CN115792217A - Quantitative bovine immunoglobulin G detection test paper and detection method based on quantum dot immunochromatography - Google Patents
Quantitative bovine immunoglobulin G detection test paper and detection method based on quantum dot immunochromatography Download PDFInfo
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Abstract
The invention relates to the technical field of quantitative detection of bovine immunoglobulin G, and discloses immunochromatography test paper which comprises an adhesive bottom lining, absorbent paper, an NC membrane, a combination pad and a sample pad, wherein the analysis membrane is attached to the middle of the adhesive bottom lining, the absorbent pad is attached to one side of the analysis membrane and forms a first combination area with the analysis membrane, the combination pad is attached to the other side of the analysis membrane and forms a second combination area with the analysis membrane, the sample pad is attached to the other side of the combination pad and forms a third combination area with the combination pad, a detection line and a quality control line are arranged on the surface of the analysis membrane, and after experiments, the QD-LF-competition method test paper can accurately quantify the bovine IgG content in a complex cow milk and a colostrum sample, which cannot be realized by an ultraviolet light splitting method; the test strip is very simple to operate, can finish detection and output results only by three steps of sample dilution, sample loading and machine reading, and can finish detection and output results in 15min, and expensive equipment and professional operation places are not needed.
Description
Technical Field
The invention relates to the technical field of quantitative detection of bovine immunoglobulin G, in particular to quantitative detection test paper and a detection method of bovine immunoglobulin G based on quantum dot immunochromatography.
Background
Immunoglobulin (Ig) is a kind of globulin with antibody activity and chemical structure similar to that of antibody, and can combine with antigen to neutralize toxicity, combine with complement to resist invading bacteria, virus and other pathogens and strengthen body's defense capacity. Bovine immunoglobulins are widely present in serum and milk, and are most abundant in colostrum (the initial secretion of the mammary gland within 72 hours after production of a cow, belonging to physiologically abnormal milk), with major classes of IgG, igA and IgM, with IgG accounting for 80%. The detection of the bovine IgG content in the bovine serum or the cow milk is helpful for the cattle farm to evaluate the passive immune effect of calves and guide the feeding of colostrums, thereby effectively improving the survival rate of the calves and reducing the morbidity; also can provide powerful product quality supervision means for dairy factories producing milk powder and dairy products; in addition, in the fields of biological pharmacy (vaccines and health products), biological cosmetics (beauty masks), daily chemicals (novel functional toothpaste) and the like, the detection of the bovine IgG content is also important for evaluating the quality safety of products. Therefore, the establishment of a rapid, accurate and convenient method for detecting the bovine IgG content can be promoted and applied in the industries of animal husbandry and veterinary, dairy products, biopharmaceuticals, cosmetics, chemicals, standard product production and the like.
At present, the detection methods of bovine IgG content mainly comprise an ultraviolet spectrophotometry, an immunoassay and an affinity chromatography, wherein the immunoassay mainly comprises an agar diffusion method, an immunoturbidimetry and an enzyme-linked immunosorbent assay, and the methods have respective advantages and disadvantages. Although the ultraviolet spectrophotometry is simple to operate and low in cost, the ultraviolet spectrophotometry is only suitable for analyzing the content of bovine IgG in standard substances, igG pure products and animal serum samples; the immunoassay method and affinity chromatography method have the advantages of accurate analysis and good repeatability, but need complex instrument and equipment for support, higher implementation cost, fussy operation and longer time consumption, and cannot be applied to basic layers.
Disclosure of Invention
The invention aims to provide quantitative bovine immunoglobulin G detection test paper and a detection method based on quantum dot immunochromatography, which achieve the purposes of realizing the quantitative detection of bovine IgG in samples such as bovine serum, cow milk or cow milk products and the like, and having good application prospects, and integrating rapidness, simplicity, accuracy and low cost.
In order to achieve the purpose, the invention provides the following technical scheme: a quantitative bovine immunoglobulin G detection test paper based on quantum dot immunochromatography comprises an immunochromatography test paper strip, wherein the immunochromatography test paper strip is a QD-LF-sandwich test paper strip and a QD-LF-competition test paper strip respectively;
the immunochromatographic test strip comprises an adhesive bottom lining, an analysis film, a combination pad, a sample pad and absorbent paper, wherein the analysis film is attached to the middle of the adhesive bottom lining, the absorbent pad is attached to one side of the analysis film and forms a first combination area with the analysis film, the combination pad is attached to the other side of the analysis film and forms a second combination area with the analysis film, the sample pad is attached to the other side of the combination pad and forms a third combination area with the combination pad, and a detection line and a quality control line are arranged on the surface of the analysis film.
Preferably, the quality control lines on the surfaces of the analysis membranes of the QD-LF-sandwich test strip and the QD-LF-competition test strip are both 2mg/mL goat anti-rabbit polyclonal antibodies, the detection line on the surface of the analysis membrane of the QD-LF-sandwich test strip is 3mg/mL rabbit anti-bovine IgG polyclonal antibodies, and the detection line on the surface of the analysis membrane of the QD-LF-competition test strip is 2mg/mL bovine IgG.
Preferably, the binding pad of the QD-LF sandwich test strip takes a QD-labeled rabbit anti-bovine IgG polyclonal antibody as a tracer, and the binding pad of the QD-LF competition test strip takes QD-labeled bovine IgG as a tracer.
A quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography, comprising the following steps:
step one, extracting bovine IgG;
step two, measuring the bovine IgG immunity and rabbit anti-bovine IgG polyclonal antibody titer;
step three, respectively diluting the goat anti-rabbit antibody, the rabbit anti-bovine IgG polyclonal antibody and the bovine IgG to the use concentration of a sprayed membrane by using 0.01 mol/L PB (phosphate buffer solution), then carrying out chromatographic membrane spraying, and drying at 37 ℃;
step four, adding 20 mu L of 0.2 g/mL quantum dots into a 1.5mL centrifuge tube, adding 500 mu L of a marking buffer solution, placing on a constant temperature oscillator, adding 15 mu L of an activating reagent, oscillating for 1h at 1400r/min, adding 0.05mg of rabbit anti-bovine IgG polyclonal antibody or bovine IgG, placing at 25 ℃ and 1400r/min, oscillating for 2h, adding bovine serum albumin with the final concentration of 1%, oscillating for 1h at 1400r/min, adding 3.88 mL of 0.01 mol/L PB (the pH value is 7.0), uniformly mixing, rapidly pouring onto glass fibers by using a capillary tube, placing at 37 ℃ for drying for 3h, drying and storing for later use;
step five, taking 2 mL of chromatography confining liquid (0.01 mol/L PB containing 1% bovine serum albumin, pH value of 7.0), pouring onto the cut glass fiber, placing at 37 ℃ for drying for 2h, and drying and storing for later use;
step six, 2, evaluation of sensitivity, linearity and precision of the test strip and field evaluation;
step seven: and (4) performing statistical analysis on the data.
Preferably, the first step is as follows:
adding 60mL of bovine serum into a beaker, dropwise adding a sodium acetate solution while stirring, and adjusting the pH value to 4.5; then dropwise adding 5mL of n-octanoic acid solution, uniformly stirring at normal temperature, placing at 4 ℃ for 1h, transferring to a 50 mL centrifuge tube, centrifuging at 12000r/min for 30min, taking supernate, filtering with filter paper, adding 1/10 volume of 0.1moL/L PBS (phosphate buffer) into the supernate, adjusting the pH value to 7.0, dropwise adding saturated ammonium sulfate solution to 50% of the total volume while stirring, placing at 4 ℃ for 2h, centrifuging at 12000r/min for 30min, and discarding the supernate;
resuspending the centrifuged precipitate with 0.01 moL/L PBS, dialyzing at 4 deg.C, and replacing dialysate for 1 time at 2h intervals until completely removing ammonia ions;
and (4) detecting the concentration of the bovine IgG by using a trace ultraviolet spectrophotometer, and freezing and storing for later use.
Preferably, the second step is as follows:
respectively immunizing 2 big-ear white rabbits with the prepared bovine IgG antigen, wherein the immunization mode is that 200mg is injected subcutaneously once, the immunization period is 2 weeks and 1 time, 4 times of immunization are carried out, after 2 weeks of 4 times of immunization, ear marginal venous blood of the 2 big-ear white rabbits is respectively taken to carry out enzyme-linked immunosorbent assay to detect the rabbit anti-bovine IgG polyclonal antibody titer in 2 parts of serum, the immunization is stopped when the dilution multiple of the serum is more than 10 ten thousand and the absorbance (ODg) of 450nm is more than 0.200, carotid blood sampling is carried out, the dilution multiple of the serum is the rabbit anti-bovine IgG polyclonal antibody titer, and the collected 2 parts of rabbit blood is centrifuged at 4 ℃ and 5000r/min for 15min to collect upper layer serum (serum numbers 1# and 2 #); and (3) extracting the rabbit anti-bovine IgG polyclonal antibody in serum by referring to a bovine IgG extraction method, measuring the concentration and the titer of the rabbit anti-bovine IgG polyclonal antibody, and freezing for later use.
Preferably, the sixth step is as follows:
sensitivity evaluation: bovine IgG was subjected to gradient dilution with 0.03 mol/L PB to obtain 0,0.1,0.3,0.5,0.7,1,3,5,7, 10, 30, 50, 70, 100. Mu.g/mL serial concentration standards, which were tested with 2 test strips, respectively, and the test was repeated 3 times with 0.03 mol/L PB as a blank. The sandwich method test strip takes a standard deviation of +3 times of the average value of a blank control detection band signal (T)/quality control band signal (C) value as a Cutoff value, and when the T/C value of a sample is more than or equal to the Cutoff value, the content of bovine IgG corresponding to the T/C value of the sample is the detection limit of the sandwich method test strip; the standard deviation of the blank control T/C value average value-3 times is used as the Cutoff value of the competition method test strip, and when the T/C value of the sample is less than or equal to the Cutoff value, the concentration of the bovine IgG corresponding to the T/C value of the sample is the detection limit of the competition method test strip;
and (3) linear evaluation: the sandwich method test strip takes lg (T/C value-Cutoff value) as a horizontal coordinate, the competition method test strip takes lg (T/C value) as a horizontal coordinate, and both the two types of test strips take lg (standard concentration) as a vertical coordinate to draw a quantitative standard curve;
precision evaluation: repeatedly testing each concentration of the standard substance to be tested for 3 times, and calculating the variation Coefficient (CV) of 3 detection values, wherein the smaller the variation coefficient is, the better the precision is;
and (3) field evaluation: selecting a test strip with better detection performance to carry out on-site detection, wherein samples comprise 50 parts of bovine colostrum (sample numbers are 1 to 50) and 2 parts of pasteurized milk (sample numbers are 51 and 52), simultaneously measuring by using a refractometer, randomly extracting 5 parts of bovine colostrum and 2 parts of pasteurized milk to carry out liquid chromatography measurement, and comparing the correlation of the bovine IgG content detected by the 3 methods to evaluate the accuracy of the test strip detection method.
Preferably, the step seven is as follows: sampling Excel software to carry out statistical analysis of test data, respectively drawing quantitative standard curves of 2 test paper for detecting bovine IgG standard substances, drawing a comparison histogram of a refractometer and an optimal test paper for detecting bovine IgG content in 52 milk samples, drawing a comparison histogram of the refractometer, the optimal test paper and a liquid chromatogram for detecting bovine IgG content in 7 milk samples, and analyzing the correlation of detection results of 3 methods by using the Excel software.
The invention provides quantitative bovine immunoglobulin G detection test paper based on quantum dot immunochromatography and a detection method. The method has the following beneficial effects:
(1) The quantum dot immunochromatographic test paper comprises an adhesive bottom lining, an analysis film, a combination pad, a sample pad and absorbent paper, wherein the analysis film is attached to the middle of the adhesive bottom lining, the absorbent pad is attached to one side of the analysis film and forms a first combination area with the analysis film, the combination pad is attached to the other side of the analysis film and forms a second combination area with the analysis film, the sample pad is attached to the other side of the combination pad and forms a third combination area with the combination pad, and a detection line and a quality control line are arranged on the surface of the analysis film. After experiments, the QD-LF-competition method test strip can accurately quantify the content of bovine IgG in complex bovine milk and colostrum samples, which cannot be realized by an ultraviolet spectroscopy; the test strip is very simple to operate, can finish detection and output results only by three steps of sample dilution, sample loading and machine reading, and can finish detection and output results in 15min, and expensive equipment and professional operation places are not needed.
(2) The quantitative detection of bovine IgG in samples such as bovine serum, cow milk or cow milk products and the like which are integrated with rapidness, simplicity, accuracy and low cost is really realized through the obtained quantum dot immunochromatographic test strip (competition method), and the method has a good application prospect.
Drawings
FIG. 1 is a perspective view of the immunochromatographic test strip of the present invention;
FIG. 2 is a structural view of the immunochromatographic test strip of the present invention;
FIG. 3 is a graph showing the results of the detection of the rabbit anti-bovine IgG polyclonal antibody titer of the present invention;
FIG. 4 is a graph showing the results of the average value of T/C values and CV values of 2 test strips for detecting bovine IgG standard samples with different concentrations;
FIG. 5 is a linear regression equation chart of 2 test strips of the present invention for detecting bovine IgG standard substances with different concentrations;
FIG. 6 is a field test result chart of a refractometer and a QD-LF competition test strip for No. 1 to No. 52 milk samples;
FIG. 7 is a graph showing the results of the QD-LF competitive method test strip, refractometer and liquid chromatography of the present invention for detecting the bovine IgG content in 7 bovine milk samples.
In the figure: 1 adhesive bottom lining, 2 analysis films, 3 combination pads, 4 sample pads, 5 absorbent paper, 6 detection lines and 7 quality control lines.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the invention.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
As shown in fig. 1 to 7, the present invention provides a technical solution: a quantitative bovine immunoglobulin G detection test paper based on quantum dot immunochromatography is characterized in that the immunochromatography test paper is a QD-LF-sandwich test paper strip and a QD-LF-competition test paper strip respectively;
the quantum dot immunochromatographic test paper comprises an adhesive bottom lining, an analysis film, a combination pad, a sample pad and absorbent paper, wherein the analysis film is attached to the middle of the adhesive bottom lining, the absorbent pad is attached to one side of the analysis film and forms a first combination area with the analysis film, the combination pad is attached to the other side of the analysis film and forms a second combination area with the analysis film, the sample pad is attached to the other side of the combination pad and forms a third combination area with the combination pad, and a detection line and a quality control line are arranged on the surface of the analysis film.
The quality control line 7 on the surface of the analysis membrane 2 of the QD-LF-sandwich test strip and the QD-LF-competition test strip are both 2mg/mL goat anti-rabbit polyclonal antibodies, the detection line on the surface of the analysis membrane 2 of the QD-LF-sandwich test strip is 3mg/mL rabbit anti-bovine IgG polyclonal antibodies, and the detection line 6 on the surface of the analysis membrane 2 of the QD-LF-competition test strip is 2mg/mL bovine IgG.
The binding pad 3 of the QD-LF-sandwich test strip takes a QD-labeled rabbit anti-bovine IgG polyclonal antibody as a tracer, and the binding pad 3 of the QD-LF-competition test strip takes QD-labeled bovine IgG as a tracer.
A quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography comprises the following steps:
step one, extracting bovine IgG:
adding 60mL of bovine serum into a beaker, dropwise adding a sodium acetate solution while stirring, and adjusting the pH value to 4.5; then adding 5mL of n-octanoic acid solution dropwise, stirring uniformly at normal temperature, placing at 4 ℃ for 1h, transferring into a 50 mL centrifuge tube, centrifuging at 12000r/min for 30min, taking supernatant, filtering with filter paper, adding 0.1moL/L PBS (phosphate buffer) with the volume of 1/10 into the supernatant, adjusting the pH value to 7.0, adding saturated ammonium sulfate solution dropwise to 50% of the total volume while stirring, placing at 4 ℃ for 2h, centrifuging at 12000r/min for 30min, and removing the supernatant;
resuspending the centrifuged precipitate with 0.01 moL/L PBS, dialyzing at 4 deg.C, and replacing dialysate for 1 time at 2h intervals until completely removing ammonia ions;
detecting the concentration of the bovine IgG by using a trace ultraviolet spectrophotometer, and freezing and storing for later use;
step two, measuring the bovine IgG immune and rabbit anti-bovine IgG polyclonal antibody titer:
respectively immunizing 2 big-ear white rabbits with the prepared bovine IgG antigen in a single subcutaneous injection mode of 200mg, wherein the immunization cycle is 1 time in 2 weeks for 4 times of immunization, after 2 weeks of the 4 th immunization, respectively taking the ear vein blood of the 2 big-ear white rabbits to perform enzyme-linked immunosorbent assay to detect the rabbit anti-bovine IgG polyclonal antibody titer in 2 parts of serum, stopping immunization when the dilution multiple of the serum is more than 10 million and the absorbance (ODg) of 450nm is more than 0.200, taking carotid blood, wherein the dilution multiple of the serum is the rabbit anti-bovine IgG polyclonal antibody titer, centrifuging the collected 2 parts of rabbit blood at 4 ℃ and 5000r/min for 15min, and collecting the upper serum (the serum numbers are 1# and 2 #); extracting rabbit anti-bovine IgG polyclonal antibody in serum by referring to a bovine IgG extraction method, measuring the concentration and titer of the rabbit anti-bovine IgG polyclonal antibody, and freezing for later use; the concentrations of the rabbit anti-bovine IgG polyclonal antibody are 34 mg/mL and 70 mg/mL respectively, and the titer of 2 rabbit anti-bovine IgG polyclonal antibodies detected by enzyme-linked immunosorbent assay is 1: 102, and the result is shown in figure 3.
Step three, respectively diluting the goat anti-rabbit antibody, the rabbit anti-bovine IgG polyclonal antibody and the bovine IgG to the use concentration of a sprayed membrane by using 0.01 mol/L PB (phosphate buffer solution), then carrying out chromatographic membrane spraying, and drying at 37 ℃;
step four, adding 20 mu L of 0.2 g/mL quantum dots into a 1.5mL centrifuge tube, adding 500 mu L of a marking buffer solution, placing on a constant temperature oscillator, adding 15 mu L of an activating reagent, oscillating for 1h at 1400r/min, adding 0.05mg of rabbit anti-bovine IgG polyclonal antibody or bovine IgG, placing at 25 ℃ and 1400r/min, oscillating for 2h, adding bovine serum albumin with the final concentration of 1%, oscillating for 1h at 1400r/min, adding 3.88 mL of 0.01 mol/L PB (the pH value is 7.0), uniformly mixing, rapidly pouring onto glass fibers by using a capillary tube, placing at 37 ℃ for drying for 3h, drying and storing for later use;
step five, taking 2 mL of chromatography confining liquid (0.01 mol/L PB containing 1% bovine serum albumin, pH value of 7.0), pouring onto the cut glass fiber, placing at 37 ℃ for drying for 2h, and drying and storing for later use;
step six, 2, test strip sensitivity, linearity and precision evaluation and field evaluation
Sensitivity evaluation: bovine IgG was subjected to gradient dilution with 0.03 mol/L PB to obtain 0,0.1,0.3,0.5,0.7,1,3,5,7, 10, 30, 50, 70, 100. Mu.g/mL serial concentration standards, which were tested with 4 test strips, respectively, and the test was repeated 3 times with 0.03 mol/L PB as a blank. The sandwich method test strip takes a standard deviation of +3 times of the average value of a blank control detection band signal (T)/quality control band signal (C) value as a Cutoff value, and when the T/C value of a sample is more than or equal to the Cutoff value, the content of bovine IgG corresponding to the T/C value of the sample is the detection limit of the sandwich method test strip; the standard deviation of the blank control T/C value average value-3 times is used as the Cutoff value of the competition method test strip, and when the T/C value of the sample is less than or equal to the Cutoff value, the concentration of the bovine IgG corresponding to the T/C value of the sample is the detection limit of the competition method test strip;
and (3) linear evaluation: the sandwich method test strip takes lg (T/C value-Cutoff value) as a horizontal coordinate, the competition method test strip takes lg (T/C value) as a horizontal coordinate, and both the two types of test strips take lg (standard concentration) as a vertical coordinate to draw a quantitative standard curve;
precision evaluation: repeatedly testing each concentration of the standard substance to be tested for 3 times, and calculating the variation Coefficient (CV) of the 3 detection values, wherein the smaller the variation coefficient is, the better the precision is;
the test paper strip sensitivity, linearity and precision results of the QD-LF-sandwich method and the QD-LF-competition method are as follows:
the Cutoff values of the 2 test strips were 0.635 and 2.864, respectively, and the detection limits were 0.1. Mu.g/mL and 0.5. Mu.g/mL, respectively. CV for the T/C value of the standards at each concentration is shown in FIG. 4. The T/C value-Cutoff value of the sandwich method test strip detection standard substance is positively correlated with the concentration of the standard substance, and the T/C value of the competition method test strip detection standard substance is negatively correlated with the concentration of the standard substance, as shown in figure 5. The QD-LF-competition test strip has the advantages of better precision, higher sensitivity and wider linear range, and is a test strip with better performance.
And (3) field evaluation: selecting a competitive test strip with better detection performance to carry out on-site detection, wherein samples comprise 50 parts of bovine colostrum (sample numbers are 1-50) and 2 parts of pasteurized milk (sample numbers are 51 and 52), simultaneously measuring by using a refractometer, randomly extracting 5 parts of bovine colostrum and 2 parts of pasteurized milk to carry out liquid chromatography measurement, and comparing the correlation of the bovine IgG content detected by the 3 methods to evaluate the accuracy of the test strip detection method; 50 parts of bovine colostrum and 2 parts of pasteurized milk are detected on site by a refractometer and a QD-LF-competition method test strip, the detection result of 50 parts of bovine colostrum shows that the detection result of the refractometer is concentrated between 20 and 80mg/mL, the detection result of the QD-LF-competition method is between 50 and 230mg/mL, the result is shown in figure 6, 5 parts of bovine colostrum and 2 parts of pasteurized milk are extracted for liquid chromatography determination, the detection data of 3 methods of 7 parts of samples are analyzed, and the correlation between the QD-LF-competition method test strip and the liquid chromatography detection value is found to be as high as 0.9759, while the correlation between the refractometer and the liquid chromatography detection result is only 0.8274, and is shown in figure 7.
Step seven: statistical analysis of data
Sampling Excel software to carry out statistical analysis of test data, respectively drawing quantitative standard curves of 2 test paper for detecting bovine IgG standard products, drawing a comparison histogram of a refractometer and an optimal test paper for detecting the bovine IgG content in 52 milk samples, drawing a comparison histogram of the refractometer, the optimal test paper and a liquid chromatogram for detecting the bovine IgG content in 7 milk samples, and analyzing the correlation of the detection results of 3 methods by using the Excel software.
In conclusion, the quantum dot immunochromatographic test strip (competitive method) obtained by the test really realizes the quantitative detection of bovine IgG in samples such as bovine serum, cow milk or cow milk products and the like which are integrated with rapidness, simplicity, accuracy and low cost, and has good application prospect.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The quantitative bovine immunoglobulin G detection test paper based on quantum dot immunochromatography comprises Quantum Dot (QD) immunochromatography test paper and is characterized in that: the immunochromatographic test strip is a QD-LF-sandwich test strip and a QD-LF-competition test strip respectively;
the immunochromatographic test paper comprises an adhesive bottom lining (1), an analysis film (2), a combination pad (3), a sample pad (4) and absorbent paper (5), wherein the analysis film (2) is attached to the middle of the adhesive bottom lining (1), the absorbent pad (5) is attached to one side of the analysis film (2) and forms a first combination area with the analysis film (2), the combination pad (3) is attached to the other side of the analysis film (2) and forms a second combination area with the analysis film (2), the sample pad (4) is attached to the other side of the combination pad (3) and forms a third combination area with the combination pad (3), and a detection line (6) and a quality control line (7) are arranged on the surface of the analysis film (2).
2. The quantitative bovine immunoglobulin G detection test paper based on quantum dot immunochromatography according to claim 1, which is characterized in that: the quality control line (7) on the surface of the analysis membrane (2) of the QD-LF-sandwich method test strip and the QD-LF-competition method test strip are both 2mg/mL goat anti-rabbit polyclonal antibodies, the detection line (6) on the surface of the analysis membrane (2) of the QD-LF-sandwich method test strip is both 3mg/mL rabbit anti-bovine IgG polyclonal antibodies, and the detection line (6) on the surface of the analysis membrane (2) of the QD-LF-competition method test strip is 2mg/mL bovine IgG.
3. The quantitative bovine immunoglobulin G detection test paper based on quantum dot immunochromatography according to claim 1, which is characterized in that: the binding pad (3) of the QD-LF-sandwich test strip takes a QD-labeled rabbit anti-bovine IgG polyclonal antibody as a tracer, and the binding pad (3) of the QD-LF-competition test strip takes QD-labeled bovine IgG as a tracer.
4. A quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography is characterized in that: the method comprises the following steps:
step one, extracting bovine IgG;
step two, measuring the bovine IgG immunity and rabbit anti-bovine IgG polyclonal antibody titer;
thirdly, diluting the goat anti-rabbit polyclonal antibody, the rabbit anti-bovine IgG polyclonal antibody and the bovine IgG with 0.01 mol/L PB buffer solution (phosphate buffer solution) to the concentration for use in membrane spraying, spraying a chromatographic membrane, and drying at 37 ℃;
step four, adding 20 mu L of 0.2 g/mL quantum dots into a 1.5mL centrifuge tube, adding 500 mu L of a marking buffer solution, placing on a constant temperature oscillator, adding 15 mu L of an activating reagent, oscillating for 1h at 1400r/min, adding 0.05mg of rabbit anti-bovine IgG polyclonal antibody or bovine IgG, placing at 25 ℃,1400r/min, oscillating for 2h, adding bovine serum albumin with the final concentration of 1%, continuing oscillating for 1h at 1400r/min, adding 3.88 mL of 0.01 mol/L PB (with the pH value of 7.0), uniformly mixing, quickly pouring onto glass fibers by using a capillary tube, placing at 37 ℃, drying for 3h, drying and storing for later use;
step five, taking 2 mL of chromatography confining liquid (0.01 mol/L PB containing 1% bovine serum albumin, pH value of 7.0), pouring onto the cut glass fiber, placing at 37 ℃ for drying for 2h, and drying and storing for later use;
step six, 2, evaluating the sensitivity, linearity and precision of the test strip and evaluating the test strip on site;
step seven: and (4) performing statistical analysis on the data.
5. The quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography according to claim 4, characterized in that: the first step is as follows:
adding 60mL of bovine serum into a beaker, dropwise adding a sodium acetate solution while stirring, and adjusting the pH value to 4.5; then dropwise adding 5mL of n-octanoic acid solution, uniformly stirring at normal temperature, placing at 4 ℃ for 1h, transferring to a 50 mL centrifuge tube, centrifuging at 12000r/min for 30min, taking supernate, filtering with filter paper, adding 1/10 volume of 0.1moL/L PBS buffer solution (phosphate buffer solution) into the supernate, adjusting the pH value to 7.0, dropwise adding saturated ammonium sulfate solution to 50% of the total volume while stirring, placing at 4 ℃ for 2h, centrifuging at 12000r/min for 30min, and discarding the supernate;
resuspending the centrifuged precipitate with 0.01 moL/L PBS, dialyzing at 4 deg.C, and replacing dialysate for 1 time at 2h intervals until completely removing ammonia ions;
and (4) detecting the concentration of the bovine IgG by using a micro ultraviolet spectrophotometer, and freezing and storing for later use.
6. The quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography according to claim 4, characterized in that: the second step is as follows:
respectively immunizing 2 big-ear white rabbits with the prepared bovine IgG antigen, wherein the immunization mode is that 200mg is injected subcutaneously once, the immunization period is 2 weeks and 1 time, 4 times of immunization are carried out, after 2 weeks of 4 times of immunization, ear marginal venous blood of the 2 big-ear white rabbits is respectively taken to carry out enzyme-linked immunosorbent assay to detect the rabbit anti-bovine IgG polyclonal antibody titer in 2 parts of serum, the immunization is stopped when the dilution multiple of the serum is more than 10 ten thousand and the absorbance (OD value) of 450nm is more than 0.200, carotid blood sampling is carried out, the dilution multiple of the serum is the rabbit anti-bovine IgG polyclonal antibody titer, and the collected 2 parts of rabbit blood is centrifuged at 4 ℃ and 5000r/min for 15min to collect upper layer serum (serum numbers 1# and 2 #); and (3) extracting the rabbit anti-bovine IgG polyclonal antibody in serum by referring to a bovine IgG extraction method, measuring the concentration and the titer of the rabbit anti-bovine IgG polyclonal antibody, and freezing for later use.
7. The quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography according to claim 1, characterized in that: the sixth step is as follows:
sensitivity evaluation: carrying out gradient dilution on bovine IgG by using 0.03 mol/L PB to obtain 0,0.1,0.3,0.5,0.7,1,3,5,7, 10, 30, 50, 70 and 100 mu g/mL series concentration standard products, respectively detecting by using 2 test strips, and repeatedly testing for 3 times by using 0.03 mol/L PB as a blank control;
the standard deviation of the blank control detection band signal (T)/quality control band signal (C) value plus 3 times is the Cutoff value, when the T/C value of the sample is larger than or equal to the Cutoff value, the content of the bovine IgG corresponding to the T/C value of the sample is the detection limit of the sandwich method test strip; the standard deviation of the blank control T/C value average value-3 times is used as the Cutoff value of the competition method test strip, and when the T/C value of the sample is less than or equal to the Cutoff value, the concentration of the bovine IgG corresponding to the T/C value of the sample is the detection limit of the competition method test strip;
and (3) linear evaluation: the sandwich method test strip takes lg (T/C value-Cutoff value) as a horizontal coordinate, the competition method test strip takes lg (T/C value) as a horizontal coordinate, and both the two types of test strips take lg (standard concentration) as a vertical coordinate to draw a quantitative standard curve;
precision evaluation: repeatedly testing each concentration of the standard substance to be tested for 3 times, and calculating the variation Coefficient (CV) of the 3 detection values, wherein the smaller the variation coefficient is, the better the precision is;
and (3) field evaluation: selecting a test strip with optimal detection performance for field detection, wherein samples comprise 50 parts of bovine colostrum (sample numbers are 1-50) and 2 parts of pasteurized milk (sample numbers are 51 and 52), simultaneously measuring by using a refractometer, randomly extracting 5 parts of bovine colostrum and 2 parts of pasteurized milk, and performing liquid chromatography measurement, and comparing the correlation of the bovine IgG content detected by the 3 methods to evaluate the accuracy of the test strip detection method.
8. The quantitative detection method of bovine immunoglobulin G based on quantum dot immunochromatography according to claim 1, characterized in that: the seventh step is as follows: sampling Excel software to carry out statistical analysis of test data, respectively drawing quantitative standard curves of 2 test paper for detecting bovine IgG standard products, drawing a comparison histogram of a refractometer and an optimal test paper for detecting the bovine IgG content in 52 milk samples, drawing a comparison histogram of the refractometer, the optimal test paper and a liquid chromatogram for detecting the bovine IgG content in 7 milk samples, and analyzing the correlation of the detection results of 3 methods by using the Excel software.
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