CN106596960A - Immunological fast detection kit for virus infection and detection method - Google Patents
Immunological fast detection kit for virus infection and detection method Download PDFInfo
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- CN106596960A CN106596960A CN201710011778.6A CN201710011778A CN106596960A CN 106596960 A CN106596960 A CN 106596960A CN 201710011778 A CN201710011778 A CN 201710011778A CN 106596960 A CN106596960 A CN 106596960A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
- G01N2333/04—Varicella-zoster virus
- G01N2333/045—Cytomegalovirus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
- G01N2333/12—Mumps virus; Measles virus
Abstract
The invention discloses an immunological fast detection kit for virus infection and a detection method, the immunological fast detection kit comprises two glass slides; 300 mu L of a hydration solution; 20 mu L of positive virus serum and 20 mu L of negative virus serum; wherein the hydration solution comprises 10g / L of bovine serum albumin, 100mL / L of glycerol and 250mg / L of a LTHYRESFUS-Anti-VMoAb conjugate PBS liquid. The immunological fast detection kit uses the immunological detection technology to diagnose the virus infection rapidly, and the detection result is high in specificity, strong in sensitivity and easy to judge by the naked eye, and the immunological fast detection kit is suitable for rapid detection in the field.
Description
Technical field
The invention belongs to technical field of bioengineering, specifically, is related to a kind of immunology quick detection of virus infection
Kit and detection method.
Background technology
Virus be a class individuality it is small, without intact cell configuration, containing single nucleic acid (DNA or RNA) type, must be in living cells
Endoparasitism and the noncellular microorganism for replicating, it is mainly made up of nucleic acid and protein coat.Viral infectious diseases are one
Class seriously threatens the disease of human health.The detection method of virus is varied, and the detection method of virus infection has：Viral divides
From culture technique, immunological method, molecular biology method, flow cytometry.The molecular Biological Detection technology commonly used at present
Mainly there are hybridization, polymerase chain reaction, biochip technology etc..Conventional immunology detection technology mainly has
EUSA, immunofluorescence technique, radioimmunoassay technique, chemiluminescence immunoassay technology, electro-chemistry immunity point
Analysis technology, surface plasma body resonant vibration immuno analytical method, protein-chip etc..These viral detection methods have his own strong points,
But have some limitations, such as sensitiveness is high, cumbersome, cycle length, it is costly the problems such as, in the urgent need to exploitation is ground
Make a kind of quick, easy, sensitive, accurate, economic viral infection detection technology.Clinically set up a kind of really effectively and fast
The diagnostic method of speed is increasingly paid close attention to by medical circle.
The content of the invention
In view of this, the present invention is directed to above-mentioned problem, there is provided a kind of immunology quick detection reagent of virus infection
Box and detection method, the present invention is using the virus infection of immunology detection technology quick diagnosis, and testing result specificity is high, sensitive
Property strong, naked eyes easily judge, be suitable for field quick detection.
In order to solve above-mentioned technical problem, the invention discloses a kind of immunology quick detection kit of virus infection,
The composition of the kit is as follows：
Wherein, rehydration solution bovine serum albumin(BSA) containing 10g/L, 100mL/L glycerine, 250mg/L THYRESFUS-Anti-V
The PBS liquid of MoAb conjugates.
Further, THYRESFUS-Anti-V MoAb conjugates are prepared by the following method and obtain：
(1) 25% glutaraldehyde is diluted to into 5% with 0.05mol/LPH9.5 carbonate buffer solutions；
(2) 1mg/mL viral monoclonal antibodies 0.5mL, plus the glutaraldehydes of 0.5ml 5% are taken, 37 DEG C of water-baths combine 2h；
(3) after adding 220.0g/L NaCl 0.1ml fully to mix, 4 DEG C of 10min precoolings are put；
(4) add cold absolute ethyl alcohol 2.4ml, overturn mixing, the 10min of 1000r/min centrifugations immediately；
(5) supernatant is abandoned, precipitation is washed once with cold 80% ethanol, centrifuge tube is inverted, drained；
(6) 0.05mo l/LPH9.5 carbonate buffer solution 0.5ml dissolution precipitation things are added；
(7) 150 μ gTHYRESFUS are dissolved in into 0.5ml 0.15mol/L NaCl, then it is molten with hydroformylation viral monoclonal antibodies
Liquid mixes；
(8) 1.0mol/LpH9.5 new bios sensing substance that show color is added, pH value is adjusted to 9.0-9.5；
(9) in 4 DEG C, 24h is combined under electromagnetic agitation；
(10) 0.1ml 0.2mol/L lysines are added, to close the aldehyde radical of residual, terminating reaction；
(11) 4 DEG C of placement 2h；
(12) reactant is eluted by Sephadex G-200 gel columns with PBS, collects the first eluting peak；Or will be anti-
Thing is answered to load bag filter, to 0.01mol/L pH7.2PBS, 4 DEG C of dialysed overnights are simultaneously collected；Concentration collection liquid is
THYRESFUS-Anti-V MoAb conjugates, THYRESFUS-Anti-V MoAb are adjusted to concentration 250mg/L with PBS, and are added
Enter final concentration 10g/L bovine serum albumin(BSA)s, 100mL/L glycerine obtains rehydration solution.
Further, positive-virus serum is the blood of the homemade viral double crush syndrome kit test positive of Jing
Clearly.
Further, negative viral serum is the blood that the homemade viral double crush syndrome kits of Jing are detected as feminine gender
Clearly.
Further, the preparation of homemade viral double crush syndrome kit and detection method are as follows：20 μ g/mL are sick
Malicious monoclonal antibody is coated with ELISA reaction plates, 37 DEG C of insulation 2h, board-washing 3 times, plus the PBS confining liquids containing 1% bovine serum albumin(BSA)
37 DEG C, 3h, PBS washes 2 times；The μ L of serum to be checked 100 of 1: 100 dilution are added, 37 DEG C are incubated 30 points, board-washing 3 times；Add potency be
1: the 1000 μ L of viral rabbit antibody 100,37 DEG C are incubated 30 points, board-washing 3 times；Add the HRP mark goat-anti rabbits that potency is 1: 1000
The μ L of antibody 100,37 DEG C are incubated 30 points, board-washing 3 times；TMB-H2O2Colour developing, range estimation and instrument judged result.
Further, virus is measles virus or cytomegalovirus.
The invention also discloses a kind of immunology quick detection method of virus infection, using exempting from for above-mentioned virus infection
Epidemiology quick detection kit, comprises the following steps：
1) react：Take on the slide of a cleaning, be respectively labeled as Positive control wells, instrument connection and negative control hole,
The rehydration solution of 12 μ L is added with liquid-transfering gun per hole, 3 μ L positive-virus serum are added toward Positive control wells, and toward instrument connection
3 μ L test serums, negative control hole are added to add 3 μ L negative viral serum, it is rapid to mix；Slide is put in a wet box, will
Wet box is put into 37 DEG C of incubators and reacts 30 minutes；
2) result judges：After 37 DEG C are reacted 30 minutes, wet box is taken out at once；With a blank sheet of paper or white foam box as the back of the body
Scape, by the change of reaction solution color, that is, judges testing result；If solution is colourless, illustrate that virus infection is negative；Such as
Fruit is redness, then illustrate that virus infection is positive.
Compared with prior art, the present invention can be obtained including following technique effect：
The one-step virus detection kit of the present invention combines coloration method using unique antigen-antibody, entirely detects
Journey is only needed 0.5 hour, substantially reduces detection time；Detection need to only use the serum of 3 μ L；Whole detection process only needs one 37
DEG C insulating box.
Certainly, the arbitrary product for implementing the present invention it is not absolutely required to while reaching all the above technique effect.
Description of the drawings
Accompanying drawing described herein is used for providing a further understanding of the present invention, constitutes the part of the present invention, this
Bright schematic description and description does not constitute inappropriate limitation of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is the result compares figure that measles virus different materials are detected in the embodiment of the present invention 2；Wherein, No. 1 hole is measles
Frozen tissue culture infection cell lysate (1: 1000), No. 2 holes are that (Jing self-control measles virus is double for measles virus positive human serum
Anti- sandwich ELISA lcits test positive), No. 3 holes are that (Jing makes measles virus double antibodies sandwich by oneself to measles virus negative serum
ELISA kit is detected as feminine gender), No. 4 holes are rubella virus positive human serum (Jing rubella virus double crush syndrome reagents
Box test positive), No. 5 hole herpes simplex virus type 1 positive serum (the homemade Herpes Simplex I double crush syndrome examinations of Jing
Agent box test positive), No. 6 holes are cytomegalovirus positive human serum (the homemade cytomegalovirus double crush syndrome examinations of Jing
Agent box test positive), No. 7 holes are normal human serum；
Fig. 2 is the result compares figure of the detection cytomegalovirus different materials in the embodiment of the present invention 3；Wherein, No. 1 hole is
Split for cytomegalovirus Tissue Culture Infectious cell in cytomegalovirus Tissue Culture Infectious cell pyrolysis liquid (1: 1000), No. 2 holes
Solution liquid (1: 8000), No. 3 holes are cytomegalovirus negative serum (the homemade cytomegalovirus double crush syndrome kits of Jing
It is detected as feminine gender), No. 4 holes are measles virus positive human serum (the homemade measles virus double crush syndrome kit detections of Jing
For the positive), No. 5 hole herpe simplex II type virus-positive serum (the homemade herpe simplex II type double crush syndrome kits of Jing
Test positive), No. 6 holes are (the homemade hepatitis B double crush syndrome kit detections of Jing of hepatitis B virus positive human serum
For the positive), No. 7 holes are normal human serum, and No. 8 holes are cytomegalovirus positive serum (the dual anti-folder of the homemade cytomegaloviruses of Jing
Heart ELISA kit test positive).
Specific embodiment
Embodiments of the present invention are described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section come solve technical problem and reach technology effect realize that process can fully understand and implement according to this.
The present invention thinking be：First with glutaraldehyde, this conventional homotype bi-functional cross-linking agent, biological response is shown
The amino of the phenyl phthalazines (THYRESFUS) of color substance 4- amino-(4 '-chlorine) -5 and viral monoclonal antibodies is picked up with five carbon bridgings
Come, obtain conjugate THYRESFUS-Anti-V MoAb, now the THYRESFUS in conjugate does not manifest color, but works as
It is sick that THYRESFUS-Anti-V MoAb viruses corresponding with sample combine to form compound THYRESFUS-Anti-V MoAb-
After poison, because biological response effect THYRESFUS manifests redness；If being presented colourless without corresponding virus reactant liquor in sample.
The immunology quick detection kit and detection method of the virus infection of embodiment 1：
The immunology quick detection kit composition of virus infection：
Rehydration solution：Bovine serum albumin(BSA) containing 10g/L (BSA), 100mL/L glycerine, 250mg/L THYRESFUS-Anti-
The PBS liquid of V MoAb conjugates.
The immunology quick detection method of virus infection：
1. react：Take on the slide of a cleaning, be respectively labeled as Positive control wells, instrument connection and negative control hole,
The rehydration solution of 12 μ L is added with liquid-transfering gun per hole, 3 μ L positive-virus serum are added toward Positive control wells, and toward instrument connection
3 μ L test serums, negative control hole are added to add 3 μ L negative viral serum, it is rapid to mix.Slide is put in a wet box, will
Wet box is put into 37 DEG C of incubators and reacts 30 minutes.
2. result judges：After 37 DEG C are reacted 30 minutes, wet box is taken out at once.With a blank sheet of paper or white foam box as the back of the body
Scape, by the change of reaction solution color, you can judge testing result.If solution is colourless, illustrate that virus infection is negative；
If redness, then illustrate that virus infection is positive.
Embodiment 2 illustrates the preparation and application of the kit by taking measles virus as an example
(1) preparation of THYRESFUS-Anti-V MoAb conjugates
(1) 25% glutaraldehyde is diluted to into 5% with 0.05mol/LPH9.5 carbonate buffer solutions (CBS)；
Number 161) (2) 1mg/mL measles virus monoclonal antibodies (Abnov Products, clone 0.5mL, plus 0.5ml are taken
5% glutaraldehyde, 37 DEG C of water-baths combine 2h；
(3) after adding 220.0g/L NaCl 0.1ml fully to mix, 4 DEG C of 10min precoolings are put；
(4) add cold absolute ethyl alcohol 2.4ml, overturn mixing, the 10min of 1000r/min centrifugations immediately；
(5) supernatant is abandoned, precipitation is washed once with cold 80% ethanol, centrifuge tube is inverted, drained；
(6) 0.05mol/LPH9.5CBS 0.5ml dissolution precipitation things are added；
(7) 150 μ gTHYRESFUS are dissolved in into 0.5ml 0.15mol/L NaCl, then it is anti-with hydroformylation measles virus monoclonal
Liquid solution mixes；
(8) 1.0mol/LpH9.5 CBS are added, adjusts pH value to 9.0-9.5；
(9) in 4 DEG C, 24h is combined under electromagnetic agitation；
(10) 0.1ml 0.2mol/L lysines are added, to close the aldehyde radical of residual, terminating reaction；
(11) 4 DEG C of placement 2h；
(12) reactant is eluted by Sephadex G-200 gel columns with PBS, collects the first eluting peak；Or will be anti-
Thing is answered to load bag filter, to 0.01mol/L pH7.2 PBS, 4 DEG C of dialysed overnights are simultaneously collected；Concentration collection liquid is
THYRESFUS-Anti-V MoAb conjugates, THYRESFUS-Anti-V MoAb are adjusted to concentration 250mg/L with PBS, and are added
Enter final concentration 10g/L bovine serum albumin(BSA)s (BSA), 100mL/L glycerine obtains measles virus detection reagent rehydration solution.
(2) measles virus kit detects serum to be checked：
Experimental procedure is as follows：
1. react：Take on the slide of a cleaning, be respectively labeled as Positive control wells, instrument connection and negative control hole,
The rehydration solution of 12 μ L is added with liquid-transfering gun per hole, 3 μ L positive-virus serum are added toward Positive control wells, and toward instrument connection
3 μ L test serums, negative control hole are added to add 3 μ L negative viral serum, it is rapid to mix.Slide is put in a wet box, will
Wet box is put into 37 DEG C of incubators and reacts 30 minutes.Wherein positive-virus serum is the homemade measles virus double crush syndromes of Jing
Kit test positive；Negative viral serum is detected as feminine gender for the homemade measles virus double crush syndrome kits of Jing.
Wherein, the preparation of homemade measles virus double crush syndrome kit and detection method are as follows：
20 μ g/mL measles virus monoclonal antibodies coating ELISA reaction plates, 37 DEG C of insulation 2h, board-washing 3 times, plus containing 1% N
37 DEG C of sero-abluminous PBS confining liquids, 3h, PBS washes 2 times；Add the μ L of serum to be checked 100 of 1: 100 dilution, 37 DEG C of insulations 30
Point, board-washing 3 times；Add measles virus rabbit antibody (potency：1: 1000) 100 μ L, 37 DEG C are incubated 30 points, board-washing 3 times；Add HRP
Mark goat anti-rabbit antibody (potency：1: 1000) 100 μ L, 37 DEG C are incubated 30 points, board-washing 3 times；TMB-H2O2Colour developing, range estimation and instrument
Judged result.
2. result judges：After 37 DEG C are reacted 30 minutes, wet box is taken out at once.With a blank sheet of paper or white foam box as the back of the body
Scape, by the change of reaction solution color, you can judge testing result.If solution is colourless, illustrate that virus infection is negative；
If redness, then illustrate that virus infection is positive.
The measles virus Suspect serum sample 7 collected is chosen, with present invention detection, there are 5 positives；This 5 positive use
The detection of measles virus double crush syndrome method, all positives；Another 2 negative Suspect serum measles virus double antibodies sandwiches
ELISA method detection, is as feminine gender；Two kinds of detection method coincidence rates 100%.
(3) specific assay
Choosing various human virus such as rubella virus, cytomegalovirus, herpes simplex virus I is used for measles virus kit
Specificity verification, as a result shows the measles virus kit of foundation to viral infection of measles positive serum and measles virus tissue training
Foster infection cell lysate (1: 1000) tests positive reaction, to rubella virus, cytomegalovirus, herpes simplex virus I etc.
Various human viral infection's positive serums and Tissue Culture Infectious cell pyrolysis liquid are detected as feminine gender, without no cross reaction (figure
1)。
(4) stability test
By with the 4 DEG C of preservations of batch measles virus kit set up, at interval of one month, as a result the positive antigen of detection showed,
4 DEG C of the measles virus kit of foundation is preserved 6 months still has higher recall rate (table 1), compares with the same antigen that compares, the
Reaction color about 50% when 6 months.It can be seen that, the measles virus stabilization of kit of foundation is good.
The testing result that 1 10 parts of measles virus positive serums of table are detected with the kit of 4 DEG C of preservation different times
| Standing time (moon) | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| Coincidence rate (%) | 100 | 100 | 100 | 90 | 80 | 80 | 70 |
(5) sensitivity tests
Take measles virus positive serum (the homemade measles virus double crush syndrome kit detections of Jing are positive) a
Make doubling dilution, respectively with the measles virus kit and homemade measles virus double crush syndrome kit detection set up,
The measles virus kit of foundation is with the visible obvious red minimum detectability to detect of directly naked eyes, and homemade measles virus is double
Using A values x+2s as positive threshold value as the minimum detectability that detects, experiment shows anti-sandwich ELISA lcits, the measles of foundation
Virus agent box and the highest dilution of homemade measles virus double crush syndrome kit detection are all 27, illustrate both
Sensitiveness quite (table 2).
Table 2 different dilution factor measles virus positive serum this kit and the testing results of ELISA method
| Dilution factor | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| ELISA method | + | + | + | + | + | + | + | - | - |
| This kit | + | + | + | + | + | + | + | - | - |
+：Detection is positive；-：Detection is negative.
Embodiment 3 illustrates the preparation and application of the kit by taking cytomegalovirus as an example
(1) preparation of THYRESFUS-Anti-V MoAb conjugates
(1) 25% glutaraldehyde is diluted to into 5% with 0.05mol/LPH9.5 carbonate buffer solutions (CBS)；
(2) (Shanghai Gu Duo bio tech ltd Products are compiled to take 1mg/mL cytomegalovirus monoclonal antibodies
Number：GD-H3000) 0.5mL, plus the glutaraldehydes of 0.5ml 5%, 37 DEG C of water-baths combine 2h；
(3) after adding 220.0g/L NaCl 0.1ml fully to mix, 4 DEG C of 10min precoolings are put；
(4) add cold absolute ethyl alcohol 2.4ml, overturn mixing, the 10min of 1000r/min centrifugations immediately；
(5) supernatant is abandoned, precipitation is washed once with cold 80% ethanol, centrifuge tube is inverted, drained；
(6) 0.05mol/LPH9.5CBS 0.5ml dissolution precipitation things are added；
(7) 150 μ gTHYRESFUS are dissolved in into 0.5ml 0.15mol/L NaCl, then it is anti-with hydroformylation measles virus monoclonal
Liquid solution mixes；
(8) 1.0mol/LpH9.5 CBS are added, adjusts pH value to 9.0-9.5；
(9) in 4 DEG C, 24h is combined under electromagnetic agitation；
(10) 0.1ml 0.2mol/L lysines are added, to close the aldehyde radical of residual, terminating reaction；
(11) 4 DEG C of placement 2h；
(12) reactant is eluted by Sephadex G-200 gel columns with PBS, collects the first eluting peak；Or will be anti-
Thing is answered to load bag filter, to 0.01mol/L pH7.2 PBS, 4 DEG C of dialysed overnights are simultaneously collected；Concentration collection liquid, as
THYRESFUS-Anti-V MoAb conjugates, with PBS THYRESFUS-Anti-V MoAb are adjusted to concentration 250mg/L, and add
Enter final concentration 10g/L bovine serum albumin(BSA)s (BSA), 100mL/L glycerine obtains cytomegalovirus virus detection reagent aquation molten
Liquid.
(2) cytomegalovirus virus kit detects serum to be checked
Experimental procedure is as follows：
1. react：Take on the slide of a cleaning, be respectively labeled as Positive control wells, instrument connection and negative control hole,
The rehydration solution of 12 μ L is added with liquid-transfering gun per hole, 3 μ L positive-virus serum are added toward Positive control wells, and toward instrument connection
3 μ L test serums, negative control hole are added to add 3 μ L negative viral serum, it is rapid to mix.Slide is put in a wet box, will
Wet box is put into 37 DEG C of incubators and reacts 30 minutes.Wherein positive-virus serum is the homemade cytomegalovirus double antibodies sandwiches of Jing
ELISA kit test positive；Wherein negative viral serum is the homemade cytomegalovirus double crush syndrome kits of Jing
It is detected as feminine gender.
The preparation and detection of the homemade cytomegalovirus double crush syndrome kits of Jing：
20 μ g/mL cytomegaloviruses monoclonal antibodies coating ELISA reaction plates, 37 DEG C of insulation 2h, board-washing 3 times, plus containing 1%
37 DEG C of the PBS confining liquids of bovine serum albumin(BSA), 3h, PBS washes 2 times；Add the μ L of serum to be checked 100 of 1: 100 dilution, 37 DEG C of insulations
30 points, board-washing 3 times；Add cytomegalovirus rabbit antibody (potency：1: 1000) 100 μ L, 37 DEG C are incubated 30 points, board-washing 3 times；Add
HRP marks goat anti-rabbit antibody (potency：1: 1000) 100 μ L, 37 DEG C are incubated 30 points, board-washing 3 times；TMB-H2O2Colour developing, range estimation and instrument
Device judged result.
2. result judges：After 37 DEG C are reacted 30 minutes, wet box is taken out at once.With a blank sheet of paper or white foam box as the back of the body
Scape, by the change of reaction solution color, you can judge testing result.If solution is colourless, cytomegalovirus disease is illustrated
Viral disease poison infection is negative；If redness, then illustrate that the poison infection of cytomegalovirus virosis is positive.
With 195 clinical human serum samples that the cytomegalovirus virus kit detection set up is collected, giant cell is detected
Virus-virus antigen positive 8；This 195 clinical human bloods are detected with homemade cytomegalovirus double crush syndrome kit
Clear sample, positive coincidence rate 100% illustrates the cytomegalovirus kit reliable in quality set up.
(3) cytomegalovirus virus kit specific assay
Choosing various human virus such as measles virus, herpes simplex virus type II, hepatitis B is used for measles virus reagent
Box specificity verification, as a result show foundation cytomegalovirus virus kit to cytomegalovirus virus infection positive serum and
Cytomegalovirus Tissue Culture Infectious cell pyrolysis liquid (1: 1000) tests positive is reacted, to measles virus, herpe simplex disease
Various human viral infection's positive serums such as malicious II types, hepatitis B and Tissue Culture Infectious cell pyrolysis liquid are detected as feminine gender,
Without no cross reaction (Fig. 2).
(4) sensitivity tests
Take (the homemade cytomegalovirus virus double crush syndrome kit detections of Jing of cytomegalovirus positive serum
It is positive) portion makees doubling dilution, respectively with the cytomegalovirus kit and homemade cytomegalovirus double antibodies sandwich set up
ELISA kit detects that the cytomegalovirus kit of foundation is with the visible obvious red minimum detection to detect of directly naked eyes
Limit, minimum detection of the homemade cytomegalovirus double crush syndrome kit using A values x+2s as positive threshold value as detection
Limit, experiment shows：What the cytomegalovirus kit of foundation and homemade cytomegalovirus double crush syndrome kit were detected
Highest dilution is all 210, illustrate both sensitiveness quite (table 3).
Table 3 different dilution factor cytomegalovirus positive serum this kit and the testing results of ELISA method
| Dilution factor | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 210 | 21 1 |
| ELISA method | + | + | + | + | + | + | + | + | + | + | - |
| This kit | + | + | + | + | + | + | + | + | + | + | - |
+：Detection is positive；-：Detection is negative.
Described above illustrates and describes some preferred embodiments of invention, but as previously mentioned, it should be understood that invention is not
Form disclosed herein is confined to, the exclusion to other embodiment is not to be taken as, and can be used for various other combinations, modification
And environment, and can be carried out by the technology or knowledge of above-mentioned teaching or association area in invention contemplated scope described herein
Change.And change that those skilled in the art are carried out and change be without departing from the spirit and scope of invention, then all should be in the appended power of invention
In the protection domain that profit is required.
Claims (7)
1. the immunology quick detection kit that a kind of virus infects, it is characterised in that the composition of the kit is as follows：
Wherein, rehydration solution bovine serum albumin(BSA) containing 10g/L, 100mL/L glycerine, 250mg/L THYRESFUS-Anti-V
The PBS liquid of MoAb conjugates.
2. the immunology quick detection kit that virus according to claim 1 infects, it is characterised in that described
THYRESFUS-Anti-V MoAb conjugates are prepared by the following method and obtain：
(1) 25% glutaraldehyde is diluted to into 5% with 0.05mol/LPH9.5 carbonate buffer solutions；
(2) 1mg/mL viral monoclonal antibodies 0.5mL, plus the glutaraldehydes of 0.5ml 5% are taken, 37 DEG C of water-baths combine 2h；
(3) after adding 220.0g/L NaCl 0.1ml fully to mix, 4 DEG C of 10min precoolings are put；
(4) add cold absolute ethyl alcohol 2.4ml, overturn mixing, the 10min of 1000r/min centrifugations immediately；
(5) supernatant is abandoned, precipitation is washed once with cold 80% ethanol, centrifuge tube is inverted, drained；
(6) 0.05mol/LPH9.5 carbonate buffer solution 0.5ml dissolution precipitation things are added；
(7) 150 μ g THYRESFUS are dissolved in into 0.5ml 0.15mol/L NaCl, then it is mixed with hydroformylation viral monoclonal antibodies solution
Close；
(8) 1.0mol/LpH9.5 new bios sensing substance that show color is added, pH value is adjusted to 9.0-9.5；
(9) in 4 DEG C, 24h is combined under electromagnetic agitation；
(10) 0.1ml 0.2mol/L lysines are added, to close the aldehyde radical of residual, terminating reaction；
(11) 4 DEG C of placement 2h；
(12) reactant is eluted by Sephadex G-200 gel columns with PBS, collects the first eluting peak；Or by reactant
Load bag filter, to 0.01mol/L pH7.2PBS, 4 DEG C of dialysed overnights are simultaneously collected；Concentration collection liquid is THYRESFUS-
Anti-V MoAb conjugates, THYRESFUS-Anti-V MoAb are adjusted to concentration 250mg/L with PBS, and add final concentration
10g/L bovine serum albumin(BSA)s, 100mL/L glycerine, obtain rehydration solution.
3. the immunology quick detection kit that virus according to claim 1 infects, it is characterised in that the positive disease
Malicious serum is the serum of the homemade viral double crush syndrome kit test positive of Jing.
4. the immunology quick detection kit that virus according to claim 1 infects, it is characterised in that the negative disease
Malicious serum is the serum that the homemade viral double crush syndrome kits of Jing are detected as feminine gender.
5. according to claim 3 or 4 virus infection immunology quick detection kit, it is characterised in that it is described from
The preparation of the viral double crush syndrome kit of system and detection method are as follows：20 μ g/mL viral monoclonal antibodies are coated with
ELISA reaction plates, 37 DEG C of insulation 2h, board-washing 3 times, plus 37 DEG C of the PBS confining liquids containing 1% bovine serum albumin(BSA), 3h, PBS washes 2
It is secondary；The μ L of serum to be checked 100 of 1: 100 dilution are added, 37 DEG C are incubated 30 points, board-washing 3 times；Add the viral rabbit that potency is 1: 1000
The μ L of antibody 100,37 DEG C are incubated 30 points, board-washing 3 times；Add potency for 1: 1000 HRP mark the μ L of goat anti-rabbit antibody 100,37 DEG C
30 points of insulation, board-washing 3 times；TMB-H2O2 develops the color, and estimates and instrument judged result.
6. the immunology quick detection kit of virus infection according to claim 5, it is characterised in that the virus is
Measles virus or cytomegalovirus.
7. the immunology quick detection method that a kind of virus infects, it is characterised in that using arbitrary right in claim 1 to 6
The immunology quick detection kit of described virus infection is required, is comprised the following steps：
1) react：Take on the slide of a cleaning, be respectively labeled as Positive control wells, instrument connection and negative control hole, in every hole
The rehydration solution of 12 μ L is added with liquid-transfering gun, 3 μ L positive-virus serum are added toward Positive control wells, and added toward instrument connection
3 μ L test serums, negative control hole adds 3 μ L negative viral serum, rapid to mix；Slide is put in a wet box, by wet box
It is put into 37 DEG C of incubators to react 30 minutes；
2) result judges：After 37 DEG C are reacted 30 minutes, wet box is taken out at once；With a blank sheet of paper or white foam box as background, lead to
The change of reaction solution color is crossed, that is, judges testing result；If solution is colourless, illustrate that virus infection is negative；If
Redness, then illustrate that virus infection is positive.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480310A (en) * | 2022-01-28 | 2022-05-13 | 内蒙金属材料研究所 | Packaging process and inactivation method for infectious virus liquid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114480310A (en) * | 2022-01-28 | 2022-05-13 | 内蒙金属材料研究所 | Packaging process and inactivation method for infectious virus liquid |
| CN114480310B (en) * | 2022-01-28 | 2023-07-14 | 内蒙金属材料研究所 | Packaging process and inactivating method for infectious virus liquid |
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