CN106290864A - A kind of human immunodeficiency virus antigen and TPPA test kit and preparation method - Google Patents
A kind of human immunodeficiency virus antigen and TPPA test kit and preparation method Download PDFInfo
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- CN106290864A CN106290864A CN201610665375.9A CN201610665375A CN106290864A CN 106290864 A CN106290864 A CN 106290864A CN 201610665375 A CN201610665375 A CN 201610665375A CN 106290864 A CN106290864 A CN 106290864A
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- antigen
- antibody
- enzyme
- reagent
- calibration solution
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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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
-
- 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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
Abstract
The invention discloses a kind of Magnetism particulate immuno chemistry luminescence method human immunodeficiency virus antigen and TPPA test kit, it includes Magneto separate reagent, enzyme marking reagent, Yin/Yang reference substance and calibration solution；Magneto separate reagent is that magnetic particle combines envelope antigen/antibody；Enzyme marking reagent is detection antigen/antibody coupling alkali phosphorus enzyme；Wherein envelope antigen and detection antigen are recombinant antigen, coated antibody and detection antibody and are Mus monoclonal antibody；Calibration solution comprises antigen to be checked and antibody；The surface of magnetic particle is hydrophobic surface, and it is modified by sulfonyl, by adsorption conjugated antigen or antibody.The test kit that the present invention provides, had both had the advantage that the antigen active loss of method for coating is little, had had again the advantage that magnetic microparticle chemiluminescence uncomplicated laundering, the range of linearity are wide；Present invention antigen losses is little, that the response time is short and uncomplicated laundering, the range of linearity are wide advantage first is combined, and makes assay can exempt from method with enzyme consistent, and the response time has had and shortens significantly.
Description
Technical field
The present invention relates to a kind of Magnetism particulate immuno chemistry luminescence method human immunodeficiency virus antigen and TPPA test kit and
Its preparation method.
Background technology
HIV (human immunodeficiency virus) measures test kit and has evolved to forth generation at present, i.e. antigen and antibody (HIV Ag/
Ab) detecting simultaneously, mostly be enzyme on the market and exempt from method, the reagent of such as ten thousand calm and peaceful Ying Kexin Chuan Deng producers, this method will be anti-
Former and antibody is fixed in ELISA Plate reacting hole by coated mode, then the sample after sample or process is added reaction
Hole, hatches and washs afterwards, has washed and has added enzyme-labelled antigen afterwards and antibody reacts, has reacted and added afterwards
Substrate solution develops the color, by the depth judgment sample yin and yang attribute of colour developing.
In general, enzyme exempts from method has being coated the impact of its biological activity of its advantage, i.e. antigen and antibody the least, thus right
The combination of antigen to be checked and antibody has good recognition reaction, but shortcoming is also apparent from: 1) owing to antigen and antibody are bags
By onboard, so being difficult to react completely when antigen to be checked with in sample and antibodies, therefore result can be had
Certain impact；2) in clean result, owing to the mode of 96 orifice plates is that washing liquid is for a cleaning being coated plane, Er Qiewei
Pore volume is less, and washing liquid flow velocity is relatively slow, so clean result is poor；3) and enzyme exempts from method due to colorimetric to be carried out, and color is deep
Shallow to determine the method range of linearity narrower, if immue quantitative detection reagent box to be researched and developed, the range of linearity is a defect, i.e. examines
Survey high level sample and HOOK effect easily occurs, then clinically some is accomplished by carrying out beyond the high level sample of the range of linearity
Dilution, rechecks, adds the complexity of detection.
Later stage has developed board-like chemiluminescent method on the basis of enzyme is exempted from, and representing producer has Ke Meidongya, for
From the point of view of the detection range of linearity, chemiluminescence exempts to be greatly improved relative to enzyme, but owing to it remains coated method,
Therefore still having inborn deficiency in terms of the abundant reaction of sample, there is reaction efficiency relatively low, incubation time is longer
Shortcoming.
The appearance of Magnetism particulate immuno chemistry luminescence method overcomes above-mentioned enzyme and exempts from and the defect of board-like chemical method, inherits again simultaneously
The advantage of above two method: 1) antigen or antibody are combined with magnetic particle, such magnetic particle just exists with the reaction of sample
A kind of being similar in homogeneous reaction system is carried out, and can shorten the response time；2) during cleaning, owing to magnetic particle soaks
Bubble is in washing liquid, and can shake mixing at high speed, and therefore clean result to be got well；3) due to magnetic bead specific surface area very
Greatly, the most minimal amount of magnetic bead is combined antigen or amount of antibody are just much larger than antigen or antibodies amount, the reagent of 96 orifice plates
HOOK concentration point will be more much higher with board-like chemiluminescence than enzyme is exempted from.
Magnetic particle is most widely used on the market two categories below: one is the activity having modified carboxyl, amino or hydroxyl etc.
The magnetic particle of group, when coupled antibody, can be combined by the effect of chemical bond and antigen or the amino of antibody or other groups；
Also there is surface to modify anti-FITC antibody or the magnetic particle of streptomycin, thus need antigen or antibody are carried out FITC or biotin
Coupling, enable antigen or antibody to be indirectly combined with magnetic particle.
Although magnetic particle has above-mentioned advantage, but also has deficiency, above two class magnetic beads are required to antigen or anti-when using
Body carries out coupling, and this represents that antigen or antibody have loss of activity in various degree, due to disparity items antibody concordance relatively
Height, conjugation sites is constant region, and activity is substantially unaffected in the variable region (identifying the region of antigen) of antibody, and disparity items
Antigen then specificity is very strong, and active region is not fixed, and the position of coupling is if active region, then the antigen of magnetic bead coupling combines
Arise that during antibody to be checked that mistake causes testing result inaccurate；Moreover, envelope antigen or antibody coupling FITC or life
It is accomplished by after thing element the most additionally adding the step that magnetic bead is hatched, causes the detection time to increase.
Under normal circumstances, the project of magnetic bead coupled antigen is needed mostly to be infectious disease project (part is for from exempting from project), the mankind
Immunodeficiency virus just belongs to this intermediate item, and therefore the accuracy of assay just seems extremely important.A kind of existing side of being coated
The antigen active little advantage of loss of method, have again magnetic microparticle chemiluminescence uncomplicated laundering, range of linearity width advantage test kit undoubtedly
Can very meet the demand in market.
Summary of the invention
The technical problem to be solved in the present invention is the defect overcoming prior art, it is provided that a kind of Magnetism particulate immuno chemistry luminescence method people
Antigen, by using a kind of magnetic particle, is damaged by para-immunity defective virus antigen and TPPA test kit and preparation method thereof first
Lose advantage little, that the response time is short and uncomplicated laundering, the range of linearity are wide to be combined, make assay can exempt from method with enzyme consistent,
Response time has had and has shortened significantly.
In order to solve above-mentioned technical problem, the invention provides following technical scheme:
One Magnetism particulate immuno chemistry luminescence method human immunodeficiency virus antigen of the present invention and TPPA test kit, it includes
Magneto separate reagent, enzyme marking reagent, Yin/Yang reference substance and calibration solution；Described Magneto separate reagent is that magnetic particle combination is coated anti-
Former/antibody；Described enzyme marking reagent is detection antigen/antibody coupling alkali phosphorus enzyme；Wherein envelope antigen and detection antigen are all attached most importance to
Group antigen, coated antibody and detection antibody are Mus monoclonal antibody；Described calibration solution comprises antigen to be checked and antibody；Described magnetic particle
Surface be hydrophobic surface, it is modified by sulfonyl, by adsorption conjugated antigen or antibody.
Another aspect of the present invention discloses a kind of Magnetism particulate immuno chemistry luminescence method human immunodeficiency virus antigen and resists
Body measurement test kit, it comprises the following steps:
S1, the preparation of Magneto separate reagent:
S11, envelope antigen and antibody (4:1) according to a certain percentage are mixed after with magnetic particle (5mg/ml) according to 1:50's
Inventory carries out mixing (0.2mg:10mg), fully mixes, sustained response 18 hours under conditions of room temperature；
S12, the mixture that step S11 prepares is placed in magnetic field and separates, wash with 0.1*PBS, wash 3 altogether
Secondary, add 0.1*PBS1ml every time；
S13, the Polyethylene Glycol 1ml of magnetic particle 1mg/ml after washing is closed, mix 2 hours；
S14, it is carried out according to the operation of step S12；
S15, magnetic particle use Magneto separate reagent buffer be diluted to 0.5mg/ml as working solution；
S2, the preparation of enzyme marking reagent:
Dialyse with 0.1*PBS after S21, general's detection antigen/antibody (4:1) according to a certain percentage mixing, then use 2IT
Activate；
S22, alkali phosphorus enzyme SMCC is activated；
S23, will activation after antigen/antibody mixture and alkali phosphorus enzyme mix, 2-8 ° react 18 hours；
S24, reactant chromatographic column is purified, collects I peak and after II peak mixes, with anti-reagent buffer general
Mixture is diluted to 0.2ug/ml and uses as working solution.
S3, Yin/Yang reference substance and calibration solution preparation process are as follows:
S31, negative controls are this project calibration solution dedicated buffering liquid；
S32, calibration solution are that calibration solution dedicated buffering liquid adds determined antigen and antibody makes final concentration be respectively 0.5ug/ml
And 1ug/ml.
S33, positive reference substance are that calibration solution dedicated buffering liquid adds determined antigen and antibody makes final concentration be respectively
0.8ug/ml and 1.5ug/ml.
The present invention is reached to provide the benefit that:
The test kit that the present invention provides, had both had the advantage that the antigen active loss of method for coating is little, had had again magnetic particle
Learn luminous uncomplicated laundering, range of linearity width advantage；By the present invention in that with a kind of magnetic particle, during antigen losses is little, reaction first
Between the wide advantage of short and uncomplicated laundering, the range of linearity be combined, make assay can exempt from method with enzyme consistent, the response time has
Shorten significantly.
Detailed description of the invention
Hereinafter the preferred embodiments of the present invention are illustrated, it will be appreciated that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
One Magnetism particulate immuno chemistry luminescence method human immunodeficiency virus antigen of the present invention and TPPA test kit, it includes
Magneto separate reagent, enzyme marking reagent, Yin/Yang reference substance and calibration solution；Described Magneto separate reagent is that magnetic particle combination is coated anti-
Former/antibody；Described enzyme marking reagent is detection antigen/antibody coupling alkali phosphorus enzyme；Wherein envelope antigen and detection antigen are all attached most importance to
Group antigen, coated antibody and detection antibody are Mus monoclonal antibody；Described calibration solution comprises antigen to be checked and antibody；Described magnetic particle
Surface be hydrophobic surface, it is modified by sulfonyl, by adsorption conjugated antigen or antibody.
Above-mentioned a kind of Magnetism particulate immuno chemistry luminescence method human immunodeficiency virus antigen and the preparation side of TPPA test kit
Method, it comprises the following steps:
S1, the preparation of Magneto separate reagent:
S11, envelope antigen and antibody (4:1) according to a certain percentage are mixed after with magnetic particle (5mg/ml) according to 1:50's
Inventory carries out mixing (0.2mg:10mg), fully mixes, sustained response 18 hours under conditions of room temperature；
S12, the mixture that step S11 prepares is placed in magnetic field and separates, wash with 0.1*PBS, wash 3 altogether
Secondary, add 0.1*PBS1ml every time；
S13, the Polyethylene Glycol 1ml of magnetic particle 1mg/ml after washing is closed, mix 2 hours；
S14, it is carried out according to the operation of step S12；
S15, magnetic particle use Magneto separate reagent buffer be diluted to 0.5mg/ml as working solution；
S2, the preparation of enzyme marking reagent:
Dialyse with 0.1*PBS after S21, general's detection antigen/antibody (4:1) according to a certain percentage mixing, then use 2IT
Activate；
S22, alkali phosphorus enzyme SMCC is activated；
S23, will activation after antigen/antibody mixture and alkali phosphorus enzyme mix, 2-8 ° react 18 hours；
S24, reactant chromatographic column is purified, collects I peak and after II peak mixes, with anti-reagent buffer general
Mixture is diluted to 0.2ug/ml and uses as working solution.
S3, Yin/Yang reference substance and calibration solution preparation process are as follows:
S31, negative controls are this project calibration solution dedicated buffering liquid；
S32, calibration solution are that calibration solution dedicated buffering liquid adds determined antigen and antibody makes final concentration be respectively 0.5ug/ml
And 1ug/ml.
S33, positive reference substance are that calibration solution dedicated buffering liquid adds determined antigen and antibody makes final concentration be respectively
0.8ug/ml and 1.5ug/ml.
In order to preferably compare, we are simultaneously by envelope antigen/antibody carboxyl magnetic bead, FITC and biotin system
Having carried out coupling, coupling is joined after completing and is respectively prepared 0.5mg/ml, 0.8ug/ml and 0.4ug/ml as working solution.
With above-mentioned 4 kinds of solid-phase reagents (antigen/antibody-sulfonyl magnetic bead, antigen/antibody-carboxyl magnetic bead, antigen/antibody-
FITC, antigen/antibody-biotin) coordinate enzyme marking reagent, Yin/Yang reference substance and the national dish of calibration solution test.
By test result it can be seen that for Detection of antigen, 4 kinds of coupling mode detection results give value consistent with national dish,
But sulfonyl magnetic bead conjugate testing result positive sample value is higher, and negative sample value is lower, hence it is evident that to resisting in sample
Former detection is more sensitive；For antibody test, the reagent test country dish effect of application sulfonyl magnetic bead is best, and same raw material
By testing result after chemical bond coupling, many false negatives and false positive occur, and sensitivity can not be measured, therefore, the present invention
In sulfonyl magnetic bead coupled antigen/antibody best to pattern detection effect.
Finally it is noted that the foregoing is only the preferred embodiments of the present invention, it is not limited to the present invention,
Although being described in detail the present invention with reference to previous embodiment, for a person skilled in the art, it still may be used
So that the technical scheme described in foregoing embodiments to be modified, or wherein portion of techniques feature is carried out equivalent.
All within the spirit and principles in the present invention, any amendment of being made, equivalent, all within the protection of the present invention.
Claims (3)
1. a human immunodeficiency virus antigen and TPPA test kit, it is characterised in that include Magneto separate reagent, enzyme mark
Reagent, Yin/Yang reference substance and calibration solution；Described Magneto separate reagent is that magnetic particle combines envelope antigen/antibody；Described enzyme
Mark reagent is detection antigen/antibody coupling alkali phosphorus enzyme；Wherein envelope antigen and detection antigen are recombinant antigen, coated antibody and
Detection antibody is Mus monoclonal antibody；Described calibration solution comprises antigen to be checked and antibody；The surface of described magnetic particle is hydrophobicity table
Face, it is modified by sulfonyl, by adsorption conjugated antigen or antibody.
2. a human immunodeficiency virus antigen and the preparation method of TPPA test kit, it is characterised in that include following
Step:
S1, the preparation of Magneto separate reagent:
S11, envelope antigen and antibody are mixed after mix with magnetic particle, fully mix, hold under conditions of room temperature
Continuous reaction；
S12, the mixture that step S11 prepares is placed in magnetic field and separates, wash with PBS；
S13, will washing after magnetic particle Polyethylene Glycol close, after mixing, the operation according to step S12 is carried out；
S14, magnetic particle is used Magneto separate reagent buffer dilute as working solution；
S2, the preparation of enzyme marking reagent:
S21, by detection antigen/antibody mix after dialyse with PBS, then activate with 2IT；
S22, alkali phosphorus enzyme SMCC is activated；
S23, will activation after antigen/antibody mixture and alkali phosphorus enzyme mix, 2-8 ° of reaction；
S24, reactant chromatographic column is purified, collects I peak and after II peak mixes, will mix with anti-reagent buffer
Use as working solution after thing dilution；
S3, Yin/Yang reference substance and calibration solution preparation process are as follows:
S31, negative controls are this project calibration solution dedicated buffering liquid；
S32, calibration solution be calibration solution dedicated buffering liquid add determined antigen and antibody make final concentration be respectively 0.5ug/ml and
1ug/ml。
S33, positive reference substance are that calibration solution dedicated buffering liquid adds determined antigen and antibody makes final concentration be respectively 0.8ug/ml
And 1.5ug/ml.
Human immunodeficiency virus antigen the most according to claim 2 and the preparation method of TPPA test kit, it is special
Levying and be, in step S21, described certain proportion is 4:1.
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Cited By (2)
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| CN109696545A (en) * | 2018-12-24 | 2019-04-30 | 郑州安图生物工程股份有限公司 | For reducing the method for matrix effect between heparin tubes different in immunoassays |
| CN110907645A (en) * | 2019-12-17 | 2020-03-24 | 郑州安图生物工程股份有限公司 | Detection kit for human immunodeficiency virus antibody |
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