CN109725152A - A kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit - Google Patents
A kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit Download PDFInfo
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- CN109725152A CN109725152A CN201811528961.4A CN201811528961A CN109725152A CN 109725152 A CN109725152 A CN 109725152A CN 201811528961 A CN201811528961 A CN 201811528961A CN 109725152 A CN109725152 A CN 109725152A
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- antiviral protein
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Abstract
The invention discloses a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kits, antiviral protein MxA antigen standard solution and concentrated cleaning solution including being coated with reaction buffer, magnetic particle solution, the second antiviral protein MxA monoclonal antibody solution, the first luminescent solution, the second luminescent solution, whole blood lysate, various concentration.The present invention has filled up the blank of the particulate chemiluminescence diagnostic reagent production of domestic antiviral protein MxA detection, with easy to operate, high sensitivity, the range of linearity is wide, result is stable, safety is good, the advantages that convenient for automating, in terms of have broad application prospects.
Description
Technical field
The invention belongs to chemiluminescence detection kit technical fields, and in particular to a kind of antiviral protein MxA particulate
Chemiluminescence immune assay detection kit.
Background technique
The molecular weight of Mx albumen is about 70~80kD, and protein structure is at least by N- terminal domains, central domain, C-
3 regions such as terminal domains composition, the N- terminal domains of about 300 amino acid composition are the active regions of GTP enzyme, about 100
The C- terminal domains of a amino acid composition are the effect areas of GTP enzyme, and central domain is made of about 150 amino acid.MxA
Albumen is connected in cytoplasm with sliding surface type endoplasmic reticulum, combines rear configuration with the nucleocapsid of virus and changes, in conjunction with MxA
The nucleocapsid of albumen is deposited on around nucleus in the form of fibrous composite, and the MxA albumen of activation makes virus lose core clothing
Shell, viral nucleic acid is to be released, and the endonuclease being present in cytoplasm quickly is degraded, therefore reaches anti-
Virus function.
When virus infection, body endogenous IFN obviously increases.MxA albumen is able to reflect body by IFN-α/beta induced generation
The case where interior IFN is expressed, facilitates discriminating bacteria sexuality dye and viral infection, secondly detects MxA protein level and is conducive to pair
IFN curative effect is assessed.Currently, the clinical diagnosis technology of antiviral protein MxA specifically includes that enzyme linked immune assay (ELISA),
But because cumbersome, time-consuming, interferes vulnerable to manual operation and extraneous factor, the degree of automation is low, and limits antiviral
The popularization and application of albumen MxA detection, make it that can not be widely used in clinical diagnosis and research work.
Summary of the invention
It is an object of the invention to overcome prior art defect, a kind of antiviral protein MxA particulate chemiluminescence is provided
Immunoassay detection kit.
Technical scheme is as follows:
A kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit, including
It is coated with reaction buffer, is the phosphate buffer of pH7.35~7.44, wherein also containing bovine serum albumin(BSA), junket
Albumen, nonionic surface active agent and preservative；
Magnetic particle solution, magnetic particle therein are coated with the first antiviral protein MxA monoclonal antibody；
Second antiviral protein MxA monoclonal antibody solution, the second antiviral protein MxA labeling of monoclonal antibody therein
There is acridinium ester；
First luminescent solution is the aqueous solution of hydrogen peroxide and nitric acid；
Second luminescent solution is the aqueous solution of sodium hydroxide and nonionic surface active agent；
Whole blood lysate is the aqueous solution of NP40 (Nonidet P40) and DOC (NaTDC)；
The antiviral protein MxA antigen standard solution of various concentration, solvent are above-mentioned coating reaction buffer；
And concentrated cleaning solution, it is the phosphate buffer of nonionic surface active agent and biological preservative；
Its application method includes the following steps:
(4) whole blood to be measured is taken, above-mentioned whole blood lysate is added wherein, simultaneously centrifugal treating is cracked, obtains supernatant；
(5) above-mentioned magnetic particle solution is added in above-mentioned supernatant to be reacted, then with above-mentioned dense after dilution
The washing of contracting cleaning solution, is subsequently added into above-mentioned second antiviral protein MxA monoclonal antibody solution and is reacted；
(6) the above-mentioned concentrated cleaning solution after the resulting material dilution of step (2) is washed, the first luminescent solution is then added
It is reacted with the second luminescent solution, measures luminous value, then pass through the antiviral protein MxA antigen mark of corresponding above-mentioned various concentration
The standard curve of quasi- product solution calculates result.
In a preferred embodiment of the invention, pure containing 0.5% (W/V) ox blood in the coating reaction buffer
Albumen, 0.5% (W/V) casein, 0.05% (W/V) nonionic surface active agent and appropriate preservative.
In a preferred embodiment of the invention, the magnetic particle is that surface is covered with hydrophilic polymer and carboxylic
The magnetic bead of base, partial size are 1.5~3um.
In a preferred embodiment of the invention, in first luminescent solution, the concentration of hydrogen peroxide is 0.5~
3wt%, the concentration of nitric acid are 0.01~0.5M.
In a preferred embodiment of the invention, in second luminescent solution, the concentration of sodium hydroxide is 0.05~
1M, the volumetric concentration of nonionic surface active agent are 0.1~2%.
In a preferred embodiment of the invention, 1% (V/V) NP40 and 0.25% is contained in the whole blood lysate
(W/V)DOC。
It is further preferred that the nonionic surface active agent is Triton X-100.
It is further preferred that the biological preservative is Proclin.
The beneficial effects of the present invention are:
1, antiviral protein MxA microparticle chemiluminescence immunoassay detection kit of the invention has filled up domestic anti-
The blank of the particulate chemiluminescence diagnostic reagent production of virus protein MxA detection, with easy to operate, high sensitivity, linearly
Range is wide, result is stable, safety is good, convenient for automation the advantages that, in terms of have broad application prospects.
2, reaction buffer of the invention provides suitable reaction condition for antigen-antibody reaction.
3, the present invention uses magnetic particle conjugated monoclonal antibodies, since particulate can be suspended in sample solution,
Have and come into full contact with using particulate surface antibody with determinand, shortens the reaction time.
4, the present invention is using acridinium ester as marker, available stabilization, high-intensitive signal.
Detailed description of the invention
Fig. 1 is the antiviral protein MxA antigen standard solution curve in the embodiment of the present invention 1.
Fig. 2 be the embodiment of the present invention 1 in antiviral protein MxA microparticle chemiluminescence immunoassay detection kit and
The Human MxA Protein ELISA of BioVendor company production is respectively to the correlation of 58 parts of infant's pattern detection results
Property analysis result figure.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment combination attached drawing.
Embodiment 1
A kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit, including coating reaction buffer,
Magnetic particle solution, second the first luminescent solution of antiviral protein MxA monoclonal antibody solution, the second luminescent solution, whole blood cracking
The antiviral protein MxA antigen standard solution and concentrated cleaning solution of liquid, various concentration.Its application method includes the following steps:
(1) whole blood to be measured is taken, above-mentioned whole blood lysate is added wherein, simultaneously centrifugal treating is cracked, obtains supernatant；
(2) above-mentioned magnetic particle solution is added in above-mentioned supernatant to be reacted, then with above-mentioned dense after dilution
The washing of contracting cleaning solution, is subsequently added into above-mentioned second antiviral protein MxA monoclonal antibody solution and is reacted；
(3) step (2) resulting material is washed with the above-mentioned concentrated cleaning solution after dilution, is then added first and shines
Liquid and the second luminescent solution are reacted, and luminous value is measured, and then pass through the antiviral protein MxA antigen of corresponding above-mentioned various concentration
The standard curve of standard solution calculates result.
Be coated with reaction buffer, be pH7.4 phosphate buffer, wherein containing 0.5% (W/V) bovine serum albumin(BSA),
0.5% (W/V) casein, 0.05% (W/V) Triton X-100 and appropriate preservative；
Magnetic particle solution, magnetic particle therein are coated with the first antiviral protein MxA monoclonal antibody and (are purchased from
Xiamen Wan Taikairui Bioisystech Co., Ltd, article No. M3353), magnetic particle is that surface is covered with hydrophilic polymer and carboxylic
The magnetic bead of base, partial size are 1.5~3nm；Preparation method are as follows: by the mass ratio of magnetic particle, EDC and NHS be 1: 1: 1, and
The 50mM MES solution that pH is 5.0 is added, makes magnetic particle concentration 4mg/mL, is placed on vertical rotary instrument and activates, it is living
Change 25 DEG C of environment temperature, time 20min；By the magnetic particle and the first antiviral protein MxA monoclonal antibody ratio after activation
Example marks the first antiviral protein MxA monoclonal antibody of 15ug for every milligram of magnetic particle, is placed on vertical rotary instrument
Label, 25 DEG C of reaction environment temperature, time 3h；Magnetic particle after reaction is washed 3 times with washing lotion, is added and contains sweet ammonia
Acid, 0.5% bovine serum albumin(BSA), the phosphate buffer that 0.05%TritonX-100, pH are 7.4, make magnetic particle concentration
For 4mg/mL, it is placed on vertical rotary instrument and terminates, 25 DEG C of reaction environment temperature, time 2h；By the magnetic particle after termination
It is washed 3 times with washing lotion, is added and contains 0.5% (W/V) bovine serum albumin(BSA), 0.5% (W/V) casein, 0.05%T (W/V)
RitonX-100, preservative, the phosphate buffer that pH is 7.4, make magnetic particle concentration 4mg/mL, 2-8 DEG C of preservation is standby
With；
Second antiviral protein MxA monoclonal antibody (is purchased from Xiamen Wan Taikairui Bioisystech Co., Ltd, article No.
M3352) solution, the second antiviral protein MxA labeling of monoclonal antibody therein have acridinium ester；Preparation method are as follows: take wait mark
Remember the second antiviral protein MxA monoclonal antibody 50ug, it is 300 μ L that the phosphate buffer containing NaCl to volume, which is added, then plus
Enter 5 μ L acridinium ester mother liquors, oscillation mixes, and room temperature is protected from light 30min；Phosphorus of the 200 μ L containing NaCl and glycine is added after reaction
Phthalate buffer, reverse 20 mixings, room temperature are protected from light 30min by hand；Product is transferred in bag filter after reaction, is dialysed
Liquid is the 20mM PBS buffer solution that pH is 7.4, and 2-8 DEG C is protected from light dialysis, changes a PBS buffer solution every 2h, is replaced 3 times altogether, with
Remove unlabelled acridinium ester；Marker is taken out, it is pure to ox blood that 10% (W/V) bovine serum albumin(BSA) is added by actual volume
Final concentration of 0.1% (V/V 1: 100) of albumen, is added isometric glycerol, and after being mixed by inversion by hand, -15 DEG C or less are protected from light guarantor
It deposits spare.
First luminescent solution is the aqueous solution of hydrogen peroxide and nitric acid, and the concentration of hydrogen peroxide is 0.5~3wt%, nitric acid
Concentration is 0.01~0.5M；
Second luminescent solution is the aqueous solution of sodium hydroxide and Triton X-100, and the concentration of sodium hydroxide is 0.05~1M,
The volumetric concentration of Triton X-100 is 0.1~2%；
Whole blood lysate is the aqueous solution of NP40 (Nonidet P40) and DOC (NaTDC), contains 1%
(V/V) NP40 and 0.25% (W/V) DOC；
The antiviral protein MxA antigen standard solution of various concentration, solvent are above-mentioned coating reaction buffer；It is made
Preparation Method includes: the high-purity antiviral protein MxA produced using Xiamen Wan Taikairui Bioisystech Co., Ltd independent research
Antigen, using contain 1% bovine serum albumin(BSA), 0.5% (W/V) casein, 0.1% (W/V) TritonX-100, preservative, pH
Phosphate buffer gradient dilution for 7.4 is 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL,
The antiviral protein of the series of 6.25ng/mL, 3.125ng/mL, 1.5625ng/mL, 0.78125ng/mL, 0.390625ng/mL
MxA antigen standard solution, draws standard curve as shown in Figure 1；
Concentrated cleaning solution is the phosphate buffer of Triton X-100 and Proclin；
Its application method includes the following steps:
(1) 10 μ L whole bloods to be measured are taken, the above-mentioned whole blood lysate of 90 μ L is added wherein, cracks and 12000rpm is centrifuged
5min obtains supernatant；
(2) the above-mentioned magnetic particle solution of 50 μ L is added in the 50 above-mentioned supernatants of μ L (with above-mentioned coating reaction buffer
It is diluted to 0.4mg/m1) 15min is reacted under conditions of 37 DEG C, it is then washed 2 times, is connect with the above-mentioned concentrated cleaning solution after dilution
Be added above-mentioned second antiviral protein MxA monoclonal antibody solution, the 37 DEG C of reaction 10min of 50 μ L；
(3) step (2) resulting material is washed 4 times with the above-mentioned concentrated cleaning solution after dilution, is then added first
Luminescent solution and the second luminescent solution are reacted, and luminous value is measured, and then by standard curve as shown in Figure 1, pass through light-emitting appearance
Automatically result is calculated.
The antiviral protein MxA microparticle chemiluminescence immunoassay detection kit and BioVendor of the present embodiment are public
The Human MxA Protein ELISA of production is taken charge of respectively to coming to the whole blood of 58 parts of infants of healthcare hospital for women & children, Xiamen City
The comparison of this testing result, as shown in table 1:
Table 1
] as shown in Fig. 2, the antiviral protein MxA microparticle chemiluminescence immunoassay detection kit of the present embodiment and
The Human MxA Protein ELISA of BioVendor company production is respectively to coming to 58 parts of babies of healthcare hospital for women & children, Xiamen City
The correlation of child's pattern detection result is 0.9061.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e.,
Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Claims (8)
1. a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit, it is characterised in that: including
It is coated with reaction buffer, is the phosphate buffer of pH7.35~7.44, wherein also containing bovine serum albumin(BSA), junket egg
White, nonionic surface active agent and preservative；
Magnetic particle solution, magnetic particle therein are coated with the first antiviral protein MxA monoclonal antibody；
Second antiviral protein MxA monoclonal antibody solution, the second antiviral protein MxA labeling of monoclonal antibody therein have a word used for translation
Pyridine ester；
First luminescent solution is the aqueous solution of hydrogen peroxide and nitric acid；
Second luminescent solution is the aqueous solution of sodium hydroxide and nonionic surface active agent；
Whole blood lysate is the aqueous solution of NP40 and DOC；
The antiviral protein MxA antigen standard solution of various concentration, solvent are above-mentioned coating reaction buffer；
And concentrated cleaning solution, it is the phosphate buffer of nonionic surface active agent and biological preservative；
Its application method includes the following steps:
(1) whole blood to be measured is taken, above-mentioned whole blood lysate is added wherein, simultaneously centrifugal treating is cracked, obtains supernatant；
(2) above-mentioned magnetic particle solution is added in above-mentioned supernatant to be reacted, is then washed with the above-mentioned concentration after dilution
Liquid washing is washed, above-mentioned second antiviral protein MxA monoclonal antibody solution is subsequently added into and is reacted；
(3) the above-mentioned concentrated cleaning solution after step (2) resulting material dilution is washed, the first luminescent solution and the is then added
Two luminescent solutions are reacted, and luminous value is measured, and then pass through the antiviral protein MxA antigen standard of corresponding above-mentioned various concentration
The standard curve of solution calculates result.
2. a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit as described in claim 1, special
Sign is: containing 0.5% (W/V) bovine serum albumin(BSA), 0.5% (W/V) casein, 0.05% in the coating reaction buffer
(W/V) nonionic surface active agent and appropriate preservative.
3. a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit as described in claim 1, special
Sign is: the magnetic particle is the magnetic bead that surface is covered with hydrophilic polymer and carboxyl, and partial size is 1.5~3um.
4. a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit as described in claim 1, special
Sign is: in first luminescent solution, the concentration of hydrogen peroxide is 0.5~3wt%, and the concentration of nitric acid is 0.01~0.5M.
5. a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit as described in claim 1, special
Sign is: in second luminescent solution, the concentration of sodium hydroxide is 0.05~1M, the volumetric concentration of nonionic surface active agent
It is 0.1~2%.
6. a kind of antiviral protein MxA microparticle chemiluminescence immunoassay detection kit as described in claim 1, special
Sign is: containing 1% (V/V) NP40 and 0.25% (W/V) DOC in the whole blood lysate.
7. a kind of antiviral protein MxA particulate chemiluminescence immunoassay point as described in any claim in claim 1 to 6
Analyse detection kit, it is characterised in that: the nonionic surface active agent is Triton X-100.
8. a kind of antiviral protein MxA particulate chemiluminescence immunoassay point as described in any claim in claim 1 to 6
Analyse detection kit, it is characterised in that: the biological preservative is Proclin.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110988363A (en) * | 2019-12-31 | 2020-04-10 | 苏州康和顺医疗技术有限公司 | Detection reagent for antiviral protein MxA and preparation method thereof |
| CN112710840A (en) * | 2020-12-15 | 2021-04-27 | 深圳天辰医疗科技有限公司 | S100 beta protein detection kit and S100 beta protein detection method |
| WO2021147453A1 (en) * | 2020-01-20 | 2021-07-29 | 北京九强生物技术股份有限公司 | Quantitative kit for myxovirus resistance protein 1 |
| CN114805586A (en) * | 2022-04-14 | 2022-07-29 | 天津华科泰生物技术有限公司 | anti-MxA monoclonal antibody composition and application thereof |
| CN116063536A (en) * | 2022-10-24 | 2023-05-05 | 河北科医生物技术有限公司 | Anti-human MxA monoclonal antibody, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110988363A (en) * | 2019-12-31 | 2020-04-10 | 苏州康和顺医疗技术有限公司 | Detection reagent for antiviral protein MxA and preparation method thereof |
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| CN116063536A (en) * | 2022-10-24 | 2023-05-05 | 河北科医生物技术有限公司 | Anti-human MxA monoclonal antibody, preparation method and application thereof |
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Application publication date: 20190507 |