CN102590518A - Immune colloidal gold test paper and method for quantitatively detecting configurable logic block (CLB) by matching with photoelectric sensor thereof - Google Patents
Immune colloidal gold test paper and method for quantitatively detecting configurable logic block (CLB) by matching with photoelectric sensor thereof Download PDFInfo
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Abstract
The invention discloses immune colloidal gold test paper and a method for quantitatively detecting a configurable logic block (CLB) by matching with a photoelectric sensor thereof. The immune colloidal gold test paper comprises absorbent paper, a nitrocellulose (NC) membrane, a colloidal gold protective pad and a glass fiber which are arranged on a support plate and overlapped partially and mutually in sequence. Two round holes are arranged on the support plate, round areas on the NC membrane corresponding to the two round holes on the support plate are coated by T zone stripe of CLB-bovine serum albumin (BAS) and C zone stripe of a goat- anti-mouse second antibody, and the colloidal gold protective pad comprises an antibody resisting clenbuterol hydrochloride and marked by colloidal gold. The method for quantitatively detecting the CLB includes the following steps: a tested immune colloidal gold test paper strip is placed in a detecting area of the photoelectric sensor to be detected; the photoelectric sensor converts light reflected by the detecting area into electric signals, and concentration of the CLB is calculated according to strength of the electric signals.
Description
Technical field
The invention belongs to monoclonal antibody technique, golden labeling antibody technology, competition immunochromatography technique and photoelectric sensor technical field, be specifically related to the method for a kind of immune colloid gold test paper and cooperation photoelectric sensor detection by quantitative CLB thereof.
Background technology
Clenobuterol hydrochloride has another name called and is clenbuterol hydrochloride, and chemical name is a broncovaleas, is a kind of β of synthetic
2-adrenoceptor agonists can significantly improve lean meat percentage behind the animal edible, but also causes simultaneously the residual of CLB in the animal food, thereby causes the generation of human poisoning.The World Health Organization (WHO) and the regulation CLB of FAO (Food and Agriculture Organization of the United Nation) maximum residue limit in the animal edible tissues are 0.01ng/g.The colloidal gold immune chromatography experiment method is utilized the antigen-antibody reaction principle; Utilize the colloid gold label tracer and be fixed on antigen or antibody complex formation on the film and be trapped and develop the color; Do not need the colour developing of specific enzyme and substrate, do not need the physisorption of the long period between the antigen-antibody yet, and judge the yin and yang attribute result according to whether developing the color; Safe and effective, simple and convenient.But the defective of method is the requirement that can not reach detection by quantitative fully.
Effectively detection method is one of joint link of control CLB abuse, and the detection method commonly used of clenobuterol hydrochloride mainly contains chromatogram analysis method and immunological analysis method etc. at present.But these methods need be carried out strict pre-service to test sample, and required instrument is shortcoming such as the costliness and the on-the-spot detection that do not suit relatively.The colloid gold immune test paper bar can detect the residual of CLB by fast qualitative, on producing, is used widely.In recent years, photoelectric sensor has been widely applied in the heavy metal that detects food and potable water, its have detect rapid, easy, carry, can realize advantage such as scene detection easily; Do not see at present residual related patent U.S. Patent No. report as yet with immune colloidal gold technique and photoelectric sensor detection by quantitative CLB.Application number is found in search through patent: 02228104.5, and fast clenbuterol hydrochloride detecting test paper strip by name; Application number: 02202033.0, clenobuterol hydrochloride test strips by name; Application number: 200510083198.5, the immune golden test paper of a kind of fast detecting clenobuterol hydrochloride residue by name, these technology can only qualitative and half-quantitative detection, can not detection by quantitative CLB.
Summary of the invention
The present invention is directed to that the present sample pre-treatments of existing clenobuterol hydrochloride is complicated, detection time is long, need the defective that large-scale instrument and traditional immunity colloidal gold test paper strip can not detection by quantitative, proposed a kind of immune colloid gold test paper and cooperated the method for photoelectric sensor detection by quantitative CLB.The present invention had both reached the requirement of detection by quantitative, also have detect rapidly, high specificity, easy and simple to handle, be easy to carry, can realize advantage such as on-the-spot detection, have good application prospects.
To achieve these goals, the present invention adopts following technical scheme:
The present invention relates to a kind of immunity colloidal gold test paper strip; Comprise spun glass, collaurum neonychium, back up pad, NC film (nitrocellulose filter) and thieving paper, said thieving paper, NC film, collaurum neonychium, spun glass are arranged on the said back up pad and overlap each other successively; Said back up pad is provided with two circular holes; On the said NC film with said back up pad on two corresponding border circular areas of circular hole T district band of being coated with CLB-BSA C district band anti-with being coated with sheep anti mouse two, said collaurum neonychium contains the antibody of the anti-clenobuterol hydrochloride of colloid gold label.Said immunity colloidal gold test paper strip concrete structure is: be initiating terminal with thieving paper; Spun glass is terminal; Back up pad is positioned at bottom, is followed successively by thieving paper, NC film, collaurum neonychium and spun glass from initiating terminal to end on it, and adjacent two parts intersection is overlapped; Said C district band is near the thieving paper end, and said T district band is near collaurum neonychium end.
Preferably, said back up pad is the PVC plate, and said circular hole is 1cm.
Preferably, the volume of the anti-antibody of clenbuteral hydrochloride that contains of said collaurum neonychium is 4～20 μ L.
Preferably, the preparation method of said collaurum neonychium is following:
A, preparation colloidal gold solution;
B, the antibody of the anti-clenobuterol hydrochloride of 4～20 μ L is added in the Tris-HCl damping fluid of 1mL, adds the 10ml colloidal gold solution in the mixing process, stir 10～60min after, transfer pH greater than 8.5 with sal tartari; Then add 100～200 μ L bovine serum albumin(BSA)s (BSA), leave standstill, centrifugal, abandon supernatant; With the cleansing solution washing, once centrifugal again, abandon supernatant; Collect red precipitate, it is added drop-wise on the neonychium uniformly, promptly get.
Preferably, said step a is specially: in 100～300mL distilled water, add 1% gold chloride 100～500 μ L; When being heated to boiling; Add 1% trisodium citrate 100～500 μ L again, low temperature is kept 3～10min after continuing to be heated to solution and being aubergine, promptly gets.
Preferably, washing agent is formulated by Macrogol 2000 10～100 μ L, sal tartari 100～300 μ L, macrogol 10～100 μ L and Tris-HCl damping fluid 10～30ml among the said step b.
Preferably, CLB-BSA that encapsulates on the said nitrocellulose filter and sheep anti mouse two anti-volumes are 2～20 μ L.
Preferably, the preparation method of said nitrocellulose filter is following:
A, preparation CLB-BSA;
B, on the said nitrocellulose filter with back up pad on the corresponding border circular areas of two circular holes drip uniformly the sheep anti mouse two anti-and CLB-BSA of 2～20 μ L, with blocking agent sealing 20～60min, distilled water flushing takes out nature and dries, and promptly gets.
Preferably, said CLB-BSA preparation method is following: get CLB10mg, the 0.2mol/L hydrochloric acid that adds the 2mL precooling is cooled to zero degree to all dissolvings, behind the 30min, adds 120mg NaNO
2Solution adds above-mentioned solution in the carbonate buffer solution of 4mL 0.1mol/L then, and at the static 1h of zero degree, with the SephadexG50 gel-purified, with the Tris-HCl buffer solution elution of the 0.05mol/L of pH7.4 ,-20 ℃ of preservations are subsequent use behind the mixing; The BSA that contains 40mg in the said carbonate buffer solution.
Preferably, said blocking agent is formulated by the glycocoll of 1～10% skimmed milk power, 2～10g/L and 0.1～0.5g/L.
The invention still further relates to the method for aforesaid immunity colloidal gold test paper strip of a kind of usefulness and photoelectric sensor detection by quantitative clenobuterol hydrochloride (CLB), comprise the steps:
A, the immunity colloidal gold test paper strip after test are placed on detection in the test section of photoelectric sensor;
B, photoelectric sensor convert the light of surveyed area reflection to electric signal, according to the intensity of electric signal, calculate the concentration of CLB.
Preferably, the immunity colloidal gold test paper strip after the test described in the step a detects after cutting into the big or small strip of 3cm * 0.9cm again.
Principle of work of the present invention is: when detecting clenobuterol hydrochloride with immunity colloidal gold test paper strip of the present invention; When not having clenobuterol hydrochloride in the solution, solution can be slowly chromatography up, the antibody of the anti-clenobuterol hydrochloride on the collaurum neonychium chromatography that also can make progress combines with antigen on the nitrocellulose filter; Show band in the T district; In conjunction with antibody antigen can continue up chromatography, when running into two when anti-, a band also can appear in the C district.When in the solution clenobuterol hydrochloride being arranged; Monoclonal antibody competition on clenobuterol hydrochloride meeting and the collaurum neonychium combines with the antigen on the nitrocellulose filter; Will demonstrate does not herein have or the band of lighter color; In conjunction with antibody antigen also can continue up chromatography, also can show a band at two anti-places, can be qualitative and half-quantitative detection target material; Test strips after will developing the color then cuts into the strip that is of a size of 3 * 0.9cm; To photoelectric sensor, detect, the detection principle of photoelectric sensor is: light is reflected after sending the test section that projects test strips from light source, and reflected light is received and converted into electric signal by current frequency converter and transmits; Data processing through single-chip microcomputer; Finally be presented on the display, and the shade of the intensity of electric signal and test strips is inverse ratio, photoelectric sensor is read the photosignal value; Intensity according to electric signal; Indirect calculation goes out the concentration of hydrochloric acid Ke Lunluo, can only be qualitative and the defective of half-quantitative detection thereby overcome traditional immune gold test paper strip, realize the detection by quantitative clenobuterol hydrochloride.
The present invention compared with prior art has following beneficial effect:
1, the preparation method is simple, can the detection by quantitative clenobuterol hydrochloride; The color development area of colloid gold immune test paper bar of preparation is that diameter is that the circular hole of 1cm has increased the colour developing area; After the colour developing area increased, the accuracy and the sensitivity that on photoelectric sensor, detect were higher;
2, method target material of the present invention is with strong points; Accuracy rate is high; Detection speed is fast; Immunity colloidal gold test paper strip after colour developing detection time on photoelectric sensor is 2min, has overcome the defective that traditional immunity colloidal gold test paper strip detects CLB, can satisfy the requirement of the detection by quantitative CLB of mechanism of department such as entry and exit, customs and supermarket;
3, have low, easy to carry, the simple to operate advantages such as large-scale instrument and equipment that do not need of preparation cost; Detection to clenobuterol hydrochloride in animal product, feed and the food has a good application prospect.
Description of drawings
Fig. 1 detects the synoptic diagram of clenobuterol hydrochloride for immunity colloidal gold test paper strip;
Fig. 2 is the color effects synoptic diagram of the clenobuterol hydrochloride of immunity colloidal gold test paper strip test variable concentrations;
Fig. 3 is the detection schematic diagram of photoelectric sensor;
Fig. 4 is the model machine figure of photoelectric sensor;
Wherein, 1 is the PVC plate, and 2 is spun glass, and 3 is the collaurum neonychium, and 4 is nitrocellulose filter, and 5 is thieving paper, and 6 is display screen, and 7 is the test section.
Embodiment
Elaborate in the face of embodiments of the invention down: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.
The preparation method of the CLB-BSA that uses in following examples is following:
Get CLB10mg, the 0.2mol/L hydrochloric acid that adds the 2mL precooling is cooled to zero degree to all dissolvings, behind the 30min, adds 120mg NaNO
2Solution; Then above-mentioned solution is added in the carbonate buffer solution of 4mL 0.1mol/L (BSA that contains 40mg), behind the mixing at the static 1h of zero degree, with the SephadexG50 gel-purified; With the Tris-HCl buffer solution elution of the 0.05mol/L of pH7.4 ,-20 ℃ of preservations are subsequent use.
The preparation of the immunity colloidal gold test paper strip of present embodiment and following with the combine method step of detection by quantitative clenobuterol hydrochloride of photoelectric sensor:
(1) preparation of colloidal gold solution
At first needed experimental apparatus acid bubble 12h prepares colloidal gold solution, measures the distilled water of 100mL with graduated cylinder; Add 1% gold chloride, 100 μ L, when being heated to boiling, add 1% trisodium citrate 100 μ L again; Be heated to solution and be aubergine, turn down the fire 3min that reburns and get final product.
(2) making of collaurum neonychium
In small beaker, the antibody of drawing 4 μ L adds in the Tris-HCl damping fluid of 1mL, in the process that stirs, adds its mixed liquor in the colloidal gold solution with the colloidal gold solution of clean pipette, extract 10mL; Behind 10min, transfer pH greater than 8.5 with sal tartari, wait a period of time after; Add BSA100 μ L, wait after a period of time, install in 6 centrifuge tubes its even branch centrifugal; Abandon its supernatant, with washing agent (draw Macrogol 2000 10 μ L, sal tartari 100 μ L, macrogol 10 μ L, Tris-HCl damping fluid 10mL is mixed with washing agent) washing; Once centrifugal again; Discard supernatant, collect red precipitate, it is added drop-wise on the collaurum neonychium uniformly.
(3) drip two anti-and antigens at the circular hole place of nitrocellulose filter
Nitrocellulose filter (is got 1% skimmed milk power 2g/L at blocking agent; Add the glycocoll of 0.1g/L, be mixed with blocking agent) in rock 10min, uses compasses to make on nitrocellulose filter that two diameters rob with washing lotion that the sheep anti mouse two of drawing 2 μ L resists as the circular hole of 1cm and the CLB-BSA conjugate is added drop-wise to the circular hole place of making uniformly; Seal 20min with blocking agent; Distilled water washes three times repeatedly, takes out nature and dries, and is subsequent use.
(4) assembling of colloid gold immune test paper bar
Get the PVC plate 1 of certain-length; Adopt the mechanical stamping punch method; The circular hole that two diameters of the equidistant making in middle part are 1cm on PVC plate 1; Alignment nitrocellulose filter 4 is pasted (on the said nitrocellulose filter 4 with said PVC plate 1 on two corresponding border circular areas of circular hole be coated with the T band of CLB-BSA and be coated with the anti-C band of sheep anti mouse two, C is with near thieving paper 5 ends, T is with near collaurum neonychium 3 ends); Paste thieving paper 5 in a side on nitrocellulose filter 4 tops, this thieving paper 5 is overlapped with nitrocellulose filter 4; Paste collaurum neonychium 3 at the opposite side of nitrocellulose filter 4 tops for thieving paper 5, this collaurum neonychium 3 is overlapped with nitrocellulose filter 4; Opposite side on collaurum neonychium 3 for nitrocellulose filter 4 sticks spun glass 2, this spun glass 2 and collaurum neonychium 3 overlap (as shown in Figure 1).The CLB titer of preparation variable concentrations detects: as shown in Figure 2, when no hydrochloric acid Clenbuterol, T is with color darker, is judged as feminine gender; And control line (C band) (a) should develop the color always; On the contrary, having under the situation of clenobuterol hydrochloride, clenobuterol hydrochloride at first can form compound with the monoclonal antibody on the gold mark pad; Thereby form competition with the CLB-BSA conjugate on the NC film; Suppress the CLB-BSA conjugate and catch the amount of clenobuterol hydrochloride gold mark monoclonal antibody, T band more shallow (b) or disappearance (c) are judged as the positive; Because two resistive connections close other binding site of antibody of clenbuteral hydrochloride, it doesn't matter with the existence of clenobuterol hydrochloride; So along with the increase of clenobuterol hydrochloride concentration, test strips detection with on color can be more and more shallow.
(5) combine photoelectric sensor detection by quantitative clenobuterol hydrochloride
Test strips after the colour developing is cut into the strip that is of a size of 3 * 0.9cm, to photoelectric sensor, detect ().The detection schematic diagram of photoelectric sensor is as shown in Figure 3, and the model machine of photoelectric sensor is as shown in Figure 4: be reflected after sending the test section 7 that projects test strips from light source, reflected light is received and is converted into electric signal by current frequency converter and transmits; Data processing through single-chip microcomputer; Finally be presented on the display screen 6, and the shade of the intensity of electric signal and test strips is inverse ratio, can only be qualitative and the defective of half-quantitative detection thereby overcome traditional immune gold test paper strip; Can reach quantitatively and detect; General naked-eye observation less than color, photoelectric sensor is also read the photosignal value, according to the intensity of electric signal; Indirect calculation goes out the concentration of hydrochloric acid Ke Lunluo, thereby can the detection by quantitative clenobuterol hydrochloride.
The preparation of the immunity colloidal gold test paper strip of present embodiment and following with the combine method step of detection by quantitative clenobuterol hydrochloride of photoelectric sensor:
(1) preparation of colloidal gold solution
Before the preparation immunity colloidal gold test paper strip, needed experimental apparatus acid bubble 15h, first-selected preparation colloidal gold solution; Measure the distilled water of 200mL with graduated cylinder; Add 1% gold chloride, 200 μ L, when being heated to boiling, add 1% trisodium citrate 300 μ L again; Be heated to solution and be aubergine, turn down the fire 5min that reburns and get final product.
(2) making of collaurum neonychium
In small beaker, the antibody of drawing 10 μ L adds in the Tris-HCl damping fluid of 1mL, in the process that stirs, adds its mixed liquor in the colloidal gold solution with the colloidal gold solution of clean pipette, extract 10mL; Behind 30min, with sal tartari bar pH greater than 8.5, wait a period of time after; Add BSA150 μ L, wait after a period of time, install in 6 centrifuge tubes its even branch centrifugal; Abandon its supernatant, with washing agent (draw Macrogol 2000 40 μ L, sal tartari 160 μ L, macrogol 40 μ L, Tris-HCl damping fluid 16mL is mixed with washing agent) washing; Once centrifugal again; Discard supernatant, collect red precipitate, it is added drop-wise on the collaurum neonychium uniformly.
(3) drip two anti-and antigens at the circular hole place of nitrocellulose filter
Nitrocellulose filter (is got 4% skimmed milk power 4g/L at blocking agent; Add the glycocoll of 0.3g/L, be mixed with blocking agent) in rock 20min, uses compasses to make on nitrocellulose filter that two diameters rob with washing lotion that the sheep anti mouse two of drawing 10 μ L resists as the circular hole of 1cm and the CLB-BSA conjugate is added drop-wise to the circular hole place of making uniformly; Seal 30min with blocking agent; Distilled water washes three times repeatedly, takes out nature and dries, and is subsequent use.
(4) assembling of colloid gold immune test paper bar
Get the PVC plate of certain-length; Adopt the mechanical stamping punch method; The circular hole that two diameters of the equidistant making in middle part are 1cm on the PVC plate; Alignment nitrocellulose filter is pasted (on the said nitrocellulose filter with said PVC plate on two corresponding border circular areas of circular hole T district band of being coated with CLB-BSA C district band anti-with being coated with sheep anti mouse two, C district band is near the thieving paper end, T district band is near collaurum neonychium end); Paste thieving paper in a side on nitrocellulose filter top, this thieving paper and nitrocellulose filter are overlapped; Paste the collaurum neonychium at the opposite side of nitrocellulose filter top for thieving paper, this collaurum neonychium and nitrocellulose filter are overlapped; Opposite side on the collaurum neonychium for nitrocellulose filter sticks spun glass, and this spun glass and collaurum neonychium are overlapped.The CLB titer of preparation variable concentrations detects.
(5) combine photoelectric sensor detection by quantitative clenobuterol hydrochloride
The good test strips in colour developing back is cut into the strip that is of a size of 3 * 0.9cm; Be put on the photoelectric sensor and detect, the shade that photoelectric sensor produces electrical signal intensity and test strips is inverse ratio, generally naked-eye observation less than color; Photoelectric sensor is also read the photosignal value; According to the intensity of electric signal, indirect calculation goes out the concentration of hydrochloric acid Ke Lunluo, thereby can the detection by quantitative clenobuterol hydrochloride.
The preparation of the immunity colloidal gold test paper strip of present embodiment and following with the combine method step of detection by quantitative clenobuterol hydrochloride of photoelectric sensor:
(1) preparation of colloidal gold solution
Before the preparation immunity colloidal gold test paper strip, needed experimental apparatus acid bubble 20h, first-selected preparation colloidal gold solution; Measure the distilled water of 100mL with graduated cylinder; Add 1% gold chloride, 400 μ L, when being heated to boiling, add 1% trisodium citrate 500 μ L again; Be heated to solution and be aubergine, turn down fire and reburn and got final product in 10 minutes.
(2) making of collaurum neonychium
In small beaker, the antibody of drawing 15 μ L adds in the Tris-HCl damping fluid of 1mL, in the process that stirs, adds its mixed liquor in the colloidal gold solution with the colloidal gold solution of clean pipette, extract 10mL; Behind 50min, with sal tartari bar pH greater than 8.5, wait a period of time after; Add bovine serum albumin(BSA) (BSA) 150 μ L, wait after a period of time, it is divided evenly to install to 6 centrifuge tubes centrifugal; Abandon its supernatant, with washing agent (draw Macrogol 2000 70 μ L, sal tartari 220 μ L, macrogol 70 μ L, Tris-HCl damping fluid 22mL is mixed with washing agent) washing; Once centrifugal again; Discard supernatant, collect red precipitate, it is added drop-wise on the collaurum neonychium uniformly.
(3) drip two anti-and antigens at the circular hole place of nitrocellulose filter
Nitrocellulose filter (is got 7% skimmed milk power 8g/L at blocking agent; Add the glycocoll of 0.4g/L, be mixed with blocking agent) in rock 40min, uses compasses to make on nitrocellulose filter that two diameters rob with washing lotion that the sheep anti mouse two of drawing 15 μ L resists as the circular hole of 1cm and the CLB-BSA conjugate is added drop-wise to the circular hole place of making uniformly; Seal 50min with blocking agent; Distilled water flushing takes out nature and dries, and is subsequent use.
(4) assembling of colloid gold immune test paper bar
Get the PVC plate of certain-length; Adopt the mechanical stamping punch method; The circular hole that two diameters of the equidistant making in middle part are 1cm on the PVC plate; Alignment nitrocellulose filter is pasted (on the said nitrocellulose filter with said PVC plate on two corresponding border circular areas of circular hole T district band of being coated with CLB-BSA C district band anti-with being coated with sheep anti mouse two, C district band is near the thieving paper end, T district band is near collaurum neonychium end); Paste thieving paper in a side on nitrocellulose filter top, this thieving paper and nitrocellulose filter are overlapped; Paste the collaurum neonychium at the opposite side of nitrocellulose filter top for thieving paper, this collaurum neonychium and nitrocellulose filter are overlapped; Opposite side on the collaurum neonychium for nitrocellulose filter sticks spun glass, and this spun glass and collaurum neonychium are overlapped.The CLB titer of preparation variable concentrations detects.
(5) combine photoelectric sensor detection by quantitative clenobuterol hydrochloride
Test strips after the colour developing is cut into the strip that is of a size of 3 * 0.9cm; Be put on the photoelectric sensor and detect, the intensity of the electric signal that photoelectric sensor produces is inverse ratio with the shade of test strips, generally naked-eye observation less than color; Photoelectric sensor is also read the photosignal value; According to the intensity of electric signal, indirect calculation goes out the concentration of hydrochloric acid Ke Lunluo, thereby can the detection by quantitative clenobuterol hydrochloride.
Embodiment 4
The preparation of the immunity colloidal gold test paper strip of present embodiment and following with the combine method step of detection by quantitative clenobuterol hydrochloride of photoelectric sensor:
(1) preparation of colloidal gold solution
Before the preparation immunity colloidal gold test paper strip, needed experimental apparatus acid bubble 24h, first-selected preparation colloidal gold solution; Measure the distilled water of 300mL with graduated cylinder; Add 1% gold chloride, 500 μ L, when being heated to boiling, add 1% trisodium citrate 400 μ L again; Be heated to solution and be aubergine, turn down fire and reburn and got final product in 8 minutes.
(2) making of collaurum neonychium
In small beaker, the antibody of drawing 20 μ L adds in the Tris-HCl damping fluid of 1mL, in the process that stirs, adds its mixed liquor in the colloidal gold solution with the colloidal gold solution of clean pipette, extract 10mL; Behind 60min, transfer pH greater than 8.5 with sal tartari, wait a period of time after; Add bovine serum albumin(BSA) (BSA) 200 μ L, wait after a period of time, install in 6 centrifuge tubes its even branch centrifugal; Abandon its supernatant, with washing agent (draw Macrogol 2000 100 μ L, sal tartari 300 μ L, macrogol 100 μ L, Tris-HCl damping fluid 30mL is mixed with washing agent) washing; Once centrifugal again; Discard supernatant, collect red precipitate, it is added drop-wise on the collaurum neonychium uniformly.
(3) drip two anti-and antigens at the circular hole place of nitrocellulose filter
Nitrocellulose filter (is got 10% skimmed milk power 10g/L at blocking agent; Add the glycocoll of 0.5g/L, be mixed with blocking agent) in rock 60min, uses compasses to make on cellulose nitrate that two diameters rob with washing lotion that the sheep anti mouse two of drawing 20 μ L resists as the circular hole of 1cm and the CLB-BSA conjugate is added drop-wise to the circular hole place of making uniformly; Seal 60min with blocking agent; Distilled water flushing takes out nature and dries, and is subsequent use.
(4) assembling of colloid gold immune test paper bar
Get the PVC plate of certain-length; Adopt the mechanical stamping punch method; The circular hole that two diameters of the equidistant making in middle part are 1cm on the PVC plate; Alignment nitrocellulose filter is pasted (on the said nitrocellulose filter with said PVC plate on two corresponding border circular areas of circular hole T district band of being coated with CLB-BSA C district band anti-with being coated with sheep anti mouse two, C district band is near the thieving paper end, T district band is near collaurum neonychium end); Paste thieving paper in a side on nitrocellulose filter top, this thieving paper and nitrocellulose filter are overlapped; Paste the collaurum neonychium at the opposite side of nitrocellulose filter top for thieving paper, this collaurum neonychium and nitrocellulose filter are overlapped; Opposite side on the collaurum neonychium for nitrocellulose filter sticks spun glass, and this spun glass and collaurum neonychium are overlapped.The CLB titer of preparation variable concentrations detects.
(5) combine photoelectric sensor detection by quantitative clenobuterol hydrochloride
Test strips after the colour developing is cut into the strip that is of a size of 3 * 0.9cm; Be put on the photoelectric sensor and detect, the electrical signal intensity that photoelectric sensor produces is inverse ratio with the shade of test strips, generally naked-eye observation less than color; Photoelectric sensor is also read the photosignal value; According to the intensity of electric signal, indirect calculation goes out the concentration of hydrochloric acid Ke Lunluo, thereby can the detection by quantitative clenobuterol hydrochloride.
In sum; Target material of the present invention is with strong points, and accuracy rate is high, and detection speed is fast; Immunity colloidal gold test paper strip after colour developing detection time on photoelectric sensor is 2min; Overcome the defective that traditional immunity colloidal gold test paper strip detects CLB, realized the detection by quantitative clenobuterol hydrochloride, also have simultaneously detect rapidly, high specificity, easy and simple to handle, be easy to carry, can realize advantage such as on-the-spot detection; Can satisfy the requirement of the detection by quantitative CLB of mechanism of department such as entry and exit, customs and supermarket, have good application prospects.
Claims (12)
1. immunity colloidal gold test paper strip; Comprise spun glass, collaurum neonychium, back up pad, NC film and thieving paper; It is characterized in that said thieving paper, NC film, collaurum neonychium, spun glass are arranged on the said back up pad and overlap each other successively; Said back up pad is provided with two circular holes; On the said NC film with said back up pad on two corresponding border circular areas of circular hole T district band of being coated with CLB-BSA C district band anti-with being coated with sheep anti mouse two, said collaurum neonychium contains the antibody of the anti-clenobuterol hydrochloride of colloid gold label.
2. immunity colloidal gold test paper strip according to claim 1 is characterized in that, said back up pad is the PVC plate, and said circular hole is 1cm.
3. immunity colloidal gold test paper strip according to claim 2 is characterized in that, the volume of the anti-antibody of clenbuteral hydrochloride that said collaurum neonychium contains is 4～20 μ L.
4. immunity colloidal gold test paper strip according to claim 3 is characterized in that, the preparation method of said collaurum neonychium is following:
A, preparation colloidal gold solution;
B, the antibody of the anti-clenobuterol hydrochloride of 4～20 μ L is added in the Tris-HCl damping fluid of 1mL, adds the 10ml colloidal gold solution in the mixing process, stir 10～60min after, transfer pH greater than 8.5 with sal tartari; Then add 100～200 μ L BSA, leave standstill, centrifugal, abandon supernatant; With the cleansing solution washing, once centrifugal again, abandon supernatant; Collect red precipitate, it is added drop-wise on the neonychium uniformly, promptly get.
5. immunity colloidal gold test paper strip according to claim 4; It is characterized in that said step a is specially: in 100～300mL distilled water, add 1% gold chloride 100～500 μ L; When being heated to boiling; Add 1% trisodium citrate 100～500 μ L again, low temperature is kept 3～10min after continuing to be heated to solution and being aubergine, promptly gets.
6. according to claim 4 or 5 described immunity colloidal gold test paper strips; It is characterized in that washing agent is formulated by Macrogol 2000 10～100 μ L, sal tartari 100～300 μ L, macrogol 10～100 μ L and Tris-HCl damping fluid 10～30ml among the said step b.
7. immunity colloidal gold test paper strip according to claim 2 is characterized in that, CLB-BSA that encapsulates on the said nitrocellulose filter and sheep anti mouse two anti-volumes are 2～20 μ L.
8. immunity colloidal gold test paper strip according to claim 7 is characterized in that, the preparation method of said nitrocellulose filter is following:
A, preparation CLB-BSA;
B, on the said nitrocellulose filter with back up pad on the corresponding border circular areas of two circular holes drip uniformly the sheep anti mouse two anti-and CLB-BSA of 2～20 μ L, with blocking agent sealing 20～60min, distilled water flushing takes out nature and dries, and promptly gets.
9. immunity colloidal gold test paper strip according to claim 8 is characterized in that, said CLB-BSA preparation method is following: get CLB10mg, the 0.2mol/L hydrochloric acid that adds the 2mL precooling is cooled to zero degree to all dissolvings, behind the 30min, adds 120mg NaNO
2Solution adds above-mentioned solution in the carbonate buffer solution of 4mL 0.1mol/L then, and at the static 1h of zero degree, with the SephadexG50 gel-purified, with the Tris-HCl buffer solution elution of the 0.05mol/L of pH7.4 ,-20 ℃ of preservations are subsequent use behind the mixing; The BSA that contains 40mg in the said carbonate buffer solution.
10. according to Claim 8 or 9 described immunity colloidal gold test paper strips, it is characterized in that said blocking agent is formulated by the glycocoll of 1～10% skimmed milk power, 2～10g/L and 0.1～0.5g/L.
11. the method with immunity colloidal gold test paper strip as claimed in claim 1 and photoelectric sensor detection by quantitative CLB is characterized in that, comprises the steps:
A, the immunity colloidal gold test paper strip after test are placed on detection in the test section of photoelectric sensor;
B, photoelectric sensor convert the light of surveyed area reflection to electric signal, according to the intensity of electric signal, calculate the concentration of CLB.
12. the method according to said immunity colloidal gold test paper strip of claim 11 and photoelectric sensor detection by quantitative CLB is characterized in that, the immunity colloidal gold test paper strip described in the step a after the test detects after cutting into the big or small strip of 3cm * 0.9cm again.
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Application publication date: 20120718 |