and toxic shock, and other various disease.Therefore staphylococcus aureus is one of modal pathogenic bacteria of often running into of laboratory, because infection of staphylococcus aureus occurrence frequency is high and serious, and staphylococcus aureus has drug resistance to numerous Conventional antibiotic, therefore in the urgent need to diagnosing fast and accurately, to take effective remedy measures to patient.In food manufacturing, finding that staphylococcus is the indicator bacteria of good microbial total body pollution level in addition, therefore needing a kind of staphylococcus aureus rapid identification method of high degree of specificity to infecting serious sample.
Latex agglutination is a kind of comparatively conventional method for quick occurred in recent years.The method utilizes the distinctive plasma-coagulase of staphylococcus aureus surface, the principle can reacted with plasma fibrin material generation specific agglutination, plasma fibrin material and latex particle covalent coupling are prepared into the latex particle of sensitization, sensitization microballoon after this is crosslinked can react with the plasma-coagulase of above-mentioned staphylococcus aureus surface, produce microballoon agglutination phenomenon, thus reach the object of Rapid identification staphylococcus aureus.The method has higher specificity, and detection time is short, and detection efficiency is high.But have detection leakage phenomenon to the minority staphylococcus aureus strains presenting Coagulase-negative Staphylococci, cause false-negative result, accuracy in detection is poor.
Summary of the invention
Technical matters to be solved by this invention is to provide staphylococcus aureus identification kit that a kind of detection speed is fast, detection sensitivity is high, accuracy in detection is good and preparation method thereof.
The technical scheme of staphylococcus aureus identification kit of the present invention comprises detection reagent R1 and negative control reagent R2, negative control reagent R2 is the potpourri of polystyrene microsphere and microballoon damping fluid, it is characterized in that detecting the potpourri that reagent R1 adopts immune polystyrene microsphere and microballoon damping fluid, immune polystyrene microsphere adopts hydroxy functionalized polystyrene microsphere and at least two kinds of differential protein couplings to form.
Preparation method of the present invention is:
1, detect the preparation method of reagent R1: in every 100ml microballoon damping fluid, add 0.5g immunity polystyrene microsphere;
2, the preparation method of negative control reagent R2: in every 100ml microballoon damping fluid, add 0.5g polystyrene microsphere;
Wherein the preparation method of microballoon damping fluid is:
1.. compound concentration is 0.1 ~ 1M, and pH value is phosphate buffer, tris damping fluid, carbonate buffer solution, the glycine buffer of 6.0 ~ 9.0;
2.. get above-mentioned a kind of damping fluid, add one or more compounds in Qu Latong series and TWEEN Series, make the compound concentration after preparation be 0.5 ~ 2%;
3.. to step 2. in add one or more ingredients substances in PEG2000, casein, gelatin, skim milk in the damping fluid for preparing, make each ingredients substance concentration after preparation be 1 ~ 5%;
4.. microballoon damping fluid step 3. prepared stirs, and the temperature environment being placed in 2 ~ 8 DEG C is preserved;
It is characterized in that:
The preparation method of immunity polystyrene microsphere is:
1.. hydroxy functionalized polystyrene microsphere is scattered in aqueous solution, makes its concentration be 2 ~ 5%;
2.. get above-mentioned solution 10ml, adding the concentration that 1 ~ 2ml newly joins is the NaIO of 0.1 ~ 0.5mol/L
4solution, mixing, 4 DEG C of temperature environments leave standstill 20 minutes;
3.. in step 2. solution, add 1 ~ 2ml, the glycol water of 0.16M, mixing, room temperature lucifuge leaves standstill 30 ~ 60 minutes;
4.. in the solution that 3. step obtains, add differential protein solution, mixing, load bag filter, slowly stir with 0.05mol/LPH7.0 carbonate buffer solution, dialyse 16 ~ 20 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway, and after mixing, differential protein concentration is 1 ~ 10mg/ml;
5.. in the solution that 4. step obtains, add NaBH4 solution 2 ~ 5ml that freshly prepared concentration is 5mg/ml, mixing, react in 4 DEG C of temperature environments after 2 hours, load bag filter, slowly stir with 0.1mol/LPH7.0 phosphate buffer, dialyse 16 ~ 20 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway;
6.. take out the reactant liquor of having dialysed, add to 20ml with 0.1mol/LPH7.0PBS, the temperature environment being placed in 2 ~ 8 DEG C is preserved.
Preferably, the preparation method of hydroxy functionalized polystyrene microsphere is:
1.. alkylated chitosan: shitosan, KOH, isopropyl alcohol mixing are placed in three-neck flask, at the uniform velocity stir, be warming up to 40 DEG C and keep 1 hour, be warming up to 60 DEG C after shitosan is alkalized, and drip halogenated hydrocarbons, constant temperature stirring reaction 2 ~ 12 hours; Wherein every milliliter of isopropyl alcohol adds 0.05 ~ 0.1g shitosan and 0.1 ~ 0.2gKOH; Every milliliter of reactant liquor is containing 0.10 ~ 0.30ml halogenated hydrocarbons;
2.. by through step reactant acetone 1. and distilled water alternately washing, until no longer include halogen ion in the distilled water washed out, after oven dry, obtain alkylated chitosan;
3.. the alkylated chitosan that 2. step is obtained, being dissolved in concentration is in 1 ~ 5% acetum, leaves standstill 16 ~ 20 hours, makes it fully dissolve dispersion, and after dissolving, the concentration of alkylated chitosan is 1 ~ 5%:
4.. step of learning from else's experience alkylated chitosan solution 3., is added drop-wise in polystyrene microsphere solution, at the uniform velocity stirs 1 ~ 10 hour under room temperature, and after homodisperse colloidal solution, centrifuging obtains hydroxy functionalized polystyrene microsphere; In hydroxy functionalized polystyrene microsphere solution, the concentration of alkylated chitosan is 0.01 ~ 0.5%, and the concentration of polystyrene microsphere is 0.1 ~ 1%.
One of advantage of the present invention is: provide a kind of staphylococcus aureus rapid identification method based on immune polystyrene microsphere agglutinating reaction newly, the method adopts Multiple detection mechanism, identify the plasma-coagulase of staphylococcus aureus surface, SPA, polysaccharide antigen, capsular antigen etc. simultaneously, improve the specificity and accuracy that detect reagent, reduce loss;
Two of advantage of the present invention is: provide a kind of hydroxy functionalized polystyrene microsphere preparation method, alkylated chitosan and polystyrene microsphere is adopted to react, be prepared into the functional polystyrene microballoon that a kind of surface distributed has high density oh group, hydroxy functionalized polystyrene microsphere and antibody materials coupling is adopted to be prepared into immune polystyrene microsphere, utilize the high density hydroxyl that hydroxy functionalized Surfaces of Polystyrene Microparticles distributes, improve the coupling efficiency of hydroxy functionalized polystyrene microsphere and differential protein, differential protein in immune Surfaces of Polystyrene Microparticles coupling or antibody materials are improved greatly, improve the detection sensitivity of reagent.
Embodiment
Staphylococcus aureus identification kit of the present invention, comprise and detect reagent R1 and negative control reagent R2, negative control reagent R2 is the potpourri of polystyrene microsphere and microballoon damping fluid, detect the potpourri that reagent R1 is immune polystyrene microsphere and microballoon damping fluid, immune polystyrene microsphere adopts hydroxy functionalized polystyrene microsphere and at least two kinds of differential protein couplings to form.Described differential protein can be the blood plasma of human or animal, the IgG immunoglobulin (Ig) of human or animal, anti-SPA monoclonal antibody or polyclonal antibody, anti-Staphylococcus aureus capsular polysaccharide antigen monoclonal antibody or polyclonal antibody, anti-Staphylococcus aureus monoclonal antibody or polyclonal antibody.
The compound method of staphylococcus aureus identification kit of the present invention:
1, detect the preparation method of reagent R1: in every 100ml microballoon damping fluid, add 0.5g immunity polystyrene microsphere;
2, the preparation method of negative control reagent R2: in every 100ml microballoon damping fluid, add 0.5g polystyrene microsphere;
Wherein the preparation method of microballoon damping fluid is:
1.. compound concentration is 0.1 ~ 1M, and pH value is phosphate buffer, tris damping fluid, carbonate buffer solution, the glycine buffer of 6.0 ~ 9.0;
2.. get above-mentioned a kind of damping fluid, add one or more compounds in Qu Latong series and TWEEN Series, make the compound concentration after preparation be 0.5 ~ 2%;
3.. to step 2. in add one or more ingredients substances in PEG2000, casein, gelatin, skim milk in the damping fluid for preparing, make each ingredients substance concentration after preparation be 1 ~ 5%;
4.. microballoon damping fluid step 2. 3. prepared with step stirs, and the temperature environment being placed in 2 ~ 8 DEG C is preserved;
The preparation method of immunity polystyrene microsphere is:
1.. hydroxy functionalized polystyrene microsphere is scattered in aqueous solution, makes its concentration be 2 ~ 5%;
2.. get above-mentioned solution 10ml, adding the concentration that 1 ~ 2ml newly joins is the NaIO of 0.1 ~ 0.5mol/L
4solution, mixing, 4 DEG C of temperature environments leave standstill 20 minutes;
3.. in step 2. solution, add 1 ~ 2ml, the glycol water of 0.16M, mixing, room temperature lucifuge leaves standstill 30 ~ 60 minutes;
4.. in the solution that 3. step obtains, add differential protein solution, mixing, load bag filter, slowly stir with 0.05mol/LPH7.0 carbonate buffer solution, dialyse 16 ~ 20 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway, and after mixing, differential protein concentration is 1 ~ 10mg/ml;
5.. in the solution that 4. step obtains, add NaBH4 solution 2 ~ 5ml that freshly prepared concentration is 5mg/ml, mixing, react in 4 DEG C of temperature environments after 2 hours, load bag filter, slowly stir with 0.1mol/LPH7.0 phosphate buffer, dialyse 16 ~ 20 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway;
6.. take out the reactant liquor of having dialysed, add to 20ml with 0.1mol/LPH7.0PBS, the temperature environment being placed in 2 ~ 8 DEG C is preserved.
The preparation method of hydroxy functionalized polystyrene microsphere is:
1.. alkylated chitosan: shitosan, KOH, isopropyl alcohol mixing are placed in three-neck flask, at the uniform velocity stir, be warming up to 40 DEG C and keep 1 hour, be warming up to 60 DEG C after shitosan is alkalized, and drip halogenated hydrocarbons, constant temperature stirring reaction 2 ~ 12 hours; Wherein every milliliter of isopropyl alcohol adds 0.05 ~ 0.1g shitosan and 0.1 ~ 0.2gKOH; Every milliliter of reactant liquor is containing 0.10 ~ 0.30ml halogenated hydrocarbons;
2.. by through step reactant acetone 1. and distilled water alternately washing, until no longer include halogen ion in the distilled water washed out, after oven dry, obtain alkylated chitosan;
3.. the alkylated chitosan that 2. step is obtained, being dissolved in concentration is in 1 ~ 5% acetum, leaves standstill 16 ~ 20 hours, makes it fully dissolve dispersion, and after dissolving, the concentration of alkylated chitosan is 1 ~ 5%:
4.. step of learning from else's experience alkylated chitosan solution 3., is added drop-wise in polystyrene microsphere solution, at the uniform velocity stirs 1 ~ 10 hour under room temperature, and after homodisperse colloidal solution, centrifuging obtains hydroxy functionalized polystyrene microsphere; In hydroxy functionalized polystyrene microsphere solution, the concentration of alkylated chitosan is 0.01 ~ 0.5%, and the concentration of polystyrene microsphere is 0.1 ~ 1%.
Preferred version prepared by hydroxy functionalized polystyrene microsphere is:
1.. the alkylation of shitosan: 5g shitosan, the mixing of 10gKOH and 100ml isopropyl alcohol are placed in three-neck flask, at the uniform velocity stir, be warming up to 40 DEG C and keep 1 hour, be warming up to 60 DEG C after shitosan is alkalized, and drip 20ml halogenated hydrocarbons, constant temperature stirring reaction 8 hours; Halogenated hydrocarbons adopts chlorinated dodecane, and the alkylated chitosan be prepared into is dodecyl shitosan;
2.. by through step reactant acetone 1. and distilled water alternately washing, until no longer include halogen ion in the distilled water washed out, after oven dry, obtain alkylated chitosan;
3.. by alkylated chitosan, being dissolved in concentration is in the acetum of 2%, leaves standstill 12 hours, makes it fully dissolve dispersion, and after dissolving, the concentration of alkylated chitosan is 1%;
4.. step of learning from else's experience alkylated chitosan solution 3., be added drop-wise in polystyrene microsphere solution, the concentration of alkylated chitosan is made to be 0.01%, the concentration of polystyrene microsphere is 0.5%, 5 hours are at the uniform velocity stirred under room temperature, after homodisperse colloidal solution, centrifuging obtains hydroxy functionalized polystyrene microsphere.
Preferred version prepared by microballoon damping fluid is:
1.. compound concentration is 1M, and pH value is the phosphate buffer of 6.0;
2.. in above-mentioned phosphate buffer, add the tween 10 of the Qu Latong and 0.5% of 1%, stir;
3.. to step 2. in add the PEG2000 of 1%, the casein of 2% in the damping fluid for preparing, stir;
4.. the cocktail buffer that 3. 2. step prepare with step is stirred, and by make microballoon damping fluid be placed in 4 DEG C temperature environment preserve.
Preferred version prepared by immunity polystyrene microsphere is:
1.. hydroxy functionalized polystyrene microsphere is scattered in aqueous solution, makes its final concentration be 3%;
2.. get above-mentioned solution 10ml, adding the concentration that 2ml newly joins is the NaIO of 0.5mol/L
4solution, mixing, 4 DEG C of temperature environments leave standstill 20 minutes;
3.. in step 2. solution, add the glycol water of 2ml, 0.16M, mixing, room temperature lucifuge leaves standstill 30 minutes;
4.. in the solution that 3. step obtains, add human plasma, anti-SPA monoclonal antibody, anti-Staphylococcus aureus capsular polysaccharide monoclonal antibody differential protein solution, the concentration of various differential protein is made to be 2mg/ml, bag filter is loaded after mixing, slowly stir with 0.05mol/LPH7.0 carbonate buffer solution, dialyse 12 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway;
5.. in the solution that 4. step obtains, add the NaBH that freshly prepared concentration is 5mg/ml
4solution 2ml, mixing, react in 4 DEG C of temperature environments after 2 hours, load bag filter, slowly stir with 0.1mol/LPH7.0 phosphate buffer, dialyse 12 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway;
6.. take out the reactant liquor of having dialysed, add to 20ml with 0.1mol/LPH7.OPBS, the temperature environment of putting 4 DEG C is preserved.
Claims (2)
1. a staphylococcus aureus identification kit, comprises and detects reagent R
1with negative control reagent R
2, negative control reagent R
2for the potpourri of polystyrene microsphere and microballoon damping fluid, it is characterized in that detecting reagent R
1adopt the potpourri of immune polystyrene microsphere and microballoon damping fluid, immune polystyrene microsphere adopts hydroxy functionalized polystyrene microsphere and at least two kinds of differential protein couplings to form;
The preparation method of above-mentioned staphylococcus aureus identification kit:
1), reagent R is detected
1preparation method: in every 100ml microballoon damping fluid, add 0.5g immunity polystyrene microsphere;
2), negative control reagent R
2preparation method: in every 100ml microballoon damping fluid, add 0.5g polystyrene microsphere;
Wherein the preparation method of microballoon damping fluid is:
1.. compound concentration is 0.1 ~ 1M, and pH value is phosphate buffer, tris damping fluid, carbonate buffer solution, the glycine buffer of 6.0 ~ 9.0;
2.. get above-mentioned a kind of damping fluid, add one or more compounds in Qu Latong series and TWEEN Series, make the compound concentration after preparation be 0.5 ~ 2%;
3.. to step 2. in add one or more ingredients substances in PEG2000, casein, gelatin, skim milk in the damping fluid for preparing, make each ingredients substance concentration after preparation be 1 ~ 5%;
4.. microballoon damping fluid step 3. prepared stirs, and the temperature environment being placed in 2 ~ 8 DEG C is preserved;
It is characterized in that:
The preparation method of immunity polystyrene microsphere is:
1.. hydroxy functionalized polystyrene microsphere is scattered in aqueous solution, makes its concentration be 2 ~ 5%;
2.. get above-mentioned solution 10ml, adding the concentration that 1 ~ 2ml newly joins is the NaIO of 0.1 ~ 0.5mol/L
4solution, mixing, 4 DEG C of temperature environments leave standstill 20 minutes;
3.. in step 2. solution, add 1 ~ 2ml, the glycol water of 0.16M, mixing, room temperature lucifuge leaves standstill 30 ~ 60 minutes;
4.. in the solution that 3. step obtains, add differential protein solution, mixing, load bag filter, slowly stir with 0.05mol/LPH7.0 carbonate buffer solution, dialyse 16 ~ 20 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway, and after mixing, differential protein concentration is 1 ~ 10mg/ml; Described differential protein solution is the blood plasma of human or animal, the IgG immunoglobulin (Ig) of human or animal, anti-SPA monoclonal antibody or polyclonal antibody, anti-Staphylococcus aureus capsular polysaccharide antigen monoclonal antibody or polyclonal antibody, anti-Staphylococcus aureus monoclonal antibody or polyclonal antibody;
5.. in the solution that 4. step obtains, add the NaBH that freshly prepared concentration is 5mg/ml
4solution 2 ~ 5ml, mixing, react in 4 DEG C of temperature environments after 2 hours, load bag filter, slowly stir with 0.1mol/LPH7.0 phosphate buffer, dialyse 16 ~ 20 hours in 4 DEG C of temperature environments, secondary dislysate is changed in midway;
6.. take out the reactant liquor of having dialysed, add to 20ml with 0.1mol/LPH7.0PBS, the temperature environment being placed in 2 ~ 8 DEG C is preserved.
2. the preparation method of staphylococcus aureus identification kit according to claim 1, is characterized in that the preparation method of hydroxy functionalized polystyrene microsphere is:
1.. alkylated chitosan: shitosan, KOH, isopropyl alcohol mixing are placed in three-neck flask, at the uniform velocity stir, be warming up to 40 DEG C and keep 1 hour, be warming up to 60 DEG C after shitosan is alkalized, and drip halogenated hydrocarbons, constant temperature stirring reaction 2 ~ 12 hours; Wherein every milliliter of isopropyl alcohol adds 0.05 ~ 0.1g shitosan and 0.1 ~ 0.2gKOH; Every milliliter of reactant liquor is containing 0.10 ~ 0.30ml halogenated hydrocarbons;
2.. by through step reactant acetone 1. and distilled water alternately washing, until no longer include halogen ion in the distilled water washed out, after oven dry, obtain alkylated chitosan;
3.. the alkylated chitosan that 2. step is obtained, being dissolved in concentration is in 1 ~ 5% acetum, leaves standstill 16 ~ 20 hours, makes it fully dissolve dispersion, and after dissolving, the concentration of alkylated chitosan is 1 ~ 5%;
4.. step of learning from else's experience alkylated chitosan solution 3., is added drop-wise in polystyrene microsphere solution, at the uniform velocity stirs 1 ~ 10 hour under room temperature, and after homodisperse colloidal solution, centrifuging obtains hydroxy functionalized polystyrene microsphere; In hydroxy functionalized polystyrene microsphere solution, the concentration of alkylated chitosan is 0.01 ~ 0.5%, and the concentration of polystyrene microsphere is 0.1 ~ 1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410057836.5A CN103852580B (en) | 2014-02-18 | 2014-02-18 | Staphylococcus aureus identification kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410057836.5A CN103852580B (en) | 2014-02-18 | 2014-02-18 | Staphylococcus aureus identification kit and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103852580A CN103852580A (en) | 2014-06-11 |
| CN103852580B true CN103852580B (en) | 2015-12-09 |
Family
ID=50860486
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410057836.5A Active CN103852580B (en) | 2014-02-18 | 2014-02-18 | Staphylococcus aureus identification kit and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103852580B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603348A (en) * | 2004-09-14 | 2005-04-06 | 清华大学 | Process for synthesis of monodisperse micron crosslinked polystyrene copolymerization microsphere |
| WO2009039854A2 (en) * | 2007-09-27 | 2009-04-02 | Dako Denmark A/S | Mhc multimers in tuberculosis diagnostics, vaccine and therapeutics |
| CN101775386A (en) * | 2010-01-26 | 2010-07-14 | 武汉工业学院 | Method for immobilizing trypsinase by chitosan microspheres |
| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| CN102695718A (en) * | 2009-12-28 | 2012-09-26 | 龟甲万株式会社 | Method for extracting staphylococcus aureus antigen, reagent for extracting staphylococcus aureus antigen, and method for testing staphylococcus aureus |
| CN102901810A (en) * | 2012-10-09 | 2013-01-30 | 北京九强生物技术股份有限公司 | Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit |
| CN102944679A (en) * | 2012-11-15 | 2013-02-27 | 北京康美天鸿生物科技有限公司 | Kit for performing retinol binding protein detection by using latex turbidimetry |
| CN103073642A (en) * | 2012-08-29 | 2013-05-01 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation process thereof |
| CN103344763A (en) * | 2013-06-18 | 2013-10-09 | 华东师范大学 | Latex-reinforced AFP detection kit and its preparation method and use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1914512A (en) * | 2003-12-30 | 2007-02-14 | 3M创新有限公司 | Method of enhancing signal detection of cell-wall components of cells |
-
2014
- 2014-02-18 CN CN201410057836.5A patent/CN103852580B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603348A (en) * | 2004-09-14 | 2005-04-06 | 清华大学 | Process for synthesis of monodisperse micron crosslinked polystyrene copolymerization microsphere |
| WO2009039854A2 (en) * | 2007-09-27 | 2009-04-02 | Dako Denmark A/S | Mhc multimers in tuberculosis diagnostics, vaccine and therapeutics |
| CN102695718A (en) * | 2009-12-28 | 2012-09-26 | 龟甲万株式会社 | Method for extracting staphylococcus aureus antigen, reagent for extracting staphylococcus aureus antigen, and method for testing staphylococcus aureus |
| CN101775386A (en) * | 2010-01-26 | 2010-07-14 | 武汉工业学院 | Method for immobilizing trypsinase by chitosan microspheres |
| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| CN103073642A (en) * | 2012-08-29 | 2013-05-01 | 深圳伯美生物医药有限公司 | CRP monoclonal antibody nanometer latex microsphere composition and preparation process thereof |
| CN102901810A (en) * | 2012-10-09 | 2013-01-30 | 北京九强生物技术股份有限公司 | Preparation method of latex particles coated with prostate specific antigen-antibody and PSA enhanced turbidimetric immunophelometry kit |
| CN102944679A (en) * | 2012-11-15 | 2013-02-27 | 北京康美天鸿生物科技有限公司 | Kit for performing retinol binding protein detection by using latex turbidimetry |
| CN103344763A (en) * | 2013-06-18 | 2013-10-09 | 华东师范大学 | Latex-reinforced AFP detection kit and its preparation method and use |
Non-Patent Citations (1)
| Title |
|---|
| 单克隆抗体标志乳胶凝集试验快速检测金黄色葡萄球菌及其耐药性;王崇义等;《中华医院感染学杂志》;20061231;第16卷(第2期);126-128 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103852580A (en) | 2014-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101216491B (en) | Mycobacterium tuberculosis detection liquid phase chip and method for making same | |
| JP5361191B2 (en) | Immunochromatographic detection method and diagnostic kit for multi-drug resistant staphylococci | |
| CN104459125A (en) | Method for rapidly detecting gram negative and positive bacteria | |
| CN102735851B (en) | Mycoplasma hyopneumoniae multi-recombination antigen ELISA (enzyme-linked immunosorbent assay) detection kit | |
| CN103529204B (en) | Detect the latex agglutination kit of milk cow Streptococcusagalactiae antibody | |
| CN105785054A (en) | Chemical-luminescent microfluidic disk for quantitative detection of procalcitonin and using method thereof | |
| CN103499685B (en) | People's urinary system cause of disease bacterium infects the preparation method of the immunofluorescence diagnostic reagent of clinical diagnosis | |
| CN103513032B (en) | Chicken nose tracheae bird bacillus hemagglutination inhibition antibody detection kit, preparation method and application | |
| CN101592660A (en) | Brucellosis indirect enzyme-linked immunosorbent assay milk humoral antibody detection kit | |
| CN101799470A (en) | Brucellosis indirect enzyme-linked immunosorbent assay antibody detection kit | |
| CN103852580B (en) | Staphylococcus aureus identification kit and preparation method thereof | |
| CN102507934B (en) | Time-resolved fluoroimmunoassay (TRFIA) of Klebsiella oxytoca | |
| CN105203769B (en) | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling | |
| AU2017223057A1 (en) | Detection of beta-hemolytic pathogens | |
| US20150079597A1 (en) | Method for capturing and concentrating a microorganism in a biological sample | |
| EP0150567A2 (en) | Process for detecting beta-hemolytic streptococcus antigens | |
| CN106802345B (en) | A kind of helicobacter pylori IgG antibody enzyme-linked immunologic diagnosis kit | |
| CN1318152A (en) | Salmonella antigen formulation and kit for detecting salmonella antibodies | |
| CN103897046A (en) | Receptor assay method and kit for detecting beta-lactam antibiotic residues | |
| RU2625031C1 (en) | Immunoenzymometric test system for animals anaerobic enterotoxemia serum diagnostics and postvaccinal immunity stress control | |
| CN101004419A (en) | Colloid selenium test paper for detecting pulpy kidney, and type of pathogenesis bacteria | |
| CN101493468A (en) | Protein suspension chip method capable of quantitatively determining yersinia pestis | |
| Ali et al. | First case report of post-operative infection due to Francisella tularensis after cardiac surgery | |
| WO2016196323A1 (en) | Serodiagnosis of melioidosis and glanders using polysaccharides | |
| CN202119709U (en) | Limulus Amebocyte Lysate box for examing endotoxin of Gram-negative bacterium infection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |