CN103048450B - The quantitative assay of high-throughout rsv protein content and detection kit thereof - Google Patents
The quantitative assay of high-throughout rsv protein content and detection kit thereof Download PDFInfo
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Abstract
The invention discloses a kind of quantitative assay and detection kit thereof of high-throughout RSV (Respiratory Syncytial Virus(RSV)) protein content, its quantitative assay comprises step: A, for non-clinical sample, its detecting step, comprises the ELISA high flux detection-phase step of the cultivation stage of Hep2 cell, RSV (Respiratory Syncytial Virus(RSV)) protein content; B, for clinical or clinical front sample, its detecting step, comprises the ELISA high flux detection-phase step of RSV (Respiratory Syncytial Virus(RSV)) protein content; Described immue quantitative detection reagent box comprises: the ELISA of RSV (Respiratory Syncytial Virus(RSV)) protein content detects reagent.The present invention changes the method for screening antiviral drugs at present, improves screening flux, shortens the screening cycle, can save the time of more than 60%.Also detect for the high-throughput quantification of RSV in clinical or non-clinical sample in a large number simultaneously.
Description
Technical field
The present invention relates to a kind of quantitative assay of virus titer, particularly relate to a kind of quantitative assay of RSV (Respiratory Syncytial Virus, Respiratory Syncytial Virus(RSV)) protein content for drug screening and biological sample inspection and high flux detection kit thereof.
Background technology
Respiratory Syncytial Virus(RSV) (RSV) is the main cause causing bronchitis and pneumonia at cradle and children in early days.This virus usually can cause and infects.Infect again and generally occur in adult.In the middle of old man with the patient of shortage immunity, the serious disease of lower respiratory tract may be caused.The usual state of an illness can continue for two to three weeks. and baby and have in the children of the excessive risk state of an illness, RSV (Respiratory Syncytial Virus(RSV)) infects the life that may jeopardize patient.Viral bronchitis is a serious infectious disease, and cause 1,000,000 people dead according to estimates every year, wherein most of case is caused by RSV (Respiratory Syncytial Virus(RSV)).First Year after birth, the baby up to 70% ~ 80% ratio will infect RSV (Respiratory Syncytial Virus(RSV)).There is no effective vaccine or small-molecule drug to be at present used for control RSV (Respiratory Syncytial Virus(RSV)) and to infect.Therefore, method is fast and efficiently used to screen the effective means that the active drug obtaining anti-RSV (Respiratory Syncytial Virus(RSV)) is research and development new drug.
In antiviral drugs screening technique, mainly use cytopathic effect (CPE) method at present.Its detection method has tetramethyl azo azoles salt (hereinafter referred to as MTT) method, or uses Cell Counting Kit-8 (CCK-8 kit) to detect.Wherein, the Cleaning Principle of mtt assay is that the succinate dehydrogenase in living cells mitochondria can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization is formed is directly proportional to cell number.And CCK-8 kit is based on WST-8[2-(2-methoxyl-4-nitre phenyl)-3-(4-nitre phenyl)-5-(2,4-disulfobenzene)-2H-tetrazolium monosodium salt] detection kit.WST-8 is a kind of compound being similar to MTT, deposits in case at beam coupling reagent, can be generated orange-yellow first a ceremonial jade-ladle, used in libation dissolve in the medium by more Intramitochondrial dehydrogenasa reduction.Cell proliferation is more much faster, then color is darker; Cytotoxicity is larger, then color is more shallow.For same cell, the depth and the cell number of color are linear, and the formazan amount namely generated is directly proportional to living cells quantity.But these CPE method sensitivity are low, and flux is also unsuitable for the screening of large quantization compound.
Enzyme linked immunosorbent assay (ELISA) is technology most widely used in enzyme immunoassay technique.Its method is that known antigen or antibody are adsorbed on surface of solid phase carriers, and the antigen-antibody reaction of enzyme labeling is carried out at solid phase surface, with washing method by the free composition eccysis in liquid phase.Conventional ELISA method has double antibody sandwich method and indirect method, and the former is for detectable antigens, and the latter is for measuring specific antibody.ELISA is that a kind of susceptibility is high, high specificity, reproducible experimental technique, due to its stable reagent, easily preserve, easy and simple to handle, result judges the factors such as more objective, have and be suitable for extensive Screening tests, the detection that may be used for again a small amount of sample, both can do qualitative test and also can do the advantages such as quantitative test, therefore, there is use value widely.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of quantitative assay of RSV (Respiratory Syncytial Virus(RSV)) protein content for high-flux medicaments sifting and biological sample inspection and detection kit thereof.The present invention changes the method for screening antiviral drugs at present, improves screening flux, shortens the screening cycle (can shorten the time of more than 60%), and the method is also for quantitative detection that is clinical or non-clinical sample simultaneously.
For solving the problems of the technologies described above, the quantitative assay of RSV (Respiratory Syncytial Virus(RSV)) protein content for high-flux medicaments sifting and biological sample inspection of the present invention, comprises step:
A, for non-clinical sample, its detecting step, comprises the ELISA high flux detection-phase step of the cultivation stage of Hep2 cell, RSV (Respiratory Syncytial Virus(RSV)) protein content;
B, for clinical or clinical front sample, its detecting step, comprises the ELISA high flux detection-phase step of RSV (Respiratory Syncytial Virus(RSV)) protein content.
Described non-clinical sample comprises: compound; Clinical sample comprises: the Nasopharyngeal swabs of patient or nasopharynx washing lotion; Clinical front sample comprises: animal model tissue extract etc.
The cultivation stage of the Hep2 cell in described A, step comprises:
1. Hep2 cell is seeded in Tissue Culture Plate and cultivates; Wherein, condition of culture is 37 DEG C, 5%CO
2, cultivate 16-24 hour;
2. detected sample (non-clinical sample, as compound) adds above-mentioned inoculation has in the culture plate of cell;
3. add in RSV (Respiratory Syncytial Virus(RSV)) to above-mentioned culture plate;
4. culture plate is after cultivating 96-120 hour, gets cells and supernatant and carries out ELISA detection.
Described Tissue Culture Plate is 384 well culture plates, and the cell density of inoculation controls at every hole 1800-3000 cell, and volume is 20-50 μ l;
Described cultivation stage 3. in, the amount adding RSV (Respiratory Syncytial Virus(RSV)) is: 50-150 times of TCID
50(Tissue cultureinfective dose, tissue culture infective dose) viral dilution liquid, volume is 10-40ul.
The ELISA detection-phase of RSV (Respiratory Syncytial Virus(RSV)) protein content in described A, B, step comprises:
1. in 384 hole ELISA Plate, add goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody, after hatching, washing, add closed reagent effect, then wash;
2. in 384 hole ELISA Plate, add testing sample (to comprise: cells and supernatant, clinical or clinical front sample) after hatching, washing, add biotin in conjunction with goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody, through hatching, washing;
3. in 384 hole ELISA Plate, add antibiotin peroxidating proteinase, after hatching, washing, after adding TMB (3,3 ', 5,5 '-Tetramethylbenzidine, tetramethyl benzidine) nitrite ion effect, then add TMB stop buffer;
4. the light absorption value of ELISA Plate is measured at 450nm wavelength place.
In described ELISA detection-phase, involved all washings use PBS-Tween 20 (PBST) to wash more than 3 times.
The step of described ELISA detection-phase 1. in, goat-anti RSV polyclonal antibody addition is: the dilution of 1: 480-1: 2000 times of goat-anti RSV polyclonal antibody (ViroStat-0601), every hole adds 30-70 μ l, and the condition of hatching is: hatch 1-3 hour for 37 DEG C; Closed reagent is the BSA (bovine serum albumin(BSA)) of 0.5%-2%, and addition is every hole 100-120 μ l, 4 DEG C spend the night or 37 DEG C hatch 2-4 hour.
The step of described ELISA detection-phase 2. in, the addition of testing sample is 10-50 μ l (sample or sample diluting liquid), and the condition that testing sample is hatched is: 25 DEG C spend the night or 37 DEG C hatch 1-3 hour; Biotin in conjunction with the addition of goat-anti RSV polyclonal antibody is: biotin is in conjunction with the dilution of 1: 2000-1: 10000 times of goat-anti RSV polyclonal antibody (ViroStat-0607), every hole adds 30-70 μ l, and the condition of hatching is: hatch 1-3 hour for 37 DEG C.
The step of described ELISA detection-phase 3. in, the addition of antibiotin peroxidating proteinase is: the dilution of 1: 1000-1: 4000 times of antibiotin peroxidating proteinase (Sigma-E2886), every hole adds 30-70 μ l, and the condition of hatching is: hatch 20-40 minute for 37 DEG C; The addition of TMB nitrite ion (Sigma-T0440) is every hole 30-60 μ l, normal temperature effect 2-15 minute; TMB stop buffer (tmb substrate stop buffer, Sigma-S5814) addition is every hole 30-60 μ l.
In addition, the invention also discloses the immue quantitative detection reagent box of a kind of RSV (Respiratory Syncytial Virus(RSV)) protein content, can be applicable to drug screening and biological sample inspection.This immue quantitative detection reagent box comprises: the ELISA of rsv protein content detects reagent, also can comprise 384 hole ELISA Plate.
Wherein, the ELISA detection reagent of rsv protein content comprises: goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody, biotin are in conjunction with goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody, antibiotin peroxidating proteinase, closed reagent, cleansing solution, TMB nitrite ion, TMB stop buffer.
Described immue quantitative detection reagent box, also comprises: Hep2 cell, RSV (Respiratory Syncytial Virus(RSV)).
The detecting step of described immue quantitative detection reagent box as described above step carries out.
The present invention is by using MBP enzyme linked immuno-adsorbent assay principle, find the narrow spectrum quantitative assay of a kind of Respiratory Syncytial Virus(RSV), this method can be used for high-throughout anti respiratory syncytial virus drug screening and quantitative detection that is clinical or non-clinical sample, and its beneficial effect had is as follows:
(1) be different from existing ELISA range of application and current triage techniques (MTT or CCK8 kit), the present invention adopts ELISA method, utilizes 384 orifice plates to carry out the screening of anti-RSV (Respiratory Syncytial Virus(RSV)) virus compound;
(2) adopt ELISA method screening medicine, have highly sensitive, high specificity, simple to operate fast, stable experiment, repeatability is high and be easy to the features such as automation mechanized operation, and can detect thousands of increment product within one day;
(3) ELISA screening is carried out by employing 384 orifice plate, greatly can improve the flux of experiment, and it detects more traditional 96 orifice plates high (as shown in Figure 1) of precision, sensitivity, the workload of up to ten thousand screening compounds can be completed experimental period, time shorten more than 60%; Original use 96 orifice plate, namely 96 samples are detected only at most, use 384 orifice plates, then once can detect 384 samples, same 384 orifice plates that use are tested, and experimental cost can be made to also reduce much, therefore, the present invention, time saving while, saves a large amount of reagent, has saved cost;
(4) the present invention is not only applicable to screening compounds, be also applicable to the detection of clinical samples detection or animal sample, and all operations all can use instrument to complete, and reduces human factor and consumptive material use.
Accompanying drawing explanation
Fig. 1 is under the same conditions, the result figure that the ELISA using 384 orifice plates and 96 orifice plates to carry out RSV detects; Wherein, horizontal ordinate is RSV viral level titre, and unit is pfu/ml, and ordinate is OD value.As for same sample, the OD value (multiple averaging value) recorded in 384 orifice plates is 1.77, and the value recorded in 96 orifice plates is then 1.55, and reading has exceeded 14%, and therefore, the ELISA detection sensitivity in 384 orifice plates is higher than 96 orifice plates.
Fig. 2 is the ELISA testing result figure of embodiment 1.
Below in conjunction with accompanying drawing and embodiment, the present invention is further detailed explanation:
Embodiment
Embodiment 1 non-clinical sample RSV (Respiratory Syncytial Virus(RSV)) is quantitative, high flux ELISA method detects
One, the cell chulture stage
(1) by Hep2 cell (ATCC-CC123) with 3000 cells, volume is that 30 μ l inoculate into 384 porocyte culture plates (Greiner-781080, the U.S.), at cell culture incubator (37 DEG C, 5%CO
2) in cultivate 16 hours;
(2) non-clinical sample (i.e. compound 1 is diluted, derive from client), according to final concentration 50 μMs, 10 μMs, 2 μMs, 0.4 μM, from mother liquor (10mM), get 300nL, 60nL, 12.5nL, 2.5nL respectively, being added to inoculation has 384 well culture plates of cell;
(3) dilute RSV (Respiratory Syncytial Virus(RSV)) (ATCC-VR26), the amount added is: 100 times of TCID
50viral dilution liquid, volume is 30 μ l;
(4) in cell culture incubator, cultivate above-mentioned 384 well culture plates after 96 hours, get cells and supernatant and carry out ELISA detection.
Two, ELISA screening compounds
(1) in 384 hole ELISA Plate (Thermo-Nunc-464718), goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody (ViroStat-0601 is added, 1: 1000 times of dilution, every hole adds 50 μ 1), hatch 1.5 hours for 37 DEG C;
(2) PBS-Tween 20 (PBST) [PBS (Thermo-Hyclone-SH30264.01), containing 0.1%Tween20 (sigma-P2287) (percent by volume)] washing 3 times are used;
(3) in 384 hole ELISA Plate, closed reagent is added---the BSA of 1% (percent by volume), addition is every hole 120 μ l, and 4 DEG C are spent the night;
(4) PBS-Tween 20 is used to wash 3 times;
(5) in 384 hole ELISA Plate, add above-mentioned cells and supernatant dilution 50 μ l (Sample Dilution 10 times of liquid), the condition that testing sample is hatched is: hatch 2 hours for 37 DEG C;
(6) PBS-Tween 20 is used to wash 3 times;
(7) in 384 hole ELISA Plate, biotin is added in conjunction with goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody (ViroStat-0607,1: 6000 times of dilution, every hole adds 50 μ l), the condition of hatching is: hatch 2 hours for 37 DEG C;
(8) PBS-Tween 20 is used to wash 3 times;
(9) in 384 hole ELISA Plate, add antibiotin peroxidating proteinase (every hole adds 50 μ l for Sigma-E2886,1: 2000 times of dilution), hatch 30 minutes for 37 DEG C;
(10) PBS-Tween 20 (PBST) is used to wash 3 times;
(11) in 384 hole ELISA Plate, add TMB (Sigma-T0440, every hole adds 50 μ l) nitrite ion, normal temperature effect after 3 minutes, then adds TMB stop buffer (Sigma-S5814, every hole adds 50 μ l);
(12) microplate reader (Molecular Device-340PC384) is utilized, at the light absorption value of OD450nm place measurement 384 hole ELISA Plate.
Be provided with corresponding contrast in above step, measurement result sees the following form 1 simultaneously:
Table 1
In table 1, vc represents negative control, and cc represents positive control, and virus only represents background.Z factor represents the major parameter of assessment method of testing quality, generally between 0.5-1, then tests credible.The cell line used is: Hep2 cell (the 14th generation), Strain is: RSV (Respiratory Syncytial Virus(RSV)) Long, and cell-seeding-density is 3000/hole.The EC50 of this compound known is at about 1.0 μMs.Known from table 1 and Fig. 2, when compound concentration is 0.4 μM, inhibiting rate (Inhibition%) is 37.37%; 2.0 μMs time, inhibiting rate is 97.91%, can infer that the EC50 of this compound is between 0.4-2.0 μM, and known results matches.
Can find out according to the above results, the compound high flux of anti-RSV (Respiratory Syncytial Virus(RSV)) can be carried out by this experimental technique, highly sensitive RSV (Respiratory Syncytial Virus(RSV)) Enzyme-Linked Immunosorbent Assay detects, to reach the object of screening medicine.Its key advantages is that flux improves and the saving of cost, can complete the screening operation of up to ten thousand compounds an experimental period, time shorten more than 60%.Original use 96 orifice plate, namely detects 96 samples only at most, uses 384 orifice plates, then once can detect 384 samples.Same use 384 orifice plate is tested, and experimental cost can be made to also reduce much.
In addition, the present embodiment 1 is equally applicable to utilize immue quantitative detection reagent box to carry out high flux detection.
Embodiment 2 non-clinical sample RSV (Respiratory Syncytial Virus(RSV)) is quantitative, high flux ELISA method detects
One, the cell chulture stage
(1) by Hep2 cell (ATCC-CC123) with 3000 cells, volume is that 30 μ l inoculate into 384 porocyte culture plates (Greiner-781080, the U.S.), at cell culture incubator (37 DEG C, 5%CO
2) in cultivate 16 hours;
(2) non-clinical sample (i.e. compound 1 is diluted, compound derives from client), according to final concentration 50 μMs, 10 μMs, 2 μMs, 0.4 μM, from mother liquor (10mM), get 300nL, 60nL, 12.5nL, 2.5nL respectively, be added to 384 well culture plates that inoculation has cell;
(3) dilute RSV (Respiratory Syncytial Virus(RSV)) (ATCC-VR26), the amount added is: 150 times of TCID
50viral dilution liquid, volume is 30 μ l;
(4) in cell culture incubator, cultivate above-mentioned 384 well culture plates after 96 hours, get cells and supernatant and carry out ELISA detection.
Two, ELISA screening compounds
(1) in 384 hole ELISA Plate (Thermo-Nunc-464718), goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody (ViroStat-0601 is added, 1: 500 times of dilution, every hole adds 50 μ l), hatch 1.5 hours for 37 DEG C;
(2) PBS-Tween 20 (PBST) [PBS (Thermo-Hyclone-SH30264.01), containing 0.1%Tween20 (sigma-P2287)] washing 3 times are used;
(3) in 384 hole ELISA Plate, add closed reagent (BSA of 1%, the amount added is every hole 120 μ l, and 4 DEG C are spent the night);
(4) PBS-Tween 20 is used to wash 3 times;
(5) in 384 hole ELISA Plate, add above-mentioned cells and supernatant dilution 50 μ l (Sample Dilution 10 times of liquid), the condition that testing sample is hatched is: hatch 2 hours for 37 DEG C;
(6) PBS-Tween 20 is used to wash 3 times;
(7) in 384 hole ELISA Plate, biotin is added in conjunction with goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody (ViroStat-0607,1: 2000 times of dilution, every hole adds 50 μ l), the condition of hatching is: hatch 2 hours for 37 DEG C;
(8) PBS-Tween 20 is used to wash 3 times;
(9) in 384 hole ELISA Plate, add antibiotin peroxidating proteinase (every hole adds 50 μ l for Sigma-E2886,1: 1000 times of dilution), hatch 30 minutes for 37 DEG C;
(10) PBS-Tween 20 (PBST) is used to wash 3 times;
(11) in 384 hole ELISA Plate, add TMB (Sigma-T0440, every hole adds 50 μ l) show liquid, normal temperature effect after 3 minutes, then adds TMB stop buffer (Sigma-S5814, every hole adds 50 μ l);
(12) microplate reader (Molecular Device-340PC384) is utilized, at the light absorption value of OD450nm place measurement 384 hole ELISA Plate.
Be provided with corresponding contrast in above step, measurement result is in table 2 simultaneously:
Table 2
In table 2, vc represents negative control, and cc represents positive control, and virus only represents background.Z factor represents the major parameter of assessment method of testing quality, generally between 0.5-1, then tests credible.The cell line used is: Hep2 cell (the 14th generation), Strain is: RSV (Respiratory Syncytial Virus(RSV)) Long, and cell-seeding-density is 3000/hole.The EC50 of this compound known is at about 1.0 μMs.Find from chart, when compound concentration is 0.4 μM, inhibiting rate (Inhibition%) is 36.92%; When 10-50 μM, inhibiting rate is 100%, can infer that the EC50 of this compound is between 0.4-2.0 μM equally, and known results matches.
As long as can find out to use within the scope of this in the above description by embodiment 1 and embodiment 2 and detect reagent, identical effect can be reached.
In addition, the present embodiment 2 is equally applicable to utilize immue quantitative detection reagent box to carry out high flux detection.
Embodiment 3 non-clinical sample RSV (Respiratory Syncytial Virus(RSV)) is quantitative, high flux ELISA method detects
One, the cell chulture stage
(1) by Hep2 cell (ATCC-CC123) with 3000 cells, volume is that 30 μ l inoculate into 384 porocyte culture plates (Greiner-781080, the U.S.), at cell culture incubator (37 DEG C, 5%CO
2) in cultivate 16 hours;
(2) non-clinical sample (i.e. compound 1 is diluted, compound derives from client), according to final concentration 50 μMs, 10 μMs, 2 μMs, 0.4 μM, from mother liquor (10mM), get 300nL, 60nL, 12.5nL, 2.5nL respectively, be added to 384 well culture plates that inoculation has cell;
(3) dilute RSV (Respiratory Syncytial Virus(RSV)) (ATCC-VR26), the amount added is: 150 times of TCID
50viral dilution liquid, volume is 30 μ l;
(4) in cell culture incubator, cultivate above-mentioned 384 well culture plates after 96 hours, get cells and supernatant and carry out ELISA detection.
Two, ELISA screening compounds
(1) in 384 hole ELISA Plate (Thermo-Nunc-464718), goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody (ViroStat-0601 is added, 1: 2000 times of dilution, every hole adds 50 μ l), hatch 1.5 hours for 37 DEG C;
(2) PBS-Tween 20 (PBST) [PBS (Thermo-Hyclone-SH30264.01), containing 0.1%Tween20 (sigma-P2287)] washing 3 times are used;
(3) in 384 hole ELISA Plate, add closed reagent (BSA of 1%, the amount added is every hole 120 μ l, and 4 DEG C are spent the night);
(4) PBS-Tween 20 is used to wash 3 times;
(5) in 384 hole ELISA Plate, add above-mentioned cells and supernatant dilution 50 μ l (Sample Dilution 10 times of liquid), the condition that testing sample is hatched is: hatch 2 hours for 37 DEG C
(6) PBS-Tween 20 is used to wash 3 times;
(7) in 384 hole ELISA Plate, biotin is added in conjunction with goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody (ViroStat-0607,1: 6000 times of dilution, every hole adds 50 μ l), the condition of hatching is: hatch 2 hours for 37 DEG C;
(8) PBS-Tween 20 is used to wash 3 times;
(9) in 384 hole ELISA Plate, add antibiotin peroxidating proteinase (every hole adds 50 μ l for Sigma-E2886,1: 4000 times of dilution), hatch 30 minutes for 37 DEG C;
(10) PBS-Tween 20 (PBST) is used to wash 3 times;
(11) in 384 hole ELISA Plate, add TMB (Sigma-T0440, every hole adds 50 μ l) show liquid, normal temperature effect after 3 minutes, then adds TMB stop buffer (Sigma-S5814, every hole adds 50 μ l);
(12) microplate reader (Molecular Device-340PC384) is utilized, at the light absorption value of OD450nm place measurement 384 hole ELISA Plate.
Be provided with corresponding contrast in above step, measurement result sees the following form 3 simultaneously:
Table 3
In table 3, vc represents negative control, and cc represents positive control, and virus only represents background.Z factor represents the major parameter of assessment method of testing quality, generally between 0.5-1, then tests credible.The cell line used is: Hep2 cell (the 14th generation), Strain is: RSV (Respiratory Syncytial Virus(RSV)) Long, and cell-seeding-density is 3000/hole.The EC50 of this compound known is at about 1.0 μMs.Find from table 3, when compound concentration is 0.4 μM, inhibiting rate (Inhibition%) is less than 50%; When 10-50 μM, inhibiting rate is 100%, can infer that the EC50 of this compound is between 0.4-2.0 μM equally, and known results matches.As long as can find out to use within the scope of this in the above description by embodiment 3 and detect reagent, identical effect can be reached.All match with embodiment 1,2.
In addition, the present embodiment 3 is equally applicable to utilize immue quantitative detection reagent box to carry out high flux detection.
Embodiment 4 clinical sample RSV (Respiratory Syncytial Virus(RSV)) is quantitative, high flux ELISA method detects
Gather the Nasopharyngeal swabs of patient or nasopharynx washing lotion as clinical sample, get 50 μ l stostes or dilution liquid measure as testing sample, carry out ELISA method direct-detection according to " two, the ELISA screening compounds " method in embodiment 1.
In the present embodiment, once can detect multiple sample.If sample does not detect in time, can be stored in carry out in the skimmed milk power of 20% frozen.
Detect these samples by ELISA, not only detection time is short, and accurately, more can the multiple sample of repeated detection, substantially increases efficiency.
The clinical front sample RSV (Respiratory Syncytial Virus(RSV)) of embodiment 5 is quantitative, high flux ELISA method detects
Clinical front sample in the present embodiment is mouse lung tissue (infecting viral and after administration mouse).
Get mouse lung tissue (about 100mg), add 1000 μ l Hanks damping fluid (Invitrogen-14025) to grind, centrifugal (4 DEG C, 800g, 5 minutes), get supernatant, get 50 μ l stoste or dilutions, carry out ELISA method direct-detection according to " two, the ELISA screening compounds " method in embodiment 1.In the present embodiment, once can detect and organize sample more.
Detect these samples by ELISA, not only detection time is short, and accurately, more can the multiple sample of repeated detection, substantially increases efficiency.
Embodiment 6 utilizes immue quantitative detection reagent box to detect
The immue quantitative detection reagent box of RSV (Respiratory Syncytial Virus(RSV)) protein content in the present embodiment, comprise: Hep2 cell, RSV (Respiratory Syncytial Virus(RSV)), the ELISA of RSV (Respiratory Syncytial Virus(RSV)) protein content detects reagent, wherein, the ELISA of RSV (Respiratory Syncytial Virus(RSV)) protein content detects reagent and comprises: goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody, biotin is in conjunction with goat-anti RSV (Respiratory Syncytial Virus(RSV)) polyclonal antibody, antibiotin peroxidating proteinase, closed reagent (comprising BSA), cleansing solution (comprising PBS-Tween 20), TMB nitrite ion, TMB stop buffer.
In addition, the kit in the present embodiment, also can comprise 384 hole ELISA Plate.
To non-clinical sample (as compound or medicine), clinical sample (Nasopharyngeal swabs or nasopharynx washing lotion as people), clinical front sample (as animal tissue), the screening of medicine and the detection of sample can be carried out according to the method in embodiment 1-5.
The invention enables the detection range of application of ELISA method from detections such as current clinical samples, expand antiviral drugs screening to; Meanwhile, the method for screening anti-RSV (Respiratory Syncytial Virus(RSV)) medicine is at present changed.
Claims (6)
1., for the quantitative assay of Respiratory Syncytial Virus(RSV) rsv protein content for high-flux medicaments sifting and biological sample inspection, comprise step:
A, for non-clinical sample, its detecting step, comprises the ELISA high flux detection-phase step of the cultivation stage of Hep2 cell, Respiratory Syncytial Virus(RSV) rsv protein content;
The cultivation stage of the Hep2 cell in described A, step comprises:
1. Hep2 cell is seeded in Tissue Culture Plate and cultivates; Wherein, condition of culture is 37 DEG C, 5%CO
2, cultivate 16-24 hour;
2. non-clinical sample being added above-mentioned inoculation has in the culture plate of cell;
3. add in Respiratory Syncytial Virus(RSV) RSV to above-mentioned culture plate;
4. culture plate is after cultivating 96-120 hour, gets cells and supernatant and carries out ELISA detection;
The ELISA detection-phase of the Respiratory Syncytial Virus(RSV) rsv protein content in described A, step comprises:
1. in 384 hole ELISA Plate, add goat-anti Respiratory Syncytial Virus(RSV) RSV polyclonal antibody, after hatching, washing, add closed reagent effect, then wash;
2. in 384 hole ELISA Plate, add testing sample after hatching, washing, add biotin in conjunction with goat-anti Respiratory Syncytial Virus(RSV) RSV polyclonal antibody, through hatching, washing;
3. in 384 hole ELISA Plate, add antibiotin peroxidating proteinase, after hatching, washing, after adding the effect of TMB nitrite ion, then add TMB stop buffer;
4. the light absorption value of ELISA Plate is measured at 450nm wavelength place.
2. quantitative assay as claimed in claim 1, is characterized in that: described non-clinical sample comprises: compound.
3. quantitative assay as claimed in claim 1, it is characterized in that: described Tissue Culture Plate is 384 well culture plates, the cell density of inoculation controls at every hole 1800-3000 cell, and volume is 20-50 μ l;
Described cultivation stage 3. in, the amount adding Respiratory Syncytial Virus(RSV) RSV is: 50-150 times of TCID
50viral dilution liquid, volume is 10-40 μ l.
4. quantitative assay as claimed in claim 1, is characterized in that: in described ELISA detection-phase, and involved all washings use PBS-Tween 20 to wash more than 3 times;
The step of described ELISA detection-phase 1. in, goat-anti Respiratory Syncytial Virus(RSV) RSV polyclonal antibody addition is: the 1:480-1:2000 dilution doubly of goat-anti Respiratory Syncytial Virus(RSV) RSV polyclonal antibody, every hole adds 30-70 μ l, and the condition of hatching is: hatch 1-3 hour for 37 DEG C;
Closed reagent is the BSA of 0.5%-2%, and the amount added is every hole 100-120 μ l, 4 DEG C spend the night or 37 DEG C hatch 2-4 hour.
5. quantitative assay as claimed in claim 1, is characterized in that: the step of described ELISA detection-phase 2. in, testing sample comprises: cells and supernatant;
The addition of testing sample is 10-50 μ l, and the condition that testing sample is hatched is: 25 DEG C spend the night or 37 DEG C hatch 1-3 hour;
Biotin in conjunction with the addition of goat-anti Respiratory Syncytial Virus(RSV) RSV polyclonal antibody is: biotin is in conjunction with the 1:2000-1:10000 dilution doubly of goat-anti Respiratory Syncytial Virus(RSV) RSV polyclonal antibody, every hole adds 30-70 μ l, and the condition of hatching is: hatch 1-3 hour for 37 DEG C.
6. quantitative assay as claimed in claim 1, it is characterized in that: the step of described ELISA detection-phase 3. in, the addition of antibiotin peroxidating proteinase is: the 1:1000-1:4000 dilution doubly of antibiotin peroxidating proteinase, every hole adds 30-70 μ l, and the condition of hatching is: hatch 20-40 minute for 37 DEG C;
The addition of TMB is every hole 30-60 μ l, normal temperature effect 2-15 minute;
The addition of TMB stop buffer is every hole 30-60 μ l.
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