CN110346557A - A kind of detection kit - Google Patents
A kind of detection kit Download PDFInfo
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- CN110346557A CN110346557A CN201910637869.XA CN201910637869A CN110346557A CN 110346557 A CN110346557 A CN 110346557A CN 201910637869 A CN201910637869 A CN 201910637869A CN 110346557 A CN110346557 A CN 110346557A
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- 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
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/543—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
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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
- G01N33/56927—Chlamydia
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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
- G01N33/56933—Mycoplasma
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention provides a kind of detection kit, which is used to detect the determinand in sample to be tested, and it includes two kinds of liquid reagents: reagent 1, includes magnetic particle, and microparticle surfaces are combined with the substance A that can be specifically bound with the determinand in sample；Reagent 2, comprising the non-magnetic microspheres with one or more colors, wherein the microsphere surface of the first color is combined with substance B, and the microsphere surface unbonded material B of remaining color, the substance B can be specifically bound with substance A, identical or different with determinand.When being detected with kit of the invention, the color quantification or sxemiquantitative that can be presented according to reaction solution judge the level of determinand.Solution colour is more intended to the first color, shows that the level of determinand is higher.Detection kit of the invention only can obtain testing result with naked eyes, be not only able to achieve quickly detection but also can guarantee that testing result is intuitive, accurate, can effectively use in especially basic health medical institutions, hygiene medical treatment mechanism.
Description
Technical field
The invention belongs to medical instruments fields, and in particular to a kind of external diagnosis reagent is especially examined for aided disease
Disconnected vitro detection reagent.
Background technique
Autoimmune disease occur with affected tissue autoreactivity lymphocyte and anti-autoantigen autoantibody,
Immunoglobulin is characterized.Systemic loupus erythematosus (Systemic Lupus Erythematosus, SLE) is autoimmune
The Typical Representative of disease often has the generation of Multi-system injuries and a variety of autoantibodies, and most outstanding show is B in pathogenesis
Lymphocyte function is hyperfunction or overactivity and a large amount of polyclonal immunoglobulins of spontaneous generation and itself for autologous tissue
Antibody.Skin is the most common afflicted organ, and skin lesion is the first cause for facilitating patient assessment, and most has prompt meaning
Clinical diagnosis foundation and curative effect judging standard.Different according to the organ of damage, clinical manifestation can have fever, skin lesion (such as face
Butterfly erythema) and the damage such as joint, kidney, liver, heart serous coat and the reduction of whole blood cells.It is more common in young woman, men and women's ratio is
1:6~9, protracted course repeatedly, poor prognosis.Worldwide, European SLE annual newly-increased disease incidence is about 25/,100,000, north
Beauteously area's disease incidence is higher.
Anti-double-chain DNA (dsDNA) antibody is generally acknowledged index relevant to SLE state of an illness activity, is to participate in lupus nephritis hair
A kind of autoantibody of the interpretation of the cause, onset and process of an illness.Anti-hCG action has more than 50 years history as the significant antibody of SLE, in diagnosis and
It is considered as an important serologic marker object in terms of the assessment of Disease activity, positive rate is 40%~90%, specificity
More than 90%, seldom occur in other autoimmune diseases and normal person, general positive rate is below 10%.Anti-dsDNA
Antibody and dsDNA have very strong affinity, and the two is caused a disease in kidney in conjunction with forming immune complex deposit.It is bright at present
Really, Anti-hCG action helps to judge SLE disease early ambulant, can be negative after disease resting stage or treatment improve, therefore anti-
DsDNA antibody can be used as the foundation of monitoring treatment SLE.
With can by detection Anti-hCG action, SLE is similar diagnosing, the various diseases in the following table 1 can also pass through inspection
Special marker is surveyed to be diagnosed.
Table 1
| Number | Disease | Marker |
| 1 | Rheumatoid arthritis RA | Anti- cyclic citrullinated peptide (CCP) antibody |
| 2 | Ankylosing spondylitis AS | Anti- HLA-B27 antibody |
| 3 | Tetanus | Anti-tetanus toxin antibody |
| 4 | Pemphigus | Anti- pemphigus antibody |
| 5 | Influenza A H1N1 | Anti-influenza A H 1 N 1 virus antibody |
| 6 | Eaton agent pneumonia | Anti- mycoplasma pneumoniae (MP) antibody |
| 7 | Chlamydia pneumonia | Anti- chlamydia pneumoniae (CP) antibody |
| 8 | Diphtheria | Diphtheria toxin antibody |
| 9 | Pertussis | Anti-PT antibody |
| 10 | Morbilli | Anti- measles virus antibody |
| 11 | Yochubio | Anti- Orientia Tsutsugamushi antibody |
| 12 | Rabies | Anti-rabies virus antibody |
| 13 | Syphilis | Anti- syphilis helicoid antibody |
| 14 | Dengue fever | Anti-dengue virus antibody |
| 15 | Rubeola | Rubella virus antibody |
| 16 | Toxoplasma | Toxoplasma Antibody |
| 17 | Giant cell | Anti-cytomegalovirus antibody |
| 18 | Herpe simplex | Anti-herpes simplex virus I/II type antibody |
At present detection Anti-hCG action common method mainly have: enzyme-linked immunization, chemoluminescence method, be immunized indirectly it is glimmering
Light method and colloidal gold method.
Enzyme-linked immunization operation is relatively complicated, and key step includes: that sample-adding → incubation → washing → enzyme marker → is incubated
It educates → washing → and adds substrate solution → incubation → plus terminate liquid → reading OD value → judging result, time-consuming is up to 1~2 hour.Separately
Outside, there are also other shortcomings, such as washing step are larger by man's activity for this method；Chromogenic substrate stability is poor, easy to pollute
It is rotten；Terminate liquid contains highly acid substance, Yi Yinqi skin burn etc..
Chemoluminescence method generally carries out on professional and complete Full-automatic chemiluminescence apparatus device, and operating procedure generally includes:
Shine on sample-adding → incubation → washing → plus luminous marker → light-emitting appearance → read result.This method has liberated manpower, and sends out
Stimulative substance, which shines, does not need the catalysis of enzyme, shortens detection time to a certain extent, but Full-automatic chemiluminescence apparatus price is high
It is expensive, and professional is needed to operate, can not effectively it carry out in the basic health medical institutions of fund and crew shortage.
In addition, enzyme-linked immunization and chemoluminescence method before sample-adding, require to be diluted sample processing, colour developing or hair
Light step is to play Cascaded amplification effect by the property of enzyme, thus while sensitivity is higher, but end value is not intuitively
Reflect the real standard of human body endoantigen or antibody.
The key step of indirect immunofluorescence includes sample-adding, and → incubation → develops a film, and → adding secondary antibody → is incubated for, and → developing a film → does
Dry → mounting → microscopy, this method are needed to be observed using fluorescence microscope, be affected by artificial subjective factor, experiment knot
Fruit has certain subjectivity.
Colloidal gold method operation is relatively easy, and detecting step mainly includes sample-adding → incubation → colour developing, time-consuming short, generally 5
~15 minutes or so.However, limitation of this method because of its solid phase reaction system, sensitivity is poor, and vulnerable to interference, accuracy is low.
In addition, although this method is able to reflect the real standard of body, can only do qualitative inspection without being diluted processing to sample
It surveys, a visual evaluation can not be done to the level of antigen/antibody.
Since enzyme-linked immunization, chemoluminescence method, indirect immunofluorescence, colloidal gold method etc. are also each specific mark in table 1
The common detection method of will object, therefore detected with these methods and be also inevitably present disadvantages mentioned above and deficiency.
In view of the above-mentioned condition of the prior art, it is desirable to provide a kind of visual quick detection kit was both able to achieve
It quickly detects, can guarantee that testing result is intuitive, accurate again, can make in especially basic health medical institutions, medical institutions at different levels
With to detect requirements of one's work under satisfaction classification diagnosis and treatment system.
Summary of the invention
It is a primary object of the present invention to overcome shortcoming and defect present in existing detection technique, providing one kind only needs letter
Single instrument can be achieved with the Visual retrieval kit of result quantitatively or semi-quantitatively even without instrument, both be able to achieve fast
Speed detection, the accuracy and specificity that can guarantee testing result again.
To achieve the goals above, the present invention provides a kind of detection kits, and the kit is for detecting sample to be tested
In determinand, it includes two kinds of liquid reagents:
Reagent 1 includes magnetic particle, and magnetic particle surface is combined with can be with the specific binding of the determinand in sample
Substance A；
Reagent 2, comprising the non-magnetic microspheres with multiple color, wherein the non-magnetic microspheres surface of the first color is combined
There is substance B, the non-magnetic microspheres surface unbonded material B of remaining color, substance B can be specifically bound with substance A.
Preferably, further comprise aggregation apparatus, the magnetic particle of dispersion gathers together using magnetism to realize magnetic
The aggregation of property particle.
Preferably, aggregation apparatus includes pedestal, at least one container cup and at least one magnet, and container cup is for accommodating
The mixed liquor of reagent 1, sample to be tested and reagent 2, container cup is detachable to be set on pedestal, and magnet is set on pedestal and magnetic
Iron is set to the lower section of container cup, for magnetic particle to be adsorbed to the bottom of container cup.
Preferably, wherein determinand be Anti-hCG action, cyclic citrullinated peptid, anti-HLA-B27 antibody, resist it is broken
Wind toxin antibody, anti-pemphigus antibody, anti-influenza A H 1 N 1 virus antibody, anti-mycoplasma pneumoniae antibody, anti-chlamydia pneumoniae
Antibody, Anti-PT antibody, anti-measles virus antibody, anti-Orientia Tsutsugamushi antibody, resists mad dog at diphtheria toxin antibody
Antiviral antibody, anti-syphilis helicoid antibody, anti-dengue virus antibody, rubella virus antibody, Toxoplasma Antibody, resist it is big and small
Any one of cellular virus antibody and anti-herpes simplex virus I/II type antibody.
Preferably, wherein substance A is dsDNA antigen, cyclic citrullinated peptide antigen, HLA-B27 antigen, tetanus toxin resist
Original, pemphigus antigen, H1N1virus antigen, mycoplasma pneumoniae antigen, chlamydia pneumoniae antigen, diphtheria toxin are anti-
Original, pertussis toxin antigen, measles virus antigens, Orientia Tsutsugamushi antigen, rabies virus antigen, TP antigen,
Dengue Virus Antigen, rubella virus antigen, toxoplasma antigen, cytomegalovirus antigen and herpes simplex virus I/II type antigen
Any one of, substance B is correspondingly anti-dsDNA monoclonal antibody, anti-cyclic citrullinated peptide monoclonal antibody, anti-HLA-B27 mono-
Clonal antibody, anti-tetanus toxin monoclone antibody, anti-pemphigus monoclonal antibody, anti-influenza A H 1 N 1 virus monoclonal are anti-
Body, anti-mycoplasma pneumoniae monoclonal antibody, anti-monoclonal antibody to Chlamydia pneumoniae, diphtheria toxin monoclone antibody, anti-one hundred days
Cough toxin monoclone antibody, anti-measles virus monoclonal antibody, anti-Orientia Tsutsugamushi monoclonal antibody, rabies poison Dan Ke
Grand antibody, anti-dengue virus monoclonal antibody, rubella virus monoclonal antibody, resists anti-microspironema pallidum monoclonal antibody
Appointing in Anti-toxoplasma Monoclonal Antibody, anti-cytomegalovirus monoclonal antibody and anti-herpes simplex virus I/II type monoclonal antibody
It is a kind of.
Preferably, wherein non-magnetic microspheres have two kinds of colors in three kinds of colors of red, green, blue, the first color
The volume ratio of microballoon and second of color microballoon is 10:1~1:1.
Preferably, wherein the first color is blue, and second of color is red.
Preferably, wherein magnetic particle and substance A by coupling mode in conjunction with, non-magnetic microspheres and substance B pass through coupling
Mode combines.
It preferably, also include colorimetric card, which makes according to the combination of the color of non-magnetic microspheres and ratio.
Preferably, positive quality control reagent and negative quality control reagent are further included, positive quality control reagent is by will be positive
The determinand of concentration, which is added in Matrix buffer, to be formulated；Negative quality control reagent directly uses above-mentioned Matrix buffer, or
Person is formulated by the way that the serum of normal person is added into above-mentioned Matrix buffer, and the serum of normal person refers to HIV, HBV, HCV
Detection is human serum that is negative and not containing determinand.
Preferably, one of sealing adhesive strip, operation instructions or a variety of are further included.
The present invention provides a kind of detection kit, which is used to detect the determinand in sample to be tested, and it includes two
Kind liquid reagent: reagent 1 includes magnetic particle, and microparticle surfaces are combined with can be with the specific binding of the determinand in sample
Substance A；Reagent 2, comprising the non-magnetic microspheres with one or more colors, wherein the microsphere surface of the first color is combined with
Substance B, the microsphere surface unbonded material B of remaining color, the substance B can be specifically bound with substance A, with determinand phase
It is same or different.When being detected with kit of the invention, the color quantification or sxemiquantitative that can be presented according to reaction solution are sentenced
The level of disconnected determinand.Solution colour is more intended to the first color, shows that the level of determinand is higher.Detection examination of the invention
Agent box only can obtain testing result with naked eyes, be not only able to achieve quickly detection but also can guarantee that testing result is intuitive, accurate, can
Effectively to be used in basic health medical institutions.
Compared with prior art, the present invention has the advantage that
1. easy to operate, detection is quickly: can directly be loaded, without carrying out advanced processing or dilution to sample to be tested.Add
Sample process can foreshorten to 3 steps, can foreshorten to 5~10 minutes from result judgement is loaded onto.
2. colour developing is simple, can sxemiquantitative: colour developing only realizes that, without in addition adding the catalysis of other substances, naked eye is with itself
It can determine whether color change.By further comparing with colorimetric card, the classification results of detection, energy can be determined by the number on colorimetric card
Really, intuitively reflect the real standard of body.
3. equipment and personnel requirement are low, the basic medical unit for being especially suitable for financial resources and shortage of manpower is used: of the invention
Kit is without instrument or simple instrument and simple training is only needed to can be operated, and greatly reduces medical institutions' cost, especially has
Work is detected conducive to Grass-roots Hospital.
Detailed description of the invention
Fig. 1~3: the color of testing example 1 of the present invention shows result.
Fig. 4~5: the color of testing example 2 of the present invention shows result.
Fig. 6: the present invention prepares the colorimetric card made in embodiment 1.
Fig. 7: the present invention prepares the colorimetric card made in embodiment 2.
Fig. 8: the structural schematic diagram of one embodiment of aggregation apparatus of the present invention.
Fig. 9: the structural schematic diagram at another visual angle of aggregation apparatus of the present invention.
Figure 10: the structural schematic diagram of one embodiment of container cup assembly of aggregation apparatus of the present invention.
Specific embodiment
In order to be easier to understand above-mentioned purpose of the invention, technical solution and effect, below to specific implementation of the invention
Mode is described in detail.
Compound used in the present invention abbreviation be it is known in the art, concrete meaning is as follows:
BSA (Bovine Serum Albumin): bovine serum albumin(BSA)
MES (2- (N-Morpholino) ethanesulfonic acid): 2- (N- morpholine) ethanesulfonic acid
SDS (Sodium Dodecyl Sulfate): lauryl sodium sulfate
EDC (1-Ethyl-3- (3-Dimethylaminopropyl) Carbodiimide): 1- ethyl -3- (3- dimethyl
Aminopropyl) carbodiimide
NHS (N-Hydroxysuccinimide): N- hydroxysuccinimide
PBS (Phosphate Buffer Saline): phosphate buffered saline solution
Term used in the present invention " substance A " is the bio-ligand for referring to specifically bind with determinand, such as anti-
Original, antibody, polypeptide, protein etc.；" substance B " be refer to substance A specifically bind bio-ligand, can with it is to be measured
Object is identical or different, such as antigen, antibody, polypeptide, protein etc..
It is for the liquid reagent 1 that convenient for explaining and stating technical solution of the present invention, the embodiment part of specification is recorded
For the liquid reagent M of the present invention, liquid reagent 2 is the liquid detection reagents of the present invention.According to a kind of embodiment, the present invention
Kit include liquid reagent 1 and liquid reagent 2 containing different particles.
The magnetic particle recorded in the present invention can contain magnetic particle for magnetic bead and magnetic porous microspheres etc..For just
In understanding and stating, section Example will explain the present invention by taking magnetic bead as an example in description of the invention.That records in the present invention is non-
Magnetic particle can not have magnetic particle for colloid micro ball, fluorescent microsphere, glue bead and colloid porous microsphere etc..For convenient for
Understand and state, section Example will explain the present invention by taking colloid micro ball as an example in description of the invention.
Testing principle: the present invention uses Immune competition method principle.By taking one of reaction sequence as an example specifically, first
First, it is incorporated in substance A on magnetic particle with debita spissitudo, then, sample to be tested is added and is reacted, combination is then added
There is the color non-magnetic microballoon of substance B, makes the substance A on both determinand and substance B competitive binding magnetic particle.To abundant
By the way that the magnetic particle dispersed in liquid phase mixed liquor gathers together after reaction, after assembling magnetic particle, part and magnetic particle
The non-magnetic particles that upper substance A reacts are gathered, and therefore, are dispersed in liquid phase mixed liquor not in conjunction with substance A
Color non-magnetic microballoon content in the solution can change, the changes of contents of color non-magnetic microballoon in the solution can show
Different color, to obtain testing result by color.The contents level of the solution colour and determinand in sample to be tested is close
Cut phase is closed, and color is more intended to be combined with the color of the non-magnetic microspheres of substance B, and the level of determinand is higher, so as to logical
Cross the content that colorimetric determines determinand.
Before illustrating specific testing example, the present invention first illustrates composition and the preparation of reagent 1 and 2.
Reagent 1: reagent 1 includes the magnetic particle that surface is combined with substance A, which is suspended in confining liquid, is led to
It is often stored in spare at 4 DEG C.
It is prepared by reagent 1: the magnetic particle of requirement is added in reaction vessel, washing buffer is added and mixes well, it
After separate magnetic particle, abandon supernatant.After repeating the washing step 2~3 times, magnetic particle is dispersed again to be suspended in washing buffer
In liquid.Immediately activation buffer is prepared, is added it in aforementioned magnetic particle suspension, being vortexed after mixing, it is magnetic micro- to separate
Grain abandons supernatant.It reuses washing buffer to wash and repeat 2~3 times, magnetic particle finally is resuspended with washing buffer, obtains
To activated magnetic particle suspension.
With washing buffer diluent materials A to required concentration, takes and be added in activated magnetic particle suspension in right amount, whirlpool
Rotation adds after mixing and liquid is quenched, and continues to separate magnetic particle after mixing, abandons supernatant, confining liquid, vortex mixing overnight is added.Point
From magnetic particle, supernatant is abandoned, confining liquid is added and is resuspended, repeats the step several times, finally magnetic particle is resuspended in confining liquid,
Obtain reagent 1.
Magnetic particle used in reagent 1 can be any with paramagnetic commercially available product, the modification group of microparticle surfaces
It can be one or more activity functional groups, including but not limited to carboxyl, amino, p-toluenesulfonyl or Streptavidin-
Biotin, preferably carboxylic group, partial size is generally between 500~5000nm, between preferably 1000~5000nm；Content is generally
1~20mg/mL, preferably 2~10mg/mL.
As long as substance A can be specifically bound with determinand, it is not particularly limited, the concentration being coupled on magnetic bead
Range is generally within 600 μ g/mL.For example, substance A is dsDNA antigen when determinand is Anti-hCG action.
Other than Anti-hCG action, the determinand in the present invention can also be that for example anti-cyclic citrullinated peptide (CCP) is anti-
Body, anti-HLA-B27 antibody, anti-tetanus toxin antibody, anti-pemphigus antibody, anti-influenza A H 1 N 1 virus antibody, anti-pneumonia
Mycoplasma (MP) antibody, anti-chlamydia pneumoniae (CP) antibody, diphtheria toxin antibody, Anti-PT antibody, anti-measles
Malicious antibody, anti-rabies virus antibody, anti-syphilis helicoid antibody, anti-dengue virus antibody, resists anti-Orientia Tsutsugamushi antibody
It is arranged in the tables 1 such as Detecting Rubella Virus Antibodies In Human Sera, Toxoplasma Antibody, anti-cytomegalovirus antibody and anti-herpes simplex virus I/II type antibody
Marker out.
Corresponding to these above-mentioned determinands, substance A can be such as cyclic citrullinated peptide (CCP), HLA-B27, broken respectively
Wind toxin element, pemphigus, H1N1virus, mycoplasma pneumoniae (MP), chlamydia pneumoniae (CP), diphtheria toxin, pertussis
It is toxin, measles virus, Orientia Tsutsugamushi, rabies viruses, microspironema pallidum, dengue fever virus, rubella virus, toxoplasma, huge
The antigens such as cell virus and herpes simplex virus I/II type.
Combination of the substance A on magnetic particle surface may include physical bond and chemical coupling, preferably coupling mode.
Confining liquid is common confining liquid in Bioexperiment, may include PBS buffer solution, Tris, Tween-20, sucrose,
BSA, calf serum etc. preferably comprise 0.05~0.5M glycine, 0.9%Nacl, 0.5~2%BSA, 0.02~0.5%
Two or more in Tween-20,0.1~0.5%Proclin 300, pH value is in the range of 6~10.
Washing buffer used in the preparation process of reagent 1 is common buffer in Bioexperiment, may include PBS
Buffer, Tris, Tween-20, MES, SDS etc. preferably comprise the buffer of 20~100nm MES, 0.001~0.1%SDS,
PH is between 6~10.Activation buffer is common buffer in Bioexperiment, may include EDC, NHS, SDS etc., preferably wraps
Buffer containing 100~500nm EDC and 100~500nm NHS, it is that liquid is commonly quenched in Bioexperiment that liquid, which is quenched, can be with
Include 2 mercapto ethanol, glycine, ethanol amine etc., one or both of preferred alcohol amine, 2 mercapto ethanol.
Reagent 2: reagent 2 includes the non-magnetic microspheres with multiple color, wherein the non-magnetic microspheres table of the first color a
Face is combined with substance B, the microsphere surface unbonded material B of remaining color.These non-magnetic microspheres are suspended in confining liquid, usually
It is stored in spare at 4 DEG C.
Prepared by reagent 2: the non-magnetic microspheres of the first color a of requirement being added in test tube, washing buffer is added
It mixes well, separates non-magnetic microspheres later, abandon supernatant.After repeating the washing step 2~3 times, non-magnetic microspheres are hanged again
Float in washing buffer.Immediately activation buffer is prepared, is added it in aforementioned non-magnetic microspheres suspension, is vortexed and mixes
After separate non-magnetic microspheres, abandon supernatant.It reuses washing buffer to wash and repeat 2~3 times, finally with washing buffer weight
Outstanding non-magnetic microspheres, obtain activated non-magnetic microspheres suspension.
With washing buffer diluent materials B to required concentration, takes and is added in activated non-magnetic microspheres suspension in right amount,
It is vortexed to add after mixing and liquid is quenched, continue to separate non-magnetic microspheres after mixing, abandon supernatant, confining liquid is added, vortex mixed
Night.Non-magnetic microspheres are separated, supernatant is abandoned, confining liquid is added and is resuspended, repeats the step several times, is finally resuspended in non-magnetic microspheres
In confining liquid.
, there are two types of when the non-magnetic microspheres of color, the non-magnetic microspheres of second of color b of requirement are added when using tool
Enter in test tube, washing buffer is added and mixes well, separates non-magnetic microspheres later, abandons supernatant.Repeat the washing step 2~3
After secondary, non-magnetic microspheres are resuspended in washing buffer.Immediately activation buffer is prepared, is added it to aforementioned non-magnetic
Property microsphere suspension liquid in, be vortexed after mixing and separate non-magnetic microspheres, abandon supernatant.Washing buffer is reused to wash and repeat 2
~3 times.
With washing buffer dilution BSA to required concentration, takes and be added in activated non-magnetic microspheres suspension in right amount, whirlpool
Rotation adds after mixing and liquid is quenched, and continues to separate non-magnetic microspheres after mixing, abandons supernatant.Confining liquid, vortex mixing overnight is added.
Non-magnetic microspheres are separated, supernatant is abandoned, confining liquid is added and is resuspended, repeats the step 2~3 time, is finally resuspended in non-magnetic microspheres
In confining liquid.
If necessary to use the third color c or more color, carried out referring to the operating procedure of second of color b.
Color combination as needed, by the non-magnetic microspheres of the first color a and second of color b, the third color c
Deng non-magnetic microspheres mix by a certain percentage, obtain reagent 2.
Non-magnetic microspheres used in reagent 2 can be with one or more activity functional groups, including but not limited to
Carboxyl, amino, paratoluenesulfonic acid base or the non-magnetic microballoon of Streptavidin-biotin, it is preferably non-magnetic with carboxyl
Property microballoon, partial size is generally between 500~5000nm, between preferably 500~2000nm；Content is generally 1~20mg/mL,
It is preferred that 2~10mg/mL.
Microballoon can have any commercially available color, and including the various microballoons with fluorescence, color combination can be 2
Kind, 3 kinds or even more kinds of.By adjusting the mixing ratio with different colours microballoon, can be realized determinand different level to
The effect of test sample this presentation different colours.According to color theory, can be mixed by red (Red) green (Green) blue (Blue) three primary colors
Various colors is synthesized, these three primary colours are easy visually to be identified.Furthermore, it is contemplated that serum sample to be measured is usually faint yellow,
In order to offset this background colour, while the color of solution after reacting being made to be easier to distinguish, three primary colours are preferably applied in combination in the present invention
In two kinds, the first color a microballoon: the volume ratio of second of color b microballoon is usually 10:1~1:1, preferably 8:1~4:1.
The present invention more preferably the first color a is blue, and second of color b is red.
As long as the substance B that the first color a microsphere surface combines can be specifically bound with substance A, without special
It limits, such as substance B can be the monoclonal antibody specifically bound with substance A, polyclonal antibody, is also possible to combine strepto-
The antibody of Avidin-Biotin label, concentration range is generally within 600 μ g/mL.When determinand is Anti-hCG action,
The preferred anti-dsDNA monoclonal antibody of substance B.
It is for example anti-cyclic citrullinated peptide (CCP) antibody, anti-HLA-B27 antibody, anti-tetanus poison in determinand of the invention
Plain antibody, anti-pemphigus antibody, anti-influenza A H 1 N 1 virus antibody, anti-mycoplasma pneumoniae (MP) antibody, anti-chlamydia pneumoniae
(CP) antibody, diphtheria toxin antibody, Anti-PT antibody, anti-measles virus antibody, anti-Orientia Tsutsugamushi antibody, anti-
Rabies virus antibodies, anti-dengue virus antibody, rubella virus antibody, Toxoplasma Antibody, resist anti-syphilis helicoid antibody
When the antibody listed in the tables 1 such as CMV antibody, anti-herpes simplex virus I/II type antibody, the preferred each of substance B
Corresponding monoclonal antibody.
Substance B may include physical bond and chemical coupling, preferably coupling mode in the combination of microsphere surface.
It confining liquid used in 2 preparation process of reagent, washing buffer, activation buffer and liquid and reagent 1 is quenched prepares
Each solution in the process is identical.
Kit of the invention may be implemented by the different liquid feedings sequence using reagent 1 and reagent 2 by a kit
The detection for carrying out determinand and the substance A in the corresponding sample to be tested of determinand simultaneously, thus, it is possible to verify testing result mutually,
It is more advantageous to the auxiliary diagnosis of disease.
If you need to detect determinand, the load procedure of kit of the invention can foreshorten to the operation of 3 steps: reagent adding 1 → plus to
Test sample sheet → reagent adding 2.After the completion of operation, by the color of observing response solution, the level of determinand can be determined.Solution face
Color is more intended to the first color a, illustrates that the non-magnetic microspheres in conjunction with substance A are fewer, and the level of determinand is higher.
If you need to detect the substance A in the corresponding sample to be tested of aforementioned determinand, it is only necessary to exchange the liquid feeding of reagent 1 and reagent 2
Sequentially, 3 steps operation: reagent adding 2 → plus sample to be tested → reagent adding 1 is equally foreshortened to.After the completion of operation, as above it is also possible to
By observing the color of solution, the level of determinand is determined.
When being detected using kit of the invention, the amount of the reagent 1 or reagent 2 that are firstly added is usually 10~
100 μ L, preferably 30~80 μ L.Sample to be tested amount and the reagent being firstly added are isometric, and the reagent being added afterwards is sample to be tested
Double volume.
The concrete operations of detection are as follows:
It is taken in a certain amount of addition reaction cup after reagent 1 or reagent 2 are mixed, isometric sample to be tested is added and mixes reaction
A few minutes take double volume that reaction cup is added, are placed on aggregation apparatus pedestal after mixing after mixing reagent 2 or reagent 1
It stands and waits colour developing.By the color of observation solution after colour developing, the level of determinand is judged.
Particularly, if sample to be tested color is deeper, detection effect may be influenced, can eliminate by once washing step should
It influences.Specifically, after reagent 1 or reagent 2 and sample to be tested mix, precipitation and separation supernatant is abandoned supernatant, is resuspended with cleaning solution,
Reagent 2 or reagent 1 are added, is placed on aggregation apparatus pedestal to stand to wait after mixing and develop the color.
According to another embodiment, kit of the invention further includes colorimetric card.
Colorimetric card and its production
The color gamut of colorimetric card depends on the color combination and the ratio of various colors microballoon of selected non-magnetic microspheres.?
In the case that the first color a of non-magnetic microspheres is blue, second of color b be red, the color of colorimetric card is arrived in pink colour
Change between bluish violet.
Production: (need to include critical using 6 known concentration ranges according to the detection reference value of the determinand of clinical detection
Concentration, negative concentration, positive concentration and clinical detection maximum concentration) gradient standard items, according to this 6 standard items in the present invention
Colour developing situation in kit prepares colour atla corresponding to the standard items of each concentration according to 0~10 range.On colorimetric card
0~2 corresponding negative range, 2~4 corresponding critical ranges, 4~10 respectively correspond different degrees of positive range.
If the corresponding number of solution colour can determine whether in sample to be tested in the range of 0~2 without determinand after reaction
Or determinand content does not have clinical meaning.If corresponding number, in the range of 2~4, sample to be tested is doubtful sample
This, needs to test again or sampling and testing again after 2~4 weeks.If corresponding number is in the range of 4~10, to test sample
Determinand content has positive clinical meaning in this.Solution colour is more intended to the first color a, shows that the level of determinand is got over
Height, the number on colorimetric card are bigger.
The gradient magnitude of colorimetric card is example herein, for explaining the producing principle of colorimetric card.
Colorimetric card can also not be used, judges testing result of the invention by instruments such as external color readings instrument.For
Quantitative detection is more accurately obtained as a result, those skilled in the art can utilize the concentration of standard items by well known method
Standard curve is made with chromatic value/absorbance value, is read on standard curve according to the chromatic value of sample to be tested or absorbance value
Sample to be tested concentration is taken, rational judgment is carried out.Instrument can be those skilled in the art understand that various instruments, including it is full-automatic
Or semi-automatic detecting instrument.
For example, the foundation of standard items definite value and standard curve can carry out as follows: according to the inspection of Anti-hCG action in the market
Range is surveyed, formulates serial standards, and according to the " measurement measured in GBT21415-2008 in-vitro diagnosis medical instrument biological sample
The meterological traceability of calibration object and control substance assignment " method carries out definite value of tracing to the source, and the standard items of concentration will be determined in this hair
It is tested, the corresponding color chromaticity values of each standard items is read on color readings instrument, according to coloration on bright kit
The concentration value of value and standard items does standard curve.
According to another embodiment, kit of the invention can also include positive quality control reagent and negative Quality Control examination
Agent.
Positive quality control reagent and preparation
A certain amount of determinand monoclonal antibody, which is added in Matrix buffer, can prepare positive quality control reagent.Matrix
Buffer can be the buffer of the compositions such as common buffer, such as PBS, calf serum, BSA in Bioexperiment, can make
With one such or several be applied in combination.
Negative quality control reagent and preparation
Above-mentioned Matrix buffer can be directly used as negative quality control reagent, can also be by being added just into Matrix buffer
The serum of ordinary person prepares negative quality control reagent.The serum of normal person refers to that HIV, HBV, HCV detection are negative and do not contain
The human serum of determinand.
In order to veritify the validity of kit, when detecting sample to be tested, 2 reaction cups additionally can be separately taken, are used respectively
Positive quality control reagent and negative quality control reagent replace sample to be tested, carry out similar detection operation.By compare positive quality control and
Whether whether color gamut corresponding with colorimetric card is consistent for the solution colour developing of negative Quality Control, effective further to verify kit.
In view of operating and using upper convenience, kit of the invention can further include magnetic separation rack,
Reaction cup, sealing adhesive strip and operation instructions.
As long as magnetic separation rack has separation that is magnetic, facilitating magnetic particle in reagent 1, do not have to other aspects
It is particularly limited to.Preferably, the pedestal for the aggregation apparatus for selecting the embodiment of the present invention to record is as magnetic separation rack.
Reaction cup is for detecting sample to be tested, as long as can be placed on magnetic separation rack, the clean transparent, capacity of cup body is fitted
It closes reaction to need, other aspects is not particularly limited.Preferably, the aggregation apparatus for selecting the embodiment of the present invention to record
Container cup is as reaction cup.
As long as sealing adhesive strip can cover rim of a cup in mixed reactant, and not have an impact to reactive material, do not have
It is particularly limited to, for example, adhesive tape can be film, has gummed paper protective layer, and the gummed paper at adhesive tape both ends is independently of the glue at intermediate position
Paper facilitates hand-held, while avoiding pollution the middle section of adhesive tape.
Operation instructions are used to disclose the relevant information of kit of the present invention, describe its application method, preservation condition, attention
Item etc..
Below for detecting Anti-hCG action, the present invention is further illustrated by specific embodiment.
Prepare embodiment 1
1) preparation of reagent 1
It takes 20 μ L magnetic particle stostes (partial size 500nm, 10%w/v) to be fitted into 2mL test tube, it is slow that 1mL MES washing is added
Fliud flushing, which is sufficiently mixed, to be placed on magnetic separation rack, and supernatant is sucked out after magnetic particle precipitating.Repeat above-mentioned washing step 2~
3 times, after the completion of washing, 1mL MES washing buffer is added, magnetic particle is resuspended.Then 12 μ L of Fresh are added thereto
EDC and 120 μ L NHS activation buffers are vortexed and on roll mixer with 100rpm mixed at room temperature 30 minutes, are placed in magnetism
Supernatant is sucked out after magnetic particle precipitating on separator frame.1mL MES washing buffer is added and cleans magnetic particle, and repeats 2
~3 times.After the completion of washing, 850 μ LMES washing buffers is added, magnetic particle is resuspended.
It is 100 μ g/mL that dsDNA, which is diluted to concentration, with MES washing buffer, and 150 μ L is taken to be added in above-mentioned suspension,
With 100rpm mixed at room temperature 1 hour on roll mixer, then it is added and liquid (purchased from Chinese medicines group) is quenched, it is small to continue mixing 1
When, it is precipitated with magnetic separation rack, supernatant is collected, for analyzing the efficiency of coupling.It is added into the magnetic particle of precipitating by 1mL
The confining liquid that 0.05M glycine, 0.9%Nacl, 0.5%BSA, 0.02%Tween-20,0.1%Proclin 300 are formed, whirlpool
Rotation mixes and mixed at room temperature is stayed overnight on roll mixer.Liquid is discarded supernatant after precipitating on magnetic separation rack within 2nd day.In addition
It states confining liquid to clean magnetic particle 2~3 times, after the completion of washing, 1mL confining liquid is added, magnetic particle is resuspended, obtain reagent 1.It will
It is stored in 4 DEG C it is spare.
2) preparation of reagent 2
It takes 20 μ L blue carboxyl latex microballoon stostes (partial size 500nm, 10%w/v) to be fitted into 2mL test tube, 1mLMES is added
Washing buffer is centrifuged 5 minutes after being sufficiently mixed with 15000rpm, and supernatant is sucked out after microballoon precipitating.Repeat above-mentioned purge step
Rapid 2~3 times, after the completion of washing, 1mL MES washing buffer is added, carboxyl latex microballoon is resuspended.Then it is added thereto fresh
The 12 μ L EDC and 120 μ L NHS activation buffers prepared are vortexed and are divided on roll mixer with 100rpm mixed at room temperature 30
Supernatant is sucked out in clock, centrifugation.1mL MES washing buffer is added and cleans carboxyl latex microballoon, and repeats 2~3 times.Washing is completed
Afterwards, 850 μ L MES washing buffers are added and microballoon is resuspended.
It is 100 μ g/mL that anti-dsDNA monoclonal antibody, which is diluted to concentration, with MES washing buffer, and 150 μ L is taken to be added to
In above-mentioned suspension, with 100rpm mixed at room temperature 1 hour on roll mixer, 50 μ L is then added, liquid is quenched (purchased from traditional Chinese medicines
Group), continue to mix 1 hour, supernatant is collected by centrifugation, for analyzing the efficiency of coupling.Into the microballoon of precipitating be added 1mL by
The confining liquid of 0.05M glycine, 0.9%NaCl, 0.5%BSA, 0.02%Tween-20 and 0.1%Proclin300 composition, whirlpool
Rotation mixes and mixed at room temperature is stayed overnight on roll mixer.2nd day centrifugation microballoon, discards supernatant liquid.The above-mentioned envelope of 1mL is added
It closes liquid to clean microballoon 2~3 times, after the completion of washing, 1mL confining liquid is added, microballoon is resuspended.Be stored in 4 DEG C it is spare.
20 μ L red carboxyl latex microballoons (partial size 500nm, 10%w/v) are taken, it is sufficiently mixed that 1mL MES washing buffer is added
With 15000rpm centrifugation 5 minutes after conjunction, supernatant is sucked out after microballoon precipitating.It repeats above-mentioned washing step 2~3 times, has washed
Microballoon is resuspended in Cheng Hou, the washing buffer that 1mL MES is added.Then the 12 μ L EDC and 120 μ of Fresh are added thereto
LNHS activation buffer is vortexed and on roll mixer with 100rpm mixed at room temperature 30 minutes, and supernatant is sucked out in centrifugation.It is added
1mL MES washing buffer cleans microballoon, and repeats 2~3 times.After the completion of washing, 850 μ LMES washing buffers are added and are resuspended
Microballoon.
It is 100 μ g/mL that BSA, which is diluted to concentration, with MES washing buffer, and 150 μ L is taken to be added in above-mentioned suspension,
With 100rpm mixed at room temperature 1 hour on roll mixer, 50 μ L is then added, liquid (purchased from Chinese medicines group) is quenched, continue mixing 1
Hour, centrifugation discards supernatant liquid.1mL is added by 0.05M glycine, 0.9%Nacl, 0.5%BSA, 0.02%Tween-20,
The confining liquid of 0.1%Proclin300 composition, vortex mixes and mixed at room temperature is stayed overnight on roll mixer.Centrifugation in 2nd day is heavy
Shallow lake microballoon, discards supernatant liquid.The above-mentioned confining liquid of 1mL is added to clean microballoon 2~3 times, after the completion of washing, 1mL confining liquid weight is added
Outstanding microballoon.
The blue carboxyl latex microballoon prepared and red carboxyl latex microsphere suspensions are uniformly mixed according to the volume ratio of 8:1
Close, as reagent 2, be stored in 4 DEG C it is spare.
3) preparation of positive quality control and negative Quality Control
Positive quality control: anti-dsDNA monoclonal antibody is diluted to 500 μ g/mL with the PBS solution containing 5%BSA, mixes and makees
For positive quality control.
Negative Quality Control: using the PBS solution containing 5%BSA as negative Quality Control.
4) production of colorimetric card
It is respectively 6 gradient standard items of 0,30,100,200,400,800IU/mL, root using Anti-hCG action concentration
According to this colour developing situation of 6 standard items in kit of the present invention, according to the standard items of 0,2,4,6,8, the 10 each concentration of production
Corresponding colour atla.
Prepare embodiment 2
Method is recorded such as preparation embodiment 1, the difference is that:
For the magnetic particle partial size used for 5000nm, dosage is 100 μ L, and the dsDNA antigen concentration of coupling is 500 μ g/mL,
For the carboxyl latex microspherulite diameter used for 2000nm, blue microballoon dosage is 100 μ L, the Anti-hCG action of coupling
Concentration is 500 μ g/mL, and red microsphere dosage is 50 μ L.
The volume ratio of blue microballoon and red microsphere is 4:1,
The confining liquid is by 0.5M glycine, 0.9%NaCl, 2%BSA, 0.5%Tween-20,0.5%Proclin 300
Composition, pH 10.
29 serum samples through clinical confirmation result are taken, the kit made from preparation Examples 1 and 2 is examined respectively
It surveys.
Testing example 1
18 samples to be tested that number is 1~18 are taken, detect the Anti-hCG action in each sample to be tested respectively.
18 reaction cups are taken, will prepare after the reagent 1 prepared in embodiment 1 mixes and 50 μ are added into each reaction cup respectively
Then 50 μ L samples to be tested are added in L into each reaction cup respectively, mix 2 minutes.The reagent 2 of preparation is mixed, respectively to each anti-
It answers and 100 μ L is added in cup, sealed with sealing adhesive strip, reaction cup is placed on magnetic separation rack after mixing and stands 2 minutes.Observation
Color in each reaction cup, as a result as shown in Figures 1 to 3, wherein No. 1 is slightly purple pink colour, No. 2 are pink colour, and No. 3 are purple
Color, No. 4 are slightly blue purple, and 5~No. 6 are bluish violet, and 7~No. 8 are pink colour, and No. 9 are slightly purple pink colour, and No. 10 are purple
Color, No. 11 are bluish violet, and No. 12 are deeper bluish violet, and 13~No. 15 are pink colour, and 16~No. 18 are bluish violet.Due to this implementation
It include pink colour, blue two kinds of colors in example, for the writing requitements convenient for distinguishing and meeting patent text, the present invention is with oblique line item generation
Table is red, dot represents blue.
In the present embodiment, anti-dsDNA monoclonal antibody is coupled on blue carboxyl latex microballoon, since blue carboxyl latex is micro-
The blue solution phase of ball dispersion is because participating in specific reaction, and in the blended liquid phase of different samples, the amount of blue carboxyl latex microballoon is not
Together, therefore the present invention with dot represents blue, represents blue by the dot filling pattern of reaction cup liquid phase delta-shaped region
The depth；Without coupling anti-dsDNA monoclonal antibody on red carboxyl latex microballoon, the present invention represents red carboxyl cream with oblique line item
The pink colour liquid phase of glue microballoon dispersion.When test result tends to pink colour, the dot blank map of reaction cup liquid phase delta-shaped region
Case represents the further shallow of blue；When test result tends to blue, the dot filling pattern of reaction cup liquid phase delta-shaped region
Represent the further deep of blue.
During real reaction, blue carboxyl latex microballoon and red carboxyl latex microballoon are dispersed in blended liquid phase
In, blue, red mixing such as figure different proportion directly form intuitive color change.Since patent text is only capable of by black
White dichromatism expression technology scheme, the present invention use lines, dot interpretation technique scheme, it is noted that this explanation cannot limit
The technical solution of present invention essence.
See Fig. 1~3, by comparing with the colorimetric card made in preparation embodiment 1, judges the knot of 1~No. 18 sample to be tested
Fruit is as follows:
1: critical, 2: negative, 3: weakly positive, 4 is positive, and 5: the middle positive, 6: positive
7: negative；8: negative；9: critical；10: weakly positive；11: the middle positive；12: positive
13: negative；14: negative；15: negative；16: positive；17: positive；18: positive
Testing example 2
11 samples to be tested that number is 19~No. 29 are taken, detect the dsDNA antigen in each sample to be tested respectively.
11 reaction cups are taken, will prepare after the reagent 2 prepared in embodiment 2 mixes and 30 μ are added into each reaction cup respectively
Then 30 μ L samples to be tested are added in L into each reaction cup respectively, mix 2 minutes.The reagent 1 of preparation is mixed, respectively to each anti-
It answers and 60 μ L is added in cup, sealed with sealing adhesive strip, reaction cup is placed on magnetic separation rack after mixing and stands 2 minutes.Observation
Color in each reaction cup, as a result as shown in Figures 4 and 5, No. 19 be the micro- purple of pink colour, No. 20 be purple powder, No. 21 be purple, 22
Number for slightly blue purple, 23~No. 24 are blue, and 25~No. 26 are bluish violet, and No. 27 are purple, and No. 28 are the micro- indigo plant of pink colour, 29
Number be pink colour.
See Fig. 4~5, by comparing with the colorimetric card made in preparation embodiment 2, judges the knot of 19~No. 29 samples to be tested
Fruit is as follows:
19: it is negative, 20: it is critical, 21: weakly positive, 22: the middle positive；23: strong positive, 24: strong positive,
25: the middle positive, 26: the middle positive, 27: weak sun, 28: it is negative, 29: negative.
The clinic of above 29 samples to be tested of comparison confirms that result and testing example 1~2 measure as a result, both discoveries
It is consistent.
Comparative example
Respectively with prepared in preparation embodiment 1 kit of the present invention, colloidal gold kit (the biological section of Shenzhen Asia brightness dragon
The production of skill limited liability company) and ELISA kit (production of Bell's bioengineering Co., Ltd, Beijing), to 30 through facing
The Anti-hCG action positive sample to be tested of bed confirmation, the physical examination sample to be tested of 50 normal persons detect.
The following table 2 summarizes and compares to the case where using three kinds of kits to detect in terms of seven.
Table 2
Although illustrating only the preparation process and inspection of the detection kit that determinand is Anti-hCG action in above embodiments
Survey as a result, but based on the reaction principle between antigen-antibody, those skilled in the art are obviously it will be understood that determinand can also be with
It is other antibody except Anti-hCG action, such as the various antibody listed in table 1, and kit of the invention can be predicted
To also have same technical effect for detecting other antibody, reason is: the size of antibody 100~200KDa it
Between, the type for changing antibody can't produce bigger effect its coupling efficiency.Moreover, because microballoon used in detection reagent
Sphere property, substance B can be coupled to any site on microsphere surface.Even at some, determinand molecular weight is larger, deposits
Under the extreme case of steric hindrance, still it can reach the coupling amount of needs by increasing the dosage of microballoon.The case where substance A
Similarly with substance B, it repeats no more.
The present invention provides a kind of liquid detection reagents, which includes the non-magnetic microspheres with multiple color, wherein
The non-magnetic microspheres surface of the first color is combined with substance B, the microsphere surface unbonded material B of remaining color, substance B with to
Survey object is identical or different, and the two competitively combines the bio-ligand that can be specifically bound with determinand.The present invention also provides
The application method of aforesaid liquid reagent, this method include trying aforesaid liquid reagent and another liquid containing magnetic particle
Agent is used cooperatively, and the aggregation of magnetic particle is carried out using a kind of aggregation apparatus.
The aggregation apparatus, including pedestal, at least one container cup and at least one magnet, container cup is for accommodating liquid
The mixed liquor of detection reagent, sample to be tested and liquid reagent M, container cup is detachable to be set on pedestal, and magnet is set to pedestal
Upper and magnet is set to the lower section of container cup, for magnetic particle to be adsorbed to the bottom of container cup.
The present invention by following embodiments explain aggregation apparatus of the present invention specific structure and how with liquid detection reagents
It is used cooperatively.
Provided by the invention a kind of for detecting the liquid reagent of determinand, which includes non-magnetic with multiple color
Property microballoon, wherein the non-magnetic microspheres surface of the first color is combined with substance B, the microsphere surface unbonded material of remaining color
B, substance B can be identical or different with determinand, and the two competitively combines the biology that can be specifically bound with determinand to match
Body.It is used cooperatively by the liquid reagent containing magnetic particle by the liquid reagent and another kind, and is filled using a kind of aggregation
Set, may be constructed a kind of Visual retrieval kit that can only obtain testing result with naked eyes, be both able to achieve quickly detection,
It can guarantee that testing result is intuitive, accurate again, can effectively be used in basic health medical institutions.
Wherein, aggregation apparatus includes pedestal, at least one container cup and at least one magnet, and container cup is for accommodating liquid
The mixed liquor of physical examination test agent, sample to be tested and liquid reagent M, container cup is detachable to be set on pedestal, and magnet, which is set to, to be held
The lower section of device cup, for magnetic bead to be adsorbed to the bottom of container cup.Wherein, the mixed liquor with magnetic bead can be combined with can for surface
With the magnetic bead and sample to be tested liquid of the bio-ligand of determinand specific binding, non-magnetic microspheres surface is combined with substance B, remaining
The microsphere surface unbonded material B of color, substance B is identical or different with determinand, the two competitively combine can with it is to be measured
The bio-ligand of object specific binding.
The aggregation apparatus of the application includes pedestal, container cup and magnet, and pedestal is reusable, and container cup is preferably one
Secondary property uses, and the magnetic bead in container cup can be adsorbed by magnet, and non-magnetic microspheres are not adsorbed by magnet, to realize separation.This Shen
Aggregation apparatus structure please is simple, at low cost, separative efficiency is high.
Fig. 8~Figure 10 is please referred to, Fig. 8 is the structural schematic diagram of one embodiment of aggregation apparatus of the application；Fig. 9 is figure
The structural schematic diagram at another visual angle of aggregation apparatus shown in 1；Figure 10 is that the container cup assembly one of aggregation apparatus shown in Fig. 8 is implemented
The structural schematic diagram of example.In the present embodiment, aggregation apparatus includes pedestal 10, container cup assembly 20 and magnet 30.
Pedestal 10 includes partition 101, side plate 102 and package board 103, and partition 101 is horizontal plate body, partition 101
Lower section is equipped with multiple accommodation grooves 104, and for accommodating magnet 30, multiple accommodation grooves 104 are set along the length direction interval of partition 101
It sets, and each magnet 30 is corresponding with the container cup of container cup assembly 20.Preferably, accommodation groove 104 is square, and magnet 30 is side
Shape magnet 30 is a kind of neodymium iron boron that surface is zinc-plated, grade N35, having a size of 7mm × 10mm × 10mm.Preferably, pedestal 10
Formed for white ABS material, in liquid phase mixed liquor disperse magnetic bead because use aggregation apparatus introduce magnetic field after be gathered in bottom of a cup after,
Convenient for observing the color of remaining liquid phase.
The top of partition 101 is equipped with the guide plate component vertical with partition 101, and guide plate component includes the of parallel interval setting
One guide plate 105 and the second guide plate 106, guide plate component include the first guide plate 105 and the second guide plate 106 of parallel interval setting.
Side plate 102 is set to the side of partition 101, and vertical with partition 101 and guide plate component, and side plate 102 is equipped with limit
Position hole 107.
Package board 103 is parallel with side plate 102, for encapsulating magnet 30.It is after magnet 30 is packed into accommodation groove 104, i.e., available
Package board 103 seals, and magnet 30 is avoided to drop out, and package board 103 can be bonded or be connected to the open face of accommodation groove 104.
For container cup assembly 20 for accommodating the mixing liquid with magnetic bead, container cup assembly 20 is detachable to be set to pedestal 10
On, the container cup of magnet 30 and 20 bottom of container cup assembly corresponds.Wherein, the mixing liquid with magnetic bead can have energy for coupling
The magnetic bead and sample to be tested liquid of enough bio-ligands specifically bound with determinand, the diameter of magnetic bead are preferably 500~5000 to receive
Rice.Container cup assembly 20 enables to the magnetic bead of the bio-ligand specifically bound with determinand to be firmly adsorbed on container cup group
The bottom of part 20.Specifically, container cup assembly 20 is combined by two adjacent container cups, container cup assembly 20 preferably one
It is body formed.Container cup assembly 20 includes the first container cup 201, second container cup 202 and linking the first container cup 201 and second
The connecting block 203 of container cup 202, connecting block 203 be in inverted V type, connecting block 203 respectively with the first container cup 201 and second container
The joining place of cup 202 is equipped with the first clamping groove 204 and the second clamping groove 205, and the first clamping groove 204 and the first guide plate 105 are clamped,
Second clamping groove 205 and the second guide plate 106 are clamped.In the present embodiment, the first container cup 201, second container cup 202 and rank
It connects block 203 to be integrally formed, integrally formed container cup assembly 20 can reduce processing cost, increase structural stability and closing
Property.Connecting block 203 is equipped with gag lever post 206 outward, and limit hole 107 is adapted to gag lever post 206.First clamping groove 204 and first
The cooperation of guide plate 105 and the cooperation of the second clamping groove 205 and the second guide plate 106 enable to container cup assembly 20 in partition
It is limited on 101 length directions, the cooperation of limit hole 107 and gag lever post enables to pedestal 10 and container cup assembly 20 whole
When inversion regardless of from.Certainly, in other embodiments, the mode of clamping or bonding can also be used by 20 He of container cup assembly
10 detachable connection of pedestal.It should be noted that gag lever post 206 may also be arranged on the first container cup 201 and/or second container
On cup 202, limit hole 107 and gag lever post 206 on side plate 102 are corresponded.This three-point fix structure of the present embodiment
Smoothly container cup assembly 20 can be fixed on pedestal 10.Being fixed on the base for container cup bottom relative close, reduces
The distance between magnet and magnetic bead enhance the magnetic force between magnetic bead and magnet, to accelerate the aggregation of magnetic bead, simultaneously also
Increase the stability of container cup and pedestal.Meanwhile one end of magnet corresponds to the bottom of a cup of container cup, such row in the present embodiment
Column can preferably utilize the magnetic force of magnet.
Those skilled in the art it is also readily appreciated that the above location structure equivalent embodiments, which should belong to
In in the protection scope of the application.Gag lever post such as is set in side plate 102, limit hole is set in connecting block 203.
Wherein, in the present embodiment, magnet 30 is by the way of it can be taken off or be placed in, and in other embodiments, can also incite somebody to action
Magnet 30 and pedestal 10 are integrally formed, and increase the stability of structure.
Preferably, the side of container cup assembly 20 is in planar, the bottom of the first container cup 201 and second container cup 202
Portion is in planar to abut with partition 101.Contact of the plane with plane enables to the 20 more stable ground holding of container cup assembly to exist
On partition 101.
In the present embodiment, container cup assembly 20 is transparent PC material, and transparent PC material is conducive to mixed in container cup
It closes liquid to be observed, the capacity of preferably each container cup is 1mL.
Preferably, the processing of rounding angle, the first clamping groove 204 are passed through in the upper surface of the first guide plate 105 and the second guide plate 106
It is handled by rounding angle with the second clamping groove 205.Clamping groove is enabled to preferably to cooperate with guide plate by the processing of rounding angle,
And reduce difficulty of processing.
It is noted that in the present embodiment, set altogether there are three container cup assembly 20, totally six containers, it once can be into
Six groups of tests of row.In other embodiments, multiple groups container cup assembly 20 can be arranged in those skilled in the art according to the actual situation, this
Application does not limit this.
The aggregation apparatus of the present embodiment has the advantage that
1. container cup assembly is in class W type, magnet can will be adsorbed with the bio-ligand that can be specifically bound with determinand
Magnetic bead is firmly adsorbed on container cup bottom, to quickly separate with non-magnetic particles.
2. pedestal is reusable, container cup is disposable, at low cost.
If washing step can be increased 3. sample to be tested color is deeper, when operating in the apparatus, can be washed with rapid dumps
Liquid shortens detection time.
4. whole device is simple in construction, easy to disassemble and assemble, easy to clean and disinfection.
Such as the testing example 1 recorded of the present invention, take 18 samples to be tested that number is 1~18, detect respectively respectively to
Anti-hCG action in test sample sheet.Firstly, it is necessary to 18 reaction cups be taken, after the mixing of reagent 1 prepared in embodiment 1 will be prepared
50 μ L are added into each reaction cup respectively, at this point it is possible to use the container cup group of aggregation apparatus as reaction cup；Then, then divide
50 μ L samples to be tested are not added into each reaction cup, at this point, the container cup using aggregation apparatus is being packed into reagent 1, sample to be tested
Reaction 3~5 minutes is mixed afterwards；Finally, mixing examination after 100 μ L are added into each reaction cup respectively after the reagent 2 of preparation is mixed
Agent 1, sample to be tested, reagent 2 mixed liquor, it is preferable that can be sealed with sealing adhesive strip, reaction cup is placed on magnetism after mixing
3~5 minutes are stood on separator frame, observes the color in each reaction cup, at this point it is possible to which the pedestal using aggregation apparatus replaces magnetic
Property separator frame, the container cup equipped with mixed liquor is placed on pedestal and carries out magnetic aggregation separation.It is, of course, preferable to use this simultaneously
The container cup of invention is as reaction cup.
It should be noted that above-described embodiment is to be combined to form container cup assembly by two adjacent container cups,
In other embodiments, three or four container cups can also be combined according to the actual situation, multiple guide plate groups are set accordingly
Part is engaged by clamping, as long as should belong to the protection scope of the application in the scope of the V-shaped setting of container cup.Below
It briefly describes three container cups and combines the case where forming container cup assembly.
Three adjacent container cups, which combine, forms container cup assembly, and container cup assembly is integrally formed.
Pedestal includes partition, and the lower section of partition is equipped with multiple accommodation grooves, for accommodating magnet, the top of partition be equipped with every
Plate vertical the first guide plate component and the second guide plate component, the first guide plate component include the first guide plate and the of parallel interval setting
Two guide plates, the second guide plate component include the third guide plate and the 4th guide plate of parallel interval setting；Container cup assembly includes the first appearance
Device cup, second container cup, third container cup, the first connecting block and the second connecting block, the first connecting block are connected in the first container cup
Between second container cup, the second connecting block is connected the first connecting block and second between second container cup and third container cup
Connecting block is in inverted V type, and the first connecting block is equipped with the first clamping groove with the joining place of the first container cup and second container cup respectively
With the second clamping groove, the first clamping groove and the first guide plate are clamped, and the second clamping groove and the second guide plate are clamped, the second connecting block difference
It is equipped with third clamping groove and the 4th clamping groove with the joining place of second container cup and third container cup, third clamping groove is led with third
Board connects, and the 4th clamping groove and the 4th guide plate are clamped.
Kit of the invention is while realizing quick detection it can be seen from the above embodiment, and can
The accuracy and specificity for guaranteeing testing result, and do not need detection device and can be achieved with the classification of testing result, be expected to for
The detection technique of medical institutions brings qualitative leap.
It will be appreciated by those skilled in the art that specific embodiment employed in the present invention is only used for explanation and illustration, and
Non- any restriction that the present invention is done.Based on content disclosed in this invention, those of ordinary skill in the art are without creation
Property labour, only by other all technical solutions of the acquisitions such as the simple replacement of conventional technical means, change, deformation, all still exist
Within the scope of protection of present invention.
Claims (10)
1. a kind of detection kit, which is characterized in that the detection kit is used to detect the determinand in sample to be tested, packet
Containing two kinds of liquid reagents:
Reagent 1 includes magnetic particle, and the magnetic particle surface is combined with can be with the specific binding of the determinand in sample
Substance A；
Reagent 2, comprising the non-magnetic microspheres with multiple color, the non-magnetic microspheres surface of the first color is combined with object
Matter B, the non-magnetic microspheres surface unbonded material B of remaining color, the substance B can be with the substance A specificity knots
It closes.
2. detection kit according to claim 1, which is characterized in that it further comprise aggregation apparatus, it will using magnetism
The magnetic particle of dispersion gathers together to realize the aggregation of magnetic particle.
3. detection kit according to claim 2, which is characterized in that the aggregation apparatus include pedestal, at least one
Container cup and at least one magnet, the container cup is for accommodating the reagent 1, the sample to be tested and the reagent 2
Mixed liquor, the container cup it is detachable be set to the pedestal on, the magnet be set to the pedestal on and the magnet
Set on the lower section of the container cup, for the magnetic particle to be adsorbed to the bottom of the container cup.
4. detection kit according to claim 1, which is characterized in that the determinand is Anti-hCG action, anti-ring melon
Propylhomoserin peptide antibody, anti-HLA-B27 antibody, anti-tetanus toxin antibody, anti-pemphigus antibody, anti-influenza A H 1 N 1 virus are anti-
Body, anti-mycoplasma pneumoniae antibody, anti-chlamydia pneumoniae (cp), diphtheria toxin antibody, Anti-PT antibody, anti-measles
Malicious antibody, anti-rabies virus antibody, anti-syphilis helicoid antibody, anti-dengue virus antibody, resists anti-Orientia Tsutsugamushi antibody
Any in Detecting Rubella Virus Antibodies In Human Sera, Toxoplasma Antibody, anti-cytomegalovirus antibody and anti-herpes simplex virus I/II type antibody
Kind.
5. detection kit according to claim 4, which is characterized in that the substance A is dsDNA antigen, cyclic citrulline
Peptide antigen, HLA-B27 antigen, tetanus toxin antigen, pemphigus antigen, H1N1virus antigen, mycoplasma pneumoniae
Antigen, chlamydia pneumoniae antigen, diphtheria toxin antigen, pertussis toxin antigen, measles virus antigens, Orientia Tsutsugamushi are anti-
Original, rabies virus antigen, TP antigen, Dengue Virus Antigen, rubella virus antigen, toxoplasma antigen, giant cell
Any one of viral antigen and herpes simplex virus I/II type antigen, substance B are correspondingly anti-dsDNA monoclonal antibody, resist
Cyclic citrullinated peptide monoclonal antibody, anti-HLA-B27 monoclonal antibody, anti-tetanus toxin monoclone antibody, anti-pemphigus Dan Ke
Grand antibody, anti-influenza A H 1 N 1 virus monoclonal antibody, anti-mycoplasma pneumoniae monoclonal antibody, anti-chlamydia pneumoniae monoclonal
Antibody, diphtheria toxin monoclone antibody, anti-pertussis toxin monoclonal antibody, anti-measles virus monoclonal antibody, anti-tsutsugamushi mite
Sick Orientia monoclonal antibody, anti-rabies monoclonal antibodies, anti-microspironema pallidum monoclonal antibody, anti-dengue virus list
Clonal antibody, rubella virus monoclonal antibody, monoclonal antibody against Toxoplasma gondii, anti-cytomegalovirus monoclonal antibody and anti-list
Any one of pure herpesviral I/II type monoclonal antibody.
6. detection kit according to claim 1, which is characterized in that the non-magnetic microspheres, which have, is selected from three kinds of red, green, blue
The volume ratio of two kinds of colors in color, the first color microballoon and second of color microballoon is 10:1~1:1.
7. detection kit according to claim 6, which is characterized in that the first described color is blue, second of face
Color is red.
8. detection kit according to claim 1, which is characterized in that the magnetic particle and substance A pass through coupling side
Formula combines, the non-magnetic microspheres and substance B by coupling mode in conjunction with.
9. described in any item detection kits according to claim 1~8, which is characterized in that it further include colorimetric card, the colorimetric card
It is made according to the combination of the color of the non-magnetic microspheres and ratio.
10. described in any item detection kits according to claim 1~8, which is characterized in that further comprise positive quality control examination
Agent and negative quality control reagent, the positive quality control reagent are prepared by the way that the determinand of positive concentration to be added in Matrix buffer
It forms；The feminine gender quality control reagent directly uses above-mentioned Matrix buffer, or by being added just into above-mentioned Matrix buffer
The serum of ordinary person is formulated, and the serum of the normal person refers to that HIV, HBV, HCV detection are negative and without containing determinand
Human serum.
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| CN201910637869.XA CN110346557B (en) | 2019-07-15 | 2019-07-15 | Detection kit |
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|---|---|---|---|
| CN201910637869.XA CN110346557B (en) | 2019-07-15 | 2019-07-15 | Detection kit |
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| CN110346557B CN110346557B (en) | 2023-03-31 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111812336A (en) * | 2020-08-10 | 2020-10-23 | 苏州康和顺医疗技术有限公司 | Detection kit for detecting coronavirus antibody and preparation method thereof |
| CN112666357A (en) * | 2020-12-31 | 2021-04-16 | 宝锐生物科技泰州有限公司 | Preparation method of anti-platelet antibody detection signal preparation, anti-platelet antibody detection signal preparation and application |
| CN112698043A (en) * | 2020-12-31 | 2021-04-23 | 宝锐生物科技泰州有限公司 | Platelet antibody detection kit, application and detection method |
| CN117192134A (en) * | 2023-09-14 | 2023-12-08 | 宁波美康盛德生物科技有限公司 | Detection kit and detection method for oxidized low-density lipoprotein |
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| CN117192134A (en) * | 2023-09-14 | 2023-12-08 | 宁波美康盛德生物科技有限公司 | Detection kit and detection method for oxidized low-density lipoprotein |
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|---|---|
| CN110346557B (en) | 2023-03-31 |
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