CN109613273A - A kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit - Google Patents
A kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit Download PDFInfo
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- CN109613273A CN109613273A CN201910064627.6A CN201910064627A CN109613273A CN 109613273 A CN109613273 A CN 109613273A CN 201910064627 A CN201910064627 A CN 201910064627A CN 109613273 A CN109613273 A CN 109613273A
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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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- 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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
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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
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
Abstract
The present invention provides a kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kits, including detection card and fluorescent microsphere working solution；The detection card include for detecting the detection card of HBsAg, the detection card for detecting HBeAg, the detection card for detecting Anti-HBs antibody, the detection card for detecting anti-HBe and detection card for detecting anti-HBc；Time-resolved fluorescence microballoon in fluorescent microsphere working solution containing label corresponding antigens or antibody.In detection process, it is incorporated in the fluorescent microsphere compound of detection zone and quality control region, under the action of exciting light, the fluorescence launched can calculate testing result by fluorescence immunity analyzer acquisition process.The present invention has the advantages that high sensitivity, high specificity, easy to operate and measurement are accurate.
Description
Technical field
The invention belongs to infectious disease protein immunization analysis technical fields, glimmering more particularly, to a kind of five indexes of hepatitis b time resolution
Light immunochromatographic measurement kit and preparation method thereof.
Background technique
Hepatitis type B virus (HBV) is a kind of virus of very little, it belongs in the viral group of thermophilic liver DNA (DNA)
A member.Virion is made of outer membrane and kernel two parts, and complete HBV particle is the spheric granules of diameter 42nm,
Its outer film thickness 7nm, is made of the membrane lipid of protein, referred to as hepatitis B surface antibody (HBsAg), is the mark of virus infection
Will.Hepatitis B surface antibody (HBsAb) is the protection antibody to hepatitis B virus immune.Its appearance indicates to HBV infection
Specific immunity is generated, is usually occurred after HBV infection convalescence or injection hepatitis B vaccine.
The diameter of viral central part is about 28nm, is the core of virus, is that virus is multiple including e antigen (HBeAg)
The mark of system.And core antigen (HBcAg), HBcAg is occupied an important position in HBV infection, he can reflect in serum: Dane
The duplication of HBV in the presence of particle and liver, and the work worked in coordination and complemented each other can be acted with other HBV serologic marker objects
With.Since core antibody (HBcAb) has very strong affinity, can be formed immune multiple promptly in conjunction with the HBcAg in serum
Object is closed, thus is difficult to measure free HBcAg in serum, therefore the blood serum designated object of hepatitis B detection is usually anti-without core
Two double including original.
The early diagnosis and prevention and treatment of hepatitis B are the important measures for avoiding infecting and propagating on a large scale, and otherwise either the state of an illness is disliked
Change or viral transmission, all can bring loss to the health of human body and property status of itself and other people.
Currently, in clinical examination, mainly with enzyme linked immunosorbent assay, colloidal gold immunity chromatography, chemoluminescence method and
The detection method of the different forms such as Magnetism particulate immuno chemistry luminescence method and reaction principle.Existing detection method is all there are some disadvantages
As detection time is long, test operation is complicated.And common colloidal gold immunochromatographimethod is although easy to operate, but will appear the anti-of detection
Answer speed excessively slow, colour developing is uneven, and testing result determines fuzzy, the bad problem of sensitivity.
Summary of the invention
In view of this, the present invention is directed to propose a kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit；It should
Kit energy fast reaction, the kit for detecting Hepatitis B virus of high sensitivity, high specificity, to realize that Hepatitis B virus is examined
It is convenient, quick to provide accurate detection result during survey.
In order to achieve the above objectives, the technical scheme of the present invention is realized as follows:
A kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit, including detection card and fluorescent microsphere work
Liquid；
The detection card includes for detecting the detection card of HBsAg (hepatitis B surface antigen), for detecting HBeAg (hepatitis B e
Antigen) detection card, the detection card for detecting Anti-HBs antibody (hepatitis B surface antibody), for detecting anti-HBe (hepatitis B e antibody)
Detection card and detection card for detecting anti-HBc (hepatitis B core antibody)；
It is described for detecting the detection card of HBsAg, including nitrocellulose filter, thereon respectively coating Anti-HBs antibody antibody and
Streptavidin is in detection zone and quality control region；
The detection card for being used to detect HBeAg, including nitrocellulose filter, are coated with anti-HBe antibody respectively thereon
With Streptavidin in detection zone and quality control region；
The detection card for being used to detect Anti-HBs antibody, including nitrocellulose filter, are coated with HBsAg and chain respectively thereon
Mould Avidin is in detection zone and quality control region；
The detection card for being used to detect anti-HBe, including nitrocellulose filter, are coated with HBeAg and chain respectively thereon
Mould Avidin is in detection zone and quality control region；
The detection card for being used to detect anti-HBc, including nitrocellulose filter, are coated with HBcAg and chain respectively thereon
Mould Avidin is in detection zone and quality control region；
The fluorescent microsphere working solution, Time resolution fluorescent microsphere and following each antibody or antigen including biotin labeling
The mixed liquid storage that the time resolution microballoon of label is respectively formed；The time resolution microballoon of antibody or antigenic mark is to have and detection
The time-resolved fluorescence microballoon of the Anti-HBs antibody antibody label of area's monoclonal antibody different loci, has and detection zone monoclonal antibody different loci
The time-resolved fluorescence microballoon of anti-HBe antibody label, the time-resolved fluorescence microballoon of HBsAg label include HBeAg label
Time-resolved fluorescence microballoon, or the time-resolved fluorescence microballoon comprising HBcAg label.
Preferably, with corresponding antibody, antigen or biotinylated derivative densimeter, the time resolution of Anti-HBs antibody antibody label
The concentration of fluorescent microsphere is 50~200 μ g/mL；The concentration of the time-resolved fluorescence microballoon of anti-HBe antibody label is 50~200 μ
g/mL；The concentration of the time-resolved fluorescence microballoon of HBsAg label is 50~200 μ g/mL；Time resolution comprising HBeAg label
The concentration of fluorescent microsphere is calculated as 50~200 μ g/mL with antigen concentration；Include the dense of the HBcAg time-resolved fluorescence microballoon marked
Degree is calculated as 50~200 μ g/mL with antigen concentration；The concentration of the Time resolution fluorescent microsphere of biotin labeling is with biotinylated derivative
Concentration is calculated as 50~300 μ g/mL；Preferably, the concentration of the time-resolved fluorescence microballoon of Anti-HBs antibody antibody label is 100 μ g/mL；
The concentration of the time-resolved fluorescence microballoon of anti-HBe antibody label is 100 μ g/mL；The time-resolved fluorescence microballoon of HBsAg label
Concentration be 100 μ g/mL；The concentration of time-resolved fluorescence microballoon comprising HBeAg label is calculated as 100 μ g/mL with antigen concentration；
The concentration of time-resolved fluorescence microballoon comprising HBcAg label is calculated as 100 μ g/mL with antigen concentration；The time of biotin labeling
The concentration of resolved fluorescence microballoon is calculated as 200 μ g/mL with biotinylated derivative concentration.
Preferably, the partial size of the time-resolved fluorescence microballoon is 100~300nm；And the lanthanide series wrapped up in microballoon
For europium, active function groups are carboxyl.
Preferably, the preparation method of the fluorescent microsphere working solution, includes the following steps,
A) activation of fluorescent microsphere:
With the pH6.0-8.0 buffer of 5-100mM, quality-volumetric concentration (solid content) of fluorescent microsphere is adjusted to
0.05%-0.2%, preferred concentration are 0.05%；It is sub- that the carbon two that 10-100 μ L concentration is 10-50mg/mL is added into system
The N- hydroxy thiosuccinimide that 10-100 μ L concentration is 10-50mg/mL is added in amine aqueous solution, ultrasound after mixing, ultrasound mixes
Afterwards, room temperature mixes 15-30 minutes；Fluorescent microsphere after centrifugal treating reaction is abandoned after supernatant with the pH5.0-6.5's of 5-100mM
MES buffer is redissolved to original volume；It is stored for future use under 2-8 DEG C of environment.
B) coupling of fluorescent microsphere and antigen/antibody albumen:
The antigen or antibody or biotin of final concentration of 50-300 μ g/mL are added into activated fluorescent microsphere solution,
Preferably, the final concentration of 80-120 μ g/mL of antibody or antigen；The final concentration of 180-220 μ g/mL of biotin；Room temperature mixes 2-4
After hour, sealer is added thereto, room temperature mixes 0.5-2 hours；Centrifugal treating abandons supernatant, and sediment, which is used, includes at least matter
The PBS or MES of the sucrose of concentration 0.1%-8% and the proclin300 of volume fraction 0.01%-0.3% are measured as dilution weight
It hangs to original and marks volume, be configured to the dense liquid storage of fluorescent microsphere of each antibody, antigen or biotin labeling；Preferably, described dilute
It is 8% that release liquid, which be mass concentration containing sucrose, the Procline that seaweed Sugar concentration is 2% and volumetric concentration is 0.05%
The 20mM MES that pH is 6.0；When preparing working solution for diluting dense storage, it is preferred that should be 0.5% comprising the mass concentration of BSA,
The mass concentration of sucrose is 0.3%, the mass concentration of trehalose is 0.2%, the volumetric concentration of Procline is 0.05%,
The PBS buffer solution that the pH that tween-20 volumetric concentration is 0.05% is 7.4.
Preferably, in step b), sealer is one of bovine serum albumin(BSA), casein, glycine or skimmed milk powder
Or several, mass concentration 0.5-1.5%；Preferably, it after antigen or antibody or biotin and fluorescent microsphere are coupled, will be coupled
The fluorescent microsphere of antibody or antigen is configured to the fluorescent microsphere of couple biotin according to the ratio that volume ratio is 1:1-3:1 respectively
Mix dense liquid storage, preferred 1:1；The ratio for mixing dense liquid storage and above-mentioned dilution 1:100-1:500 by volume is configured to
Microballoon working solution；Preferably, 1:200.
Preferably, the detection zone respectively corresponds coated Anti-HBs antibody, anti-HBe, HBsAg, HBeAg, HBcAg concentration
For 0.5-2.0mg/mL；The coated Streptavidin concentration of quality control region is in 0.5-2.0mg/mL.
Five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography reagent as described above is prepared the present invention also provides a kind of
The method of box, the preparation of preparation and fluorescent microsphere working solution including detection card；
It is described detection card preparation include the following steps,
1) preparation of detection zone and quality control region；
By corresponding antibody or antigen and Streptavidin (SA) respectively with the end of 0.5~2mg/mL and 0.2~1mg/mL
It is to uniformly spray in nitrocellulose filter in the buffer of 0.5%-1% trehalose containing mass concentration that even concentration, which is scattered in,
Detection zone and quality control region, and baking and curing under the conditions of 35-50 DEG C.
2) detection card assembling
Blotting paper, the nitrocellulose filter containing detection zone and quality control region, blood filter membrane, sample pad are successively overlapped into (1-2mm)
Be pasted on bottom plate, cut into the strip of 3-4mm, be fitted into getting stuck.HBsAg detection card is respectively prepared, HBeAg detection blocks, is anti-
HBs detection card, anti-HBe detection card, anti-HBc detection card.
Preferably, by corresponding antibody or antigen and Streptavidin respectively with the final concentration of 0.8mg/mL and 0.5mg/mL
Be dispersed in containing mass concentration be 1% trehalose buffer in, uniformly spray in nitrocellulose filter detection zone and
Quality control region, and baking and curing under the conditions of 35~50 DEG C.
In the present invention, detection blocks the blotting paper, nitrocellulose filter, blood filter membrane, sample pad by being successively pasted on bottom plate
It is formed with getting stuck, is coated with detection zone and quality control region on nitrocellulose filter；It is corresponding anti-containing label in fluorescent microsphere working solution
Former or antibody time-resolved fluorescence microballoon.In detection process, it is incorporated in the fluorescent microsphere compound of detection zone and quality control region,
Under the action of exciting light, the fluorescence launched can calculate testing result by fluorescence immunity analyzer acquisition process.
The detection box obtained present invention simultaneously provides kit as described above or preparation method as described above is quantifying
Detect the application in the reagent of the content of Anti-HBs antibody, anti-HBe, HBsAg, HBeAg, anti-HBc.
Compared with the existing technology, kit of the present invention, has the advantage that
(1) kit of the present invention uses time-resolved fluoroimmunoassay and chromatographs, and specificity and sensitivity reach
98% or more.
(2) kit of the present invention operates convenient and quick using the method for lateral chromatography.The time point is had both
The convenient and quick advantage of the high sensitivity and lateral chromatography distinguished.
Detailed description of the invention
Fig. 1 is that Magnetism particulate immuno chemistry luminescence method and this method kit detect HBsAb results relevance；
Fig. 2 is that Magnetism particulate immuno chemistry luminescence method and this method kit detect HBsAg results relevance；
Fig. 3 is that Magnetism particulate immuno chemistry luminescence method and this method kit detect HBeAb results relevance；
Fig. 4 is that Magnetism particulate immuno chemistry luminescence method and this method kit detect HBeAg results relevance；
Fig. 5 is that Magnetism particulate immuno chemistry luminescence method and this method kit detect HBcAb results relevance.
Specific embodiment
In addition to being defined, technical term used in following embodiment has universal with those skilled in the art of the invention
The identical meanings of understanding.Test reagent used in following embodiment is unless otherwise specified conventional biochemical reagent；It is described
Experimental method is unless otherwise specified conventional method.
All number marks, such as pH, temperature, time, concentration, including range, are all approximations.It is to be understood that although
Term " about " is all added before always not describing all number marks explicitly.While it will also be understood that, although always not clear
Narration, reagent described herein is only example, and equivalent is known in the art.
Below with reference to embodiment, the present invention will be described in detail.
One, the preparation of detection card
(1) preparation of detection zone and quality control region；
HBsAg detection card: Anti-HBs antibody antibody and SA is evenly dispersed with the final concentration of 0.8mg/mL and 0.5mg/mL respectively
In the buffer for being 1% trehalose containing mass concentration, the detection zone in nitrocellulose filter and Quality Control are uniformly sprayed respectively
Area, and baking and curing under the conditions of 37 DEG C.
HBeAg detection card: anti-HBe antibody and SA is evenly dispersed with the final concentration of 0.8mg/mL and 0.5mg/mL respectively
In the buffer for being 1% trehalose containing mass concentration, the detection zone in nitrocellulose filter and Quality Control are uniformly sprayed respectively
Area, and baking and curing under the conditions of 37 DEG C.
Detection of Anti-HBs card: HBsAg and SA are dispersed in respectively with the final concentration of 0.8mg/mL and 0.5mg/mL and contained
In the buffer of 1% trehalose of mass concentration, the detection zone and quality control region in nitrocellulose filter are uniformly sprayed respectively, and
Baking and curing under the conditions of 37 DEG C.
Anti- HBe detection card: HBeAg and SA are dispersed in respectively with the final concentration of 0.8mg/mL and 0.5mg/mL and contained
Mass concentration be 1% trehalose buffer in, uniformly spray the detection zone and quality control region in nitrocellulose filter respectively, and
Baking and curing under the conditions of 37 DEG C.
Anti- HBc detection card: HBcAg and SA are dispersed in respectively with the final concentration of 0.8mg/mL and 0.5mg/mL and contained
Mass concentration be 1% trehalose buffer in, uniformly spray the detection zone and quality control region in nitrocellulose filter respectively, and
Baking and curing under the conditions of 37 DEG C.
(2) detection card assembling
Blotting paper, nitrocellulose filter (containing detection zone and quality control region), blood filter membrane, sample pad are successively overlapped into (1-2mm)
Be pasted on bottom plate, cut into the strip of 3-4mm, be fitted into getting stuck.HBsAg detection card is respectively prepared, HBeAg detection blocks, is anti-
HBs detection card, anti-HBe detection card, anti-HBc detection card.
Two, the preparation of fluorescent microsphere labelled antibody (or antigen)
(1) activation of fluorescent microsphere
Taking the fluorescent microsphere that 50 μ L initial concentrations (solid content i.e. quality-volumetric concentration) is 1%, (buying is in Bangs
Laboratories, Inc.FCEU003), be added to 950 μ L pH be 6.0 20mM MES buffer in, mix, 12000g from
Heart 15min.Supernatant is abandoned, above-mentioned MES buffer is resuspended to 1mL.
It is sequentially added into each 50 μ L of EDC and NHS of 10mg/mL, after room temperature mixes 20min, 12000g is centrifuged 15min,
Supernatant is abandoned, is redissolved with above-mentioned MES buffer to 1mL, is placed in 2-8 DEG C and stores for future use.
(2) fluorescent microsphere and antibody (or antigen or biotin) are coupled
A certain amount of antibody (antigen or biotin) is added into above-mentioned solution, makes final concentration of 100 μ of antibody/antigen
G/mL, the final concentration of 200 μ g/mL of biotin.Room temperature mixes 2 hours.
(3) coupling is terminated
Terminate liquid BSA is added into coupling system, making its final concentration (mass concentration) is 1%, and it is small that room temperature continuously mixes 1
Afterwards, 12000g is centrifuged 15min, abandons supernatant, is 8% with mass concentration containing sucrose, and seaweed Sugar concentration is that 2% and volume are dense
The 20mM MES buffer that the pH for the Procline that degree is 0.05% is 6.0, sediment is resuspended to 1mL, it is micro- to obtain fluorescence
The dense liquid storage of ball labelled antibody (antigen or biotin).
(4) preparation of working solution
Mass concentration of the preparation containing BSA is 0.5%, the mass concentration of sucrose is 0.3%, the mass concentration of trehalose is
0.2%, the PBS buffering that the pH that the volumetric concentration of Procline is 0.05%, tween-20 volumetric concentration is 0.05% is 7.4
Liquid.
The dense storage of the fluorescent microsphere that each antigen-antibody marks is respectively with the dense storage of the fluorescent microsphere of biotin labeling by volume
Mixed than 1:1, and with the mass concentration of above-mentioned BSA be 0.5%, the mass concentration of sucrose is 0.3%, the mass concentration of trehalose
The PBS buffering that the pH that volumetric concentration for 0.2%, Procline is 0.05%, tween-20 volumetric concentration is 0.05% is 7.4
Liquid is as dilution, by obtained mark fluorescent containing antigen-antibody microballoon and biotin labeling fluorescent microsphere volume than for 1:1's
Dense liquid storage is mixed, is diluted according to dense liquid storage is mixed with dilution volume ratio for 1:200, microballoon working solution is obtained, by 100 μ
The packing of L/ branch, or be placed in a reagent bottle and store, 100 μ L/ branch is distributed into before to be used for detecting.
The application method of kit of the present invention
All detection components and sample to be examined are placed at room temperature, balance to room temperature.
In use, taking the above-mentioned detection card prepared and fluorescent microsphere working solution, 25 μ L sample to be examined are added to above-mentioned
It prepares and is packed as in the working solution of 100 μ L, 100 μ L of mixing sample is added into detection card well after mixing.After 15min,
It will test card insertion and enter fluorescence immunity analyzer, detected.Read testing result.It detects 120 sample results and has been achieved with
The hepatitis B 5 magnetic microparticle chemiluminescence kits detection of Bo Aosaisi (Tianjin) the Biotechnology Co., Ltd production of registration certificate
As a result it is compared.
Analyze its sensitivity, specificity and test correlation.
Sensitivity=A/ (A+C) × 100%；
Specificity=D/ (B+D) × 100%；
Statistical result is as follows:
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit, it is characterised in that: block including detection and glimmering
Light microballoon working solution；
The detection card includes for detecting the detection card of HBsAg, the detection card for detecting HBeAg, for detecting Anti-HBs antibody
Detection card, the detection card for detecting anti-HBe and detection card for detecting anti-HBc；
The detection card for being used to detect HBsAg, including nitrocellulose filter, are coated with Anti-HBs antibody antibody and strepto- respectively thereon
Avidin is in detection zone and quality control region；
The detection card for being used to detect HBeAg, including nitrocellulose filter, are coated with anti-HBe antibody and chain respectively thereon
Mould Avidin is in detection zone and quality control region；
The detection card for being used to detect Anti-HBs antibody, including nitrocellulose filter are coated with HBsAg and strepto- parent respectively thereon
With element in detection zone and quality control region；
The detection card for being used to detect anti-HBe, including nitrocellulose filter are coated with HBeAg and strepto- parent respectively thereon
With element in detection zone and quality control region；
The detection card for being used to detect anti-HBc, including nitrocellulose filter are coated with HBcAg and strepto- parent respectively thereon
With element in detection zone and quality control region；
The fluorescent microsphere working solution, Time resolution fluorescent microsphere and following each antibody or antigenic mark including biotin labeling
The mixed liquid storage that is respectively formed of time resolution microballoon；The time resolution microballoon of antibody or antigenic mark is to have and detection zone list
The time-resolved fluorescence microballoon of the Anti-HBs antibody antibody label of anti-different loci, has the anti-HBe with detection zone monoclonal antibody different loci
The time-resolved fluorescence microballoon of antibody label, the time-resolved fluorescence microballoon of HBsAg label, the time point comprising HBeAg label
Distinguish fluorescent microsphere, or the time-resolved fluorescence microballoon comprising HBcAg label.
2. five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit according to claim 1, it is characterised in that: with
Corresponding antibody, antigen or biotinylated derivative densimeter, the concentration for the time-resolved fluorescence microballoon that Anti-HBs antibody antibody marks are
50~200 μ g/mL；The concentration of the time-resolved fluorescence microballoon of anti-HBe antibody label is 50~200 μ g/mL；HBsAg label
The concentration of time-resolved fluorescence microballoon is 50~200 μ g/mL；Comprising HBeAg label time-resolved fluorescence microballoon concentration with
Antigen concentration is calculated as 50~200 μ g/mL；The concentration of time-resolved fluorescence microballoon comprising HBcAg label is calculated as with antigen concentration
50~200 μ g/mL；The concentration of the Time resolution fluorescent microsphere of biotin labeling is calculated as 50~300 μ with biotinylated derivative concentration
g/mL；Preferably, the concentration of the time-resolved fluorescence microballoon of Anti-HBs antibody antibody label is 100 μ g/mL；Anti-HBe antibody label
The concentration of time-resolved fluorescence microballoon is 100 μ g/mL；The concentration of the time-resolved fluorescence microballoon of HBsAg label is 100 μ g/mL；
The concentration of time-resolved fluorescence microballoon comprising HBeAg label is calculated as 100 μ g/mL with antigen concentration；Comprising HBcAg label when
Between the concentration of resolved fluorometric microballoon 100 μ g/mL are calculated as with antigen concentration；The concentration of the Time resolution fluorescent microsphere of biotin labeling
200 μ g/mL are calculated as with biotinylated derivative concentration.
3. five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit according to claim 1, it is characterised in that: institute
The partial size for stating time-resolved fluorescence microballoon is 100~300nm；And the lanthanide series wrapped up in microballoon is europium, active function groups are
Carboxyl.
4. five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit according to claim 1, it is characterised in that: institute
The preparation method for the fluorescent microsphere working solution stated, includes the following steps,
A) activation of fluorescent microsphere:
With the pH6.0-8.0 buffer of 5-100mM, quality-volumetric concentration of fluorescent microsphere is adjusted to 0.05%-0.2%, it is excellent
The concentration of choosing is 0.05%；The Carbodiimide solution that 10-100 μ L concentration is 10-50mg/mL is added into system, ultrasound mixes
The N- hydroxy thiosuccinimide that 10-100 μ L concentration is 10-50mg/mL is added afterwards, after ultrasound mixes, room temperature mixes 15-30
Minute；Fluorescent microsphere after centrifugal treating reaction, is redissolved with the MES buffer of the pH5.0-6.5 of 5-100mM to original after abandoning supernatant
Volume；It is stored for future use under 2-8 DEG C of environment.
B) coupling of fluorescent microsphere and antigen/antibody albumen:
The antigen or antibody or biotin of final concentration of 50-300 μ g/mL are added into activated fluorescent microsphere solution, preferably
, the final concentration of 80-120 μ g/mL of antibody or antigen；The final concentration of 180-220 μ g/mL of biotin；Room temperature mixes 2-4 hours
Afterwards, sealer is added thereto, room temperature mixes 0.5-2 hours；Centrifugal treating, abandons supernatant, and sediment is used dense including at least quality
Spend 0.1%-8% sucrose and volume fraction 0.01%-0.3% proclin300 PBS or MES as dilution be resuspended to
Original label volume, is configured to the dense liquid storage of fluorescent microsphere of each antibody, antigen or biotin labeling；Preferably, the dilution
It is 8% for mass concentration containing sucrose, the pH for the Procline that seaweed Sugar concentration is 2% and volumetric concentration is 0.05% is
6.0 20mM MES；When preparing working solution for diluting dense storage, it is preferred that should be 0.5%, sucrose comprising the mass concentration of BSA
Mass concentration be 0.3%, the mass concentration of trehalose is 0.2%, the volumetric concentration of Procline is 0.05%, tween-20
The PBS buffer solution that the pH that volumetric concentration is 0.05% is 7.4.
5. five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit according to claim 4, it is characterised in that: step
It is rapid b) in, sealer is one or more of bovine serum albumin(BSA), casein, glycine or skimmed milk powder, mass concentration
For 0.5-1.5%；Preferably, after antigen or antibody or biotin and fluorescent microsphere are coupled, by coupled antibody or the fluorescence of antigen
Microballoon is configured to mix dense liquid storage with the fluorescent microsphere of couple biotin according to the ratio that volume ratio is 1:1-3:1 respectively, preferably
1:1；The ratio for mixing dense liquid storage and above-mentioned dilution 1:100-1:500 by volume is configured to fluorescent microsphere working solution；
Preferably, 1:200.
6. five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit according to claim 1, which is characterized in that institute
The detection zone stated respectively corresponds coated Anti-HBs antibody, anti-HBe, HBsAg, HBeAg, HBcAg concentration is 0.5-2.0mg/mL；Matter
The coated Streptavidin concentration in area is controlled in 0.5-2.0mg/mL.
7. a kind of prepare five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography reagent as described in any one of claims 1 to 6
The method of box, it is characterised in that: the preparation of preparation and fluorescent microsphere working solution including detection card；
It is described detection card preparation include the following steps,
1) preparation of detection zone and quality control region；
Corresponding antibody or antigen and Streptavidin is uniform with the final concentration of 0.5~2mg/mL and 0.2~1mg/mL respectively
Be scattered in is to uniformly spray the detection zone in nitrocellulose filter in the buffer of 0.5%-1% trehalose containing mass concentration
And quality control region, and baking and curing under the conditions of 35-50 DEG C.
2) detection card assembling
Blotting paper, the nitrocellulose filter containing detection zone and quality control region, blood filter membrane, sample pad are successively overlapped into the viscous of (1-2mm)
It is affixed on bottom plate, the strip of 3-4mm is cut into, is fitted into getting stuck.HBsAg detection card, HBeAg detection card, Anti-HBs antibody is respectively prepared
Detection card, anti-HBe detection card, anti-HBc detection card.
8. preparation method as claimed in claim 7, it is characterised in that:
Corresponding antibody or antigen and Streptavidin are dispersed in respectively with the final concentration of 0.8mg/mL and 0.5mg/mL
Be containing mass concentration uniformly spray the detection zone and quality control region in nitrocellulose filter in the buffer of 1% trehalose, and
Baking and curing under the conditions of 35-50 DEG C.
9. the detection box that kit as described in any one of claims 1 to 6 or 7~8 described in any item preparation methods obtain
Application in the reagent of the content of quantitative detection Anti-HBs antibody, anti-HBe, HBsAg, HBeAg, anti-HBc.
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Application publication date: 20190412 |