CN103323599B - Time resolved immunofluorescence detection kit for rabies virus nucleoprotein, and preparation method thereof - Google Patents
Time resolved immunofluorescence detection kit for rabies virus nucleoprotein, and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a time resolved immunofluorescence detection kit for rabies virus nucleoprotein, and a preparation method thereof. The content of rabies virus nucleoprotein in a sample is quantificationally determined by using principles of a double-antibody sandwich method and time resolved immunofluorescence. The detection kit comprises a standard, a coating reaction plate, a lanthanide ion marked anti-rabies virus nucleoprotein monoclonal antibody, an analysis buffer, a washing liquid and a fluorescence enhancing liquid, wherein a first anti-rabies virus nucleoprotein monoclonal antibody is adsorbed on the coating reaction plate, and a second anti-rabies virus nucleoprotein monoclonal antibody is marked with the lanthanide ion. A preparation method comprises: adding a to-be measured sample in the coating reaction plate, incubating, washing, adding the lanthanide ion marked second anti-rabies virus nucleoprotein monoclonal antibody, incubating, washing, adding the fluorescence enhancing liquid, and determining in a time resolved immunofluorescence detector. The technical scheme of the invention has advantages of high sensitivity, good singularity, simple operation, wide detection scope and the like.
Description
Technical field
The present invention relates to virus protein technical field of immunoassay, particularly relate to a kind of Rables virus glycoprotein gene time resolution immunofluorescence detection agent box and preparation method thereof.
Background technology
Rabies viruses (Rabies Virus, RV) belongs to Rhabdoviridae, Lyssavirus, and form is bullet shaped, long 180nm, diameter 75nm, and its head is semisphere, and end is often flush end, and virion is made up of shell and core two parts.Rabies viruses genome is made up of 11928 ~ 11932 nucleotide, is the strand RNA of strand, non-segmented negative, containing 5 large open reading frame, encode 5 kinds of structure eggs from.Genome from 3 ' end to 5 ' hold put in order as N, P, M, G and L gene, encode viral nucleoprotein (nucleoprotein respectively, NP), phosphoprotein (phosphoprotein, NS), membrane matrix albumen (matrix protein, MP), glycoprotein (glycoprotein, and transcribe large protein (polymerase, LP) GP).Research in recent years shows, the immune response that hydrophobia induction body produces neutralizing antibody is main relevant with glycoprotein and nucleoprotein.Nucleoprotein is One's name is legion in virion, accounts for 36% of virus protein total amount.Nucleoprotein gene sequence relative constancy, is albumen the most stable in rabies viruses, can be used for detectable antigens.Nucleoprotein energy inducing protective immunity is the principal ingredient of induction rabies cellular immunity.In addition, nucleoprotein can also promote the generation of neutralizing antibody, suppresses the intercellular propagation of rabies viruses and in intracellular breeding.Therefore, nucleoprotein is one of indispensable composition in hydrophobia component, is the important indicator evaluating vaccine vigor.
Rables virus glycoprotein gene content is one of primary quality measure of rabies vacciness, and in the development process of rabies vacciness, normal needs carry out the detection of antigenic content in time, main by NIH method (rabies vacciness Determination method) mensuration in prior art.The method determination period is long, complex operation, is also subject to the restriction that international animal protection association uses animal used as test, is not suitable for the monitoring of each step in rabies vacciness purification process.Utilize Enzyme-linked Immunosorbent Assay (ELISA) method to detect Rables virus glycoprotein gene to have been reported, but ELISA kit can only carry out qualitative or half-quantitative detection, can not provide quantitatively accurate.Therefore, seek can the experimental technique of accurate quantification carry out alternative NIH method or ELISA method imperative.
The novel on-radiation immunolabelling technique that Time-resolved Fluoroimmunoassay (TRFIA) is is feature with rare earth ion complex labelled antigen or antibody, nucleic acid probe and cell etc.In traditional fluoroimmunoassay, the problem of high background cannot well solve always.Background is mainly from two parts, and first is scattered light, comprises and analyzes the scattering on solid phase material surface used, the Rayleigh of solvent molecule and Raman scattering etc.; Second is the non-specific fluorescence that in sample, various coexisting substances produces, and comprise the fluorescence of haemocyanin, serological compounds as bilirubinic fluorescence, the life-span of these background fluorescences above-mentioned is all between 1-10ns.The most outstanding feature of rare earth compounding compared with common organic fluorescence label is the fluorescence lifetime (more than 100us) that it is extremely grown, so adopt time-resolved fluorometry technology, only measure the fluorescence that long-life phosphors material sends, and do not measure the fluorescence that short life fluorescent material sends, just can eliminate background well, greatly improve and measure sensitivity.Time-resolved fluorometry overcomes the shortcomings such as ELISA enzyme marker instability, the easy inactivation of enzyme, sensitivity is not high, non-specific signals is made to be reduced to negligible degree, reach high signal to noise ratio (S/N ratio), thus considerably more than the sensitivity that radioactive isotope can reach.TRFIA also has that label prepares easy, storage time length, no radioactivity pollute, detection is reproducible, operating process is short, standard curve range is wide, by the interference of sample natural fluorescence and the advantage such as range of application is very extensive, become a new milestone of after radiommunoassay label development, become one of analysis means the most promising in biomedical research and clinical ultramicron biochemical investigation.There is no prior art report utilizes TRFIA standard measure to detect Rables virus glycoprotein gene.
Summary of the invention
For above-mentioned defect of the prior art, technical matters solved by the invention is to provide a kind of Rables virus glycoprotein gene time resolution immunofluorescence detection agent box and preparation method thereof, this kit quantitatively can detect Rables virus glycoprotein gene, detection sensitivity is high, simple to operate, operating process is short.
In order to solve the problems of the technologies described above, on the one hand, the invention provides a kind of Rables virus glycoprotein gene time resolution immunofluorescence detection agent box, comprise standard items, wrap by rabies poison nucleoprotein monoclonal antibody, analysis buffer, cleansing solution, the fluorescence enhancement solution of reaction plate, lanthanide ion mark;
Described bag is the microwell plate being adsorbed with the first rabies poison nucleoprotein monoclonal antibody by reaction plate;
The rabies poison nucleoprotein monoclonal antibody of described lanthanide ion mark is the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark;
Described analysis buffer is for diluting and stablize the rabies poison nucleoprotein monoclonal antibody of described lanthanide ion mark.
Wherein, described first rabies poison nucleoprotein monoclonal antibody adopts hybridoma cell strain F022 to be prepared from, and the preserving number of hybridoma cell strain F022 is CCTCC NO:C201244; Described second rabies poison nucleoprotein monoclonal antibody adopts hybridoma cell strain F028 to be prepared from, and the preserving number of hybridoma cell strain F028 is CCTCC NO:C201245.
Preferably, described analysis buffer is made up of 50mmol/L Tris-HCl, 1.5% PEG6000,0.2% BSA, 0.1% Proclin300,0.02% N of IgG, 0.1% Tween 20 and 0.84% NaCl, and pH is 7.8.
Preferably, described cleansing solution is made up of 1.25mmol/L Tris-HCl, 2.5% Tween 20 and 21%NaCl, and pH is 7.8, dilutes 25 times during use.
Preferably, described fluorescence enhancement solution contains beta-diketone compounds or aromatic amine compounds.More preferably, described fluorescence enhancement solution is made up of beta-diketon class, trioctylphosphine oxide, Triton X-200, acetic acid and Potassium Hydrogen Phthalate, and pH is 2. 0 ~ 3. 2.
Preferably, described lanthanide ion is Eu
3+, Tb
3+, Sm
3+, Dy
3+in at least one.
On the other hand, present invention also offers a kind of method preparing Rables virus glycoprotein gene time resolution immunofluorescence detection agent box, it is characterized in that comprising the steps:
A. preparation bag is by reaction plate: be added on microwell plate by the first rabies poison nucleoprotein monoclonal antibody being buffered liquid dilution with bag, bag described in low temperature removes after placing is buffered liquid, add confining liquid, described confining liquid is removed after cold sealing, microwell plate described in washing, vacuum is drained, freezen protective;
B. the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark is prepared: join in lanthanide ion labelled reagent by the second rabies poison nucleoprotein monoclonal antibody, mixing, packing, vacuum freeze drying is preserved;
C. calibration object is prepared: with the Rabies Vaccine Potency national standard of buffer 0,10,20,50,100,500 mEU/ml series concentration;
D. analysis buffer, cleansing solution, fluorescence enhancement solution is prepared.
Preferably, the second rabies poison nucleoprotein monoclonal antibody described in step b and the mass ratio of described lanthanide ion are 5:1.
As the improvement to technique scheme, purifying is carried out to the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark in step b, and is diluted to the dilutability that select linear is good, sensitivity is lower.
In addition, present invention also offers a kind of using method of Rables virus glycoprotein gene time resolution immunofluorescence detection agent box, it is characterized in that comprising the steps:
A. use detection kit before 1 hour, the rabies poison nucleoprotein monoclonal antibody deionized water dissolving marked by lanthanide ion, then dilutes by analysis buffer, makes markers work liquid;
B. added standard items and testing sample at bag successively by reaction plate hole, shaking at room temperature hatches 1 h, removes supernatant, repeatedly washs, pat dry with cleansing solution;
C. add the markers work liquid prepared in step a, shaking at room temperature hatches 1 h, removes supernatant, repeatedly washs, pat dry with cleansing solution;
D. add fluorescence enhancement solution, the slow level of lucifuge shakes 5 min;
E. on time-resolved fluorescence detector by programme and measure.
The bag mentioned in technique scheme is buffered liquid, confining liquid, microwell plate etc. and is not specifically described, and can adopt prior art well-known in the art.
The principle that present invention utilizes double antibody sandwich method and time resolution immunofluorescence quantitatively detects the content of Rables virus glycoprotein gene in sample, bag reaction plate is adsorbed with the first rabies poison nucleoprotein monoclonal antibody, and lanthanide ion marks the second rabies poison nucleoprotein monoclonal antibody.The present invention can realize the quantitative detection of Rables virus glycoprotein gene, and detection kit is simple for production, the storage time is long, detection sensitivity is high, operating process is short, can be widely used in the quantitative detection of rabies viruses associated sample.
Accompanying drawing explanation
Fig. 1 is the typical curve of embodiment 3 based on a kind of Rables virus glycoprotein gene time-resolved fluoroimmunoassay of the present invention detection kit standard items.
Fig. 2 is embodiment 4 based on the correlativity comparison diagram of a kind of Rables virus glycoprotein gene of the present invention time-resolved fluoroimmunoassay detection kit and domestic ELISA kit measurement result.
Embodiment
Rables virus glycoprotein gene time resolution immunofluorescence detection agent box is application double-antibody sandwich fluoroimmunoassay, adopt the reaction plate of a strain rabies poison nucleoprotein monoclonal antibody bag quilt, add standard items and testing sample, nucleoprotein in sample is combined into antibody-nucleoprotein complex with the monoclonal antibody of wrapping quilt, add another strain rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark and the sequestrant of lanthanide ion and fluorescence enhancement solution again, form antibody-nucleoprotein-antibody-chelator-lanthanide ion compound.When lanthanide ion sends strong fluorescence in fluorescence enhancement solution after nucleoprotein monoclonal antibody is dissociated, fluorescence intensity is directly proportional to the nuclear protein concentrations in sample, and reference standard curve can determine the amount of antigen nucleoprotein in sample.
Rables virus glycoprotein gene time resolution immunofluorescence detection agent box of the present invention comprises:
1) standard items
2) bag is by reaction plate
3) the rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark
4) analysis buffer
5) cleansing solution
6) fluorescence enhancement solution
Above-mentioned standard items are the hydrophobia effect national standard of the series concentration of preparation.In an embodiment of the present invention, standard items are adopted and are prepared with the following method: with by 2 g/L BSA(bovine serum albumin(BSA)s), 1 g/L NaN
3with 50 mmol/l Tris-Hcl form, pH is the damping fluid of 7.8, Rabies Vaccine Potency national standard (the 7th batch) is mixed with the standard solution of 0,10,20,50,100 and 500 mEU/ml series concentration.
Above-mentioned bag is the microwell plate being adsorbed with the first rabies poison nucleoprotein monoclonal antibody by reaction plate.During specific implementation, bag in the embodiment of the present invention is adopted by reaction plate and is prepared with the following method: (our company adopts the self-control of conventional hybridization knurl method by the monoclonal antibody of a strain rabies poison nucleoprotein to use the carbonate buffer solution of pH 9.6,50 mmol/L, be numbered: F022 preserving number is: CCTCC NO:C201244, depositary institution: China typical culture collection center, preservation date: on April 5th, 2012) be coated in microwell plate, close final vacuum with confining liquid and drain.
The rabies poison nucleoprotein monoclonal antibody of above-mentioned lanthanide ion mark is the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark.The rabies poison nucleoprotein antibody of lanthanide ion mark is adopted and is prepared with the following method: (our company adopts the self-control of conventional hybridization knurl method to select the monoclonal antibody of another strain rabies poison nucleoprotein, be numbered: F028 preserving number is: CCTCC NO:C201245, depositary institution: China typical culture collection center, preservation date: on April 5th, 2012) carry out lanthanide ion mark, the mass ratio of monoclonal antibody and lanthanide ion label is 5:1.Mark rate is too high, and impact is labeled the immunocompetence of monoclonal antibody; Mark rate is too low, and signal intensity is inadequate, reduces detection sensitivity.
Above-mentioned analysis buffer is for diluting and stablize the rabies poison nucleoprotein monoclonal antibody of described lanthanide ion mark.Wherein, analysis buffer is by 50mmol/L Tris-HCl, 1.5% PEG6000(Macrogol 6000), 0.2% BSA, 0.1% Proclin300(antiseptic 300), 0.02% N of IgG, 0.1% Tween 20(polysorbas20) and 0.84% NaCl form, pH is 7.8.
Above-mentioned cleansing solution adds bag by the sample after hatching in reaction plate hole and the second rabies poison nucleoprotein monoclonal antibody of being marked by lanthanide ion for washing.Wherein, cleansing solution (25x) is made up of 1.25mmol/L Tris-HCl, 2.5% Tween 20 and 21%NaCl, and pH is 7.8, dilutes 25 times during use.
Above-mentioned fluorescence enhancement solution is the chelate of lanthanide ion, lanthanide ion and its sequestrant, as
After beta-diketon body ketone or aromatic amine part form complex, Absorbable rod ultraviolet light, then sends very strong metallic ion characteristic fluorescence.Preferably, fluorescence-enhancing agent draws logical 200 by beta-diketon body, trioctylphosphine oxide (TOPO), Triton200(song), acetic acid and Potassium Hydrogen Phthalate form, pH is 2. 0 ~ 3. 2.
At present, TRFIA utilizes the trivalent ion of lanthanide series metal and chelate thereof as fluorescence labeling, the lanthanide series often had is europium (Eu), terbium (Tb), samarium (Sm) and dysprosium (Dy), that the most frequently used is Eu, Tb, lanthanide series label is more stable, 1 ~ 2 year can be preserved, overcome the shortcoming that isotope, enzyme mark etc. are unstable.
The using method of above-mentioned Rables virus glycoprotein gene time resolution immunofluorescence detection agent box is:
1) added standard items and testing sample at bag successively by reaction plate hole, shaking at room temperature hatches 1 h, removes supernatant, washs repeatedly, pat dry with cleansing solution;
2) add the rabies poison nucleoprotein monoclonal antibody with the lanthanide ion mark of analysis buffer dilution again, shaking at room temperature hatches 1 h, washs repeatedly with cleansing solution;
3) in hole, add fluorescence enhancement solution, slow level shakes 5 min;
4) on time-resolved fluorescence detector by mensuration of programming.
Below in conjunction with specific embodiment, the present invention is described further.
1. the preparation of detection kit of the present invention
(1) by the carbonate buffer solution of the preparation pH9.6 of reaction plate, 50 mmol/L, by the anti-nucleoprotein monoclonal antibody of a strain, (our company adopts the self-control of conventional hybridization knurl method to bag, be numbered: F022 preserving number is: CCTCC NO:C201244) be diluted to 3 μ g/ml, then every hole 100 μ l joins in 96 microwell plates, 4 DEG C are spent the night, sop up coating buffer, add the confining liquid (pH is 7.0) be made up of 15% sucrose, 10% calf serum, PBS damping fluid, close for 4 DEG C and spend the night, sop up confining liquid, washing final vacuum is drained ,-20 DEG C of freezen protective.
(2) preparation of the rabies poison nucleoprotein monoclonal antibody of europium mark
Antibody Concentration: (our company adopts the self-control of conventional hybridization knurl method by another strain rabies poison nucleoprotein monoclonal antibody of 0.5mg, be numbered: F028 preserving number is: CCTCC NO:C201245) add 0.2ml mark damping fluid (50mmol/L Na2CO3, pH 9.0), after mixing, with the G-50 centrifuge tube 10000rpm centrifugal 5min of Millipore company with filter membrane, repeated washing 5 times again, add 50 μ l and mark damping fluid, leave standstill 2min, reversing collected by centrifugation antibody, fixing fabric structure is at 50 μ about l;
Antibody labeling: add 0.1mg Eu
3+labelled reagent (PerKin Elmer Products, numbering: 1244-302) in the rabies poison nucleoprotein monoclonal antibody concentrated, fully mix, 25 DEG C of shaken overnight;
Labelled antibody purifying: antibody Sephadex G-50 chromatographic column (1x30cm) marked is carried out separation and purification, after eluent (the 50mmol/l Tris-HCl solution containing 0.9% NaCl) wash-out, collect eluent (1ml/ pipe), absorbance (A280nm) is measured by pipe, merge peak pipe, survey protein content and calculate mark rate;
Determine dilutability: to and labelled antibody after pipe carry out dilutability and grope, select linear is better, the dilutability that sensitivity is lower; With continuous sample-adding gun packing labelled antibody, 1.0 ml/ bottles, vacuum freeze drying, 2 DEG C-8 DEG C preservations.
(3) standard items preparation
With containing 2 g/L BSA and 1 g/L NaN
350 mmol/l Tris-Hcl damping fluids (pH7.8), Rabies Vaccine Potency national standard (the 7th batch) is mixed with the standard solution of 0,10,20,50,100 and 500 mEU/ml series concentration.
(4) analysis buffer is prepared
Prepare the analysis buffer be made up of 50mmol/L Tris-HCl, 1.5% PEG6000,0.2% BSA, 0.1% Proclin300,0.02% N of IgG, 0.1% Tween 20 and 0.84% NaCl, pH is 7.8.
(5) cleansing solution is prepared
Prepare the cleansing solution (25x) be made up of 1.25mmol/L Tris-HCl, 2.5% Tween 20 and 21%NaCl, pH is 7.8.
(6) fluorescence enhancement solution is prepared
Prepare the fluorescence enhancement solution be made up of 15 μm of ol/L β-naphthoyltrifluoroacetones (β-NTA), 50 μm of ol/L trioctylphosphine oxide (TOPO), 0.1% Triton X-100,0.6% acetic acid, adjusting its pH with appropriate Potassium Hydrogen Phthalate is pH 2. 0 ~ 3. 2.
2. the using method of detection kit of the present invention
(1) preparation of reagent
Work cleansing solution: by 50 mL cleansing solutions (25x) and the mixing of 1200 mL deionized waters, as cleansing solution;
Labelled antibody working fluid: use last hour by every bottle of labelled antibody 1 mL deionized water dissolving, by analysis buffer with 1:100 dilution proportion doubly, as labelled antibody working fluid.
(2) operation steps
1) 100 μ l normative reference product or testing sample (testing sample can be by the animal secretions of rabies virus infection or tissue extract containing product in the middle of the culture of rabies virus antigen composition, rabies vacciness and finished product, suspection) is added in the every hole of reaction plate successively to bag, shaking at room temperature hatches 1 h, supernatant is abandoned in suction, wash 4 times with work cleansing solution, pat dry;
2) every hole kind adds the labelled antibody working fluid that 100 μ l have diluted, and shaking at room temperature hatches 1 h, inhales and abandons supernatant, wash 6 times, pat dry with cleansing solution;
3) every hole adds 100 μ l fluorescence enhancement solutions, and level slowly shakes 5 min;
4) on time-resolved fluorescence detector by mensuration of programming.
3. the methodology calibrating of detection kit of the present invention
Examine and determine the detection kit be prepared in embodiment according to manufacture conventional in this area and vertification regulation, result is as follows:
1) accuracy
Sample 1, sample 2, sample 3 are pressed 1:200 respectively, the dilution proportion of 1:400,1:800 and 1:1600, each sample dilution does 3 multiple holes, and replication 3 times, n=3 × 3, calculate its measured value, actual value and accuracy.
Table 1 kit accuracy test result (n=3*3)
2) sensitivity and linear measurement range analysis
Be used as sample with zero normative reference product and measure 8 times, calculate its fluorescent value and standard deviation.The concentration value that the fluorescent value substitution typical curve equation adding 2 times of standard deviation gained with its fluorometric assay mean value calculates is for its sensitivity, and the present embodiment sensitivity for analysis is 1.0 mEU/ml after measured.Standard items are diluted to variable concentrations measure, recording the typical curve range of linearity is 5 ~ 1000mEU/ml.
3) precision (CV%)
By the detection kit in the present embodiment, standard items A-F 6 points are measured, respectively establish 10 multiple holes.The variation within batch coefficient (CV%) of result the present embodiment kit is 2.7% ~ 9.3%, and interassay coefficient of variation (CV%) is 3.5% ~ 12.2%, and meet the requirement of kit vertification regulation, precision is good.
Table 2 withinrun precision test result (
n=10)
Table 3 betweenrun precision test result (three batches,
n=3*10)
4) specificity
The sucrose of variable concentrations, human albumin, bovine serum albumin, gelatin, PBS solution are used as testing sample, use the present embodiment kit measurement, result shows all reactionless.
4. the present embodiment detection kit compares with domestic ELISA kit measurement result
ELISA(nucleoprotein with prepared by kit and Wuhan Biological Products Inst. of the present embodiment) kit originally detects 30 increments simultaneously.With the result of the present embodiment kit measurement for horizontal ordinate, ELISA(nucleoprotein prepared by Wuhan Biological Products Inst.) result of kit measurement is that ordinate does regretional analysis, dependent equation is: Y=0.732X+0.655, and correlation coefficient r=0.962(as shown in Figure 2).Result shows ELISA(nucleoprotein prepared by the present embodiment kit measurement result and Wuhan Biological Products Inst.) kit measurement result has high correlation.
Above disclosedly be only preferred embodiment of the present invention, for one of ordinary skill in the art, according to the thought of content of the present invention, all will change in specific embodiments and applications.In sum, this description should not be construed as limitation of the present invention, and all any changes done according to design philosophy of the present invention are all within protection scope of the present invention.
Claims (8)
1. a Rables virus glycoprotein gene time resolution immunofluorescence detection agent box, comprises standard items, wraps by rabies poison nucleoprotein monoclonal antibody, analysis buffer, cleansing solution, the fluorescence enhancement solution of reaction plate, lanthanide ion mark;
Described bag is the microwell plate being adsorbed with the first rabies poison nucleoprotein monoclonal antibody by reaction plate;
The rabies poison nucleoprotein monoclonal antibody of described lanthanide ion mark is the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark;
Described analysis buffer is for diluting and stablize the rabies poison nucleoprotein monoclonal antibody of described lanthanide ion mark;
Described first rabies poison nucleoprotein monoclonal antibody adopts hybridoma cell strain F022 to be prepared from, and the preserving number of hybridoma cell strain F022 is CCTCC NO:C201244; Described second rabies poison nucleoprotein monoclonal antibody adopts hybridoma cell strain F028 to be prepared from, and the preserving number of hybridoma cell strain F028 is CCTCC NO:C201245.
2. detection kit as claimed in claim 1, it is characterized in that: described analysis buffer is made up of 50mmol/L Tris-HCl, 1.5% PEG6000,0.2% BSA, 0.1% Proclin300,0.02% N of IgG, 0.1% Tween 20 and 0.84% NaCl, and pH is 7.8; Described cleansing solution is made up of 1.25mmol/L Tris-HCl, 2.5% Tween 20 and 21%NaCl, and pH is 7.8, dilutes 25 times during use.
3. detection kit as claimed in claim 1, is characterized in that: described fluorescence enhancement solution contains beta-diketone compounds or aromatic amine compounds.
4. detection kit as claimed in claim 3, it is characterized in that: described fluorescence enhancement solution is made up of beta-diketon body, trioctylphosphine oxide, Triton X-200, acetic acid and Potassium Hydrogen Phthalate, pH is 2. 0 ~ 3. 2.
5. the detection kit as described in any one of claim 1 to 4, is characterized in that: described lanthanide ion is Eu
3+, Tb
3+, Sm
3+, Dy
3+in at least one.
6. prepare a method for Rables virus glycoprotein gene time resolution immunofluorescence detection agent box as claimed in claim 1, it is characterized in that comprising the steps:
A. preparation bag is by reaction plate: be added on microwell plate by the first rabies poison nucleoprotein monoclonal antibody being buffered liquid dilution with bag, bag described in low temperature removes after placing is buffered liquid, add confining liquid, described confining liquid is removed after cold sealing, microwell plate described in washing, vacuum is drained, freezen protective;
B. the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark is prepared: join in lanthanide ion labelled reagent by the second rabies poison nucleoprotein monoclonal antibody, mixing, packing, vacuum freeze drying is preserved;
C. calibration object is prepared: with the Rabies Vaccine Potency national standard of buffer 0,10,20,50,100,500 mEU/ml series concentration;
D. analysis buffer, cleansing solution, fluorescence enhancement solution is prepared.
7. prepare the method for Rables virus glycoprotein gene time resolution immunofluorescence detection agent box as claimed in claim 6, it is characterized in that: the second rabies poison nucleoprotein monoclonal antibody described in step b and the mass ratio of described lanthanide ion are 5:1.
8. prepare the method for Rables virus glycoprotein gene time resolution immunofluorescence detection agent box as claimed in claim 6, it is characterized in that: purifying is carried out to the second rabies poison nucleoprotein monoclonal antibody of lanthanide ion mark in step b, and is diluted to the dilutability that select linear is better, sensitivity is lower.
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