CN111896749A - Bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method - Google Patents
Bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, preparation method, kit and detection method Download PDFInfo
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Abstract
The invention provides a bovine milk immunoglobulin (IgG) double-antibody sandwich colloidal gold immunochromatographic test strip, a preparation method, a kit and a detection method. The colloidal gold immunochromatographic test strip is characterized by comprising a bottom plate, a water absorption pad, a chromatographic membrane, a conjugate release pad and a sample pad, wherein the water absorption pad, the chromatographic membrane, the conjugate release pad and the sample pad are overlapped on the PVC bottom plate, the conjugate release pad is coated with a colloidal gold-labeled bovine lactoferrin antibody a compound, the chromatographic membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-bovine lactoferrin antibody b, and the quality control line is coated with goat anti-mouse IgG. The invention also provides a preparation method of the chromatography test strip, a kit comprising the reagent strip and a detection method of the kit. The technical scheme of the invention is prepared based on the principles of double-antibody sandwich colloidal gold color development and gold-labeled immunoassay quantitative analysis, and can be used for rapidly and quantitatively detecting the bovine lactoferrin (IgG) content.
Description
Technical Field
The invention relates to the field of detection reagent strips and kits, in particular to a bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, a preparation method of the reagent strip, a kit comprising the reagent strip and a method for detecting by using the kit.
Background
Lactoferrin (Lactoferrin, LF) is an iron-binding glycoprotein with a molecular weight of about 80kDa, a member of the glycoprotein family, first isolated from bovine milk in 1938 until 1960, where Johansson et al isolated Lactoferrin from human milk, mainly found in human and animal milk, is an important non-heme iron-binding glycoprotein in milk. Lactoferrin belongs to basic protein, can be specifically or non-specifically combined with metal cations, biological macromolecules and the like, is a novel natural nutritional protein, has various amino acids required by a human body, has a series of biological functions, can participate in iron transportation and promote iron absorption, and also has various functions of resisting bacteria, sterilizing, resisting viruses, regulating body immunity and the like, so that the lactoferrin is widely used in the fields of medicine and food, and is particularly used as a food additive for dairy products and formula food thereof. Due to the fact that dairy products are numerous and have different qualities, a detection method which is convenient, rapid and accurate in quantification is urgently needed.
At present, the main detection methods of lactoferrin are High Performance Liquid Chromatography (HPLC), electrophoresis and enzyme-linked immunosorbent assay (ELISA), and the HPLC method has the characteristics of high accuracy, repeatability and accurate quantification, but the instrument is complex, the sample needs to be pretreated, and the detection time is long; the electrophoresis method mainly comprises polyacrylamide gel electrophoresis (SDS-PAGE) and high-efficiency capillary electrophoresis (HPCE), and the SDS-PAGE method has high accuracy and good repeatability, but has complicated operation steps and long detection time; the HPCE method is simple to operate, but the result repeatability is poor; the ELISA method has high flux, is simple and convenient in steps compared with an instrument method, is time-consuming and low in sensitivity, is easy to generate false negative, and is not suitable for rapid on-site screening; the colloidal gold immunochromatographic assay is widely applied to the field of medicine and disease detection at present, but no report is made in bovine lactoferrin detection.
Disclosure of Invention
The invention aims to overcome the defects of the field of bovine lactoferrin detection in the prior art and the defect that high-throughput treatment cannot be realized, and provides the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, the preparation method, the kit and the detection method, which can realize rapid quantitative detection of bovine lactoferrin in a sample, greatly shorten the detection time and realize rapid and accurate screening of a large batch of samples.
In order to achieve the purpose, the invention adopts the following technical scheme:
the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip comprises a bottom plate, a water absorption pad, a chromatographic membrane, a conjugate release pad and a sample pad, wherein the water absorption pad, the chromatographic membrane, the conjugate release pad and the sample pad are overlapped on the bottom plate, the conjugate release pad is coated with a colloidal gold-labeled bovine lactoferrin antibody a compound, the chromatographic membrane is provided with a detection line and a quality control line, the detection line is coated with an anti-bovine lactoferrin antibody b, and the quality control line is coated with goat anti-mouse IgG.
Preferably, the detection line on the chromatographic membrane is coated with the anti-bovine lactoferrin antibody b with the concentration of 0.5-2mg/mL, and the quality control line on the chromatographic membrane is coated with the goat anti-mouse IgG with the concentration of 0.5-1 mg/mL.
Preferably, the anti-bovine lactoferrin antibody a and the anti-bovine lactoferrin antibody b are both one of an anti-bovine lactoferrin monoclonal antibody, an anti-bovine lactoferrin polyclonal antibody or an anti-bovine lactoferrin recombinant antibody.
Preferably, the anti-bovine lactoferrin antibody a and the anti-bovine lactoferrin antibody b are both anti-bovine lactoferrin monoclonal antibodies.
The test strip realizes detection based on a double-antibody sandwich principle, the bovine lactoferrin antibody compound marked by the colloidal gold and immobilized on the conjugate release pad and the anti-bovine lactoferrin antibody of the detection line respectively identify different antigenic determinants of bovine lactoferrin, when a sample to be detected contains bovine lactoferrin, the conjugate release pad is firstly used for forming a bovine lactoferrin-colloidal gold-bovine lactoferrin antibody a compound, so that the bovine lactoferrin-colloidal gold-bovine lactoferrin antibody a compound is combined with the anti-bovine lactoferrin antibody b on the detection line and is developed on the detection line, and the quality control line is developed all the time to ensure that the test strip is effective. When the sample to be detected does not contain bovine lactoferrin, only the quality control line is developed.
The application also provides a preparation method of the colloidal gold-labeled bovine lactoferrin antibody a compound, which comprises the following steps:
10mL of the calcined colloidal gold solution was added to the solution at 0.1M K2CO3Regulating the pH value of the solution to 7.5, dropwise adding 1mL of bovine lactoferrin antibody solution with the concentration of 110 mu g/mL under magnetic stirring, uniformly mixing, and standing for 1 h;
and (3) dropwise adding 1.3mL of boric acid buffer solution containing 10% BSA and 1% PEG into the solution after standing, uniformly mixing, standing for 1h, centrifuging at 13500r/min for 30min, discarding the supernatant, re-dissolving with the boric acid buffer solution, repeatedly centrifuging once, and finally precipitating to a constant volume of 1mL with 1% BSA and 5% sucrose, namely the colloidal gold-labeled bovine lactoferrin antibody a compound concentrated solution.
Preferably, the colloidal gold is 20-60nm colloidal gold particles obtained by firing chloroauric acid under reduction of sodium citrate.
Preferably, the colloidal gold particles are 20 to 60 nm.
The invention also provides a preparation method of the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, which comprises the following steps:
the conjugate release pad is coated with a bovine lactoferrin monoclonal antibody a marked by colloidal gold, the concentration of the bovine lactoferrin monoclonal antibody a is 0.5-1mg/mL, and the bovine lactoferrin monoclonal antibody a is obtained by spraying and drying a gold spraying and membrane scratching instrument;
The invention also provides a double-antibody sandwich colloidal gold immunochromatographic kit for quantitatively detecting bovine lactoferrin, wherein the kit comprises the colloidal gold immunochromatographic test strip and a gold-labeled immunoassay analyzer.
The invention also provides a detection method based on the quantitative detection of the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic kit, which comprises the following steps:
(1) placing the colloidal gold immunochromatographic test paper horizontally; a series of samples containing bovine lactoferrin concentration gradients are sucked, and 4 drops of each sample with each concentration gradient are respectively dripped onto a sample pad;
(2) the sample is specifically combined with the bovine lactoferrin antibody a complex labeled by the colloidal gold on the conjugate release pad through chromatography;
(3) further passing the bovine lactoferrin in the sample and the bovine lactoferrin antibody compound marked by the colloidal gold through a detection line and a quality control line;
(4) standing for 10-15 min;
(5) the color development intensity of the detection line and the quality control line is measured by a gold-labeled immunoassay analyzer, so that the bovine lactoferrin content in the sample is accurately quantified.
The application quantitative determination bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic kit adopts the colloidal gold immunochromatographic test strip to combine with a gold-labeled immunoassay analyzer, can realize rapid quantitative determination of bovine lactoferrin in a sample, greatly shortens the detection time, and can realize rapid and accurate screening of large-batch samples. The detection sensitivity is 100ppb, compared with the conventional high performance liquid chromatography and ELISA detection kit method, the kit provided by the invention can obtain results within 10-15min, has the characteristics of rapidness, convenience, sensitivity and the like, and is suitable for screening large-batch samples.
Drawings
FIG. 1 is a schematic diagram of a colloidal gold immunochromatographic test strip-based method for detecting bovine lactoferrin content in the present invention.
FIG. 2 is a result determination chart of the colloidal gold immunochromatographic test strip of the present invention.
FIG. 3 is a result chart of the colloidal gold immunochromatographic test strip for detecting bovine lactoferrin at different concentrations.
FIG. 4 is a diagram of the results of the detection of the milk sample by the colloidal gold immunochromatographic test paper at different dilution times.
FIG. 5 is a graph showing the measurement results of the detection of the practical sample of the present invention.
The figure is marked with: 1. a water absorbent pad; 2. a chromatographic membrane; 3. a quality control line; 4. detecting lines; 5. a conjugate release pad; 6. a sample pad; 7. PVC bottom plate.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
Example one
As shown in fig. 1, the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip provided by this embodiment includes a base plate, and a water absorption pad 1, a chromatography membrane 2, a conjugate release pad 5, and a sample pad 6 that are overlapped on a PVC base plate, where the conjugate release pad is coated with a bovine lactoferrin antibody a complex labeled with colloidal gold, and the chromatography membrane is provided with a detection Line (TestLine, T Line) and a quality Control Line (Control Line, C Line), the detection Line is coated with an anti-bovine lactoferrin antibody b, and the quality Control Line is coated with goat anti-mouse IgG.
The detection line on the chromatographic membrane is coated with an anti-bovine lactoferrin antibody b with the concentration of 0.5-2 mg/mL. The quality control line on the chromatographic membrane is coated with goat anti-mouse IgG with the concentration of 0.5-1 mg/mL.
The anti-bovine lactoferrin antibody a and the anti-bovine lactoferrin antibody b are both one of anti-bovine lactoferrin monoclonal antibodies or anti-bovine lactoferrin polyclonal antibodies or anti-bovine lactoferrin recombinant antibodies. In this embodiment, the anti-bovine lactoferrin antibody a and the anti-bovine lactoferrin antibody b are both anti-bovine lactoferrin monoclonal antibodies.
The test strip realizes detection based on a double-antibody sandwich principle, the bovine lactoferrin antibody compound marked by the colloidal gold and immobilized on the conjugate release pad and the bovine lactoferrin antibody of the detection line respectively identify different antigenic determinants of bovine lactoferrin, and when a sample to be detected contains bovine lactoferrin, a bovine lactoferrin-colloidal gold-bovine lactoferrin antibody a compound is formed on the conjugate release pad at first, so that the bovine lactoferrin antibody b is combined with the bovine lactoferrin antibody b on the detection line.
FIG. 2 shows the test strip result of the rapid test of the present application, wherein the test strip is colored in the test line, and the quality control line is colored all the time to ensure the test strip to be effective. When the sample to be detected does not contain bovine lactoferrin, only the quality control line is developed.
In a specific embodiment, the positive sample is selected from plain milk and the negative sample is selected from Youmian.
In a specific example, the added concentration of the bovine lactoferrin sample standard in the negative sample is 100, 200, 500, 1000, 2000, 5000, and 10000 μ g/L, and the detection result is shown in fig. 3.
Example two
The application also provides a preparation method of the colloidal gold-labeled bovine lactoferrin antibody a compound, which comprises the following steps:
10mL of the calcined colloidal gold solution was added to the solution at 0.1M K2CO3Regulating the pH value of the solution to 7.5, dropwise adding 1mL of bovine lactoferrin antibody solution with the concentration of 110 mu g/mL under magnetic stirring, uniformly mixing, and standing for 1 h;
and (3) dropwise adding 1.3mL of boric acid buffer solution containing 10% BSA and 1% PEG into the solution after standing, uniformly mixing, standing for 1h, centrifuging at 13500r/min for 30min, discarding the supernatant, re-dissolving with the boric acid buffer solution, repeatedly centrifuging once, and finally precipitating to a constant volume of 1mL with 1% BSA and 5% sucrose, namely the colloidal gold-labeled bovine lactoferrin antibody a compound concentrated solution.
The colloidal gold is 20-60nm colloidal gold particles obtained by burning chloroauric acid under the reduction of sodium citrate. The colloidal gold particles are 20-60 nm.
EXAMPLE III
The embodiment provides a preparation method of a bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip, which comprises the following steps:
the conjugate release pad is coated with a bovine lactoferrin monoclonal antibody a marked by colloidal gold, the concentration of the bovine lactoferrin monoclonal antibody a is 0.5-1mg/mL, and the bovine lactoferrin monoclonal antibody a is obtained by spraying and drying a gold spraying and membrane scratching instrument;
Example four
The embodiment of the invention provides a double-antibody sandwich colloidal gold immunochromatographic kit for quantitatively detecting bovine lactoferrin, which comprises the colloidal gold immunochromatographic test strip and a gold-labeled immunoassay analyzer.
The kit provided by the embodiment is prepared based on the principles of double-antibody sandwich colloidal gold color development and gold-labeled immunoassay quantitative analyzer quantification, can be used for rapidly and quantitatively detecting the bovine lactoferrin content, and has the detection sensitivity of 100 ppb.
EXAMPLE five
The invention also provides a detection method based on the quantitative detection of the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic kit, which comprises the following steps:
(1) placing the colloidal gold immunochromatographic test paper horizontally; a series of samples containing bovine lactoferrin concentration gradients are sucked, and 4 drops of each sample with each concentration gradient are respectively dripped onto a sample pad;
(2) the sample is specifically combined with the bovine lactoferrin antibody a complex labeled by the colloidal gold on the conjugate release pad through chromatography;
(3) further passing the bovine lactoferrin in the sample and the bovine lactoferrin antibody compound marked by the colloidal gold through a detection line and a quality control line;
(4) standing for 10-15 min;
(5) the color development intensity of the detection line and the quality control line is measured by a gold-labeled immunoassay analyzer, so that the bovine lactoferrin content in the sample is accurately quantified.
In one specific use example listed in this example, the positive sample is selected from plain milk and the negative sample is selected from Youmian. The adding concentration of the bovine lactoferrin sample standard in the negative sample is 100, 200, 500, 1000, 2000, 5000 and 10000 mug/L, and the detection result is shown in figure 3.
FIG. 4 is a diagram of the results of the detection of the milk sample by the colloidal gold immunochromatographic test paper at different dilution times. Sample matrix effect: measuring 2mL of liquid milk sample stored at 4 ℃, diluting the sample with ultrapure water at a ratio of 1:10, 1:100 and 1:1000 respectively when the sample is recovered to room temperature, testing by using the colloidal gold immunochromatography test strip, and selecting the lowest dilution multiple capable of detecting positive reaction as the dilution multiple in the detection of the actual sample.
The result is judged as shown in figure 2, and after the sample to be detected is added for 10-15min, if the T line is not developed, the C line is developed, which shows that the milk sample does not contain bovine lactoferrin, and the result is negative; if the T line and the C line are colored, the milk sample contains bovine lactoferrin, the result is positive, the color development intensity of the test strip can be read by combining a gold-labeled immunoassay analyzer, and the test strip color development intensity is compared with a standard curve to accurately quantify the bovine lactoferrin content in the sample; if the C line does not appear, the reagent or the test paper is invalid.
This example provides a method for demonstrating the accurate quantitative determination of bovine lactoferrin (IgG) content in bovine colostrum samples using the colloidal gold immunochromatographic kit of the present invention.
The operation steps are the same as those in the fifth embodiment, after 9 parts of bovine colostrum sample is cooled to room temperature, diluted by ultrapure water according to a certain proportion, and detected by colloidal gold test strips and high performance liquid chromatography respectively. The results are shown in FIG. 5. Wherein "+" indicates that two red bands appear in the detection result, and "-" indicates that the detection result only has one red band on the C line.
Claims (10)
1. The bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip is characterized by comprising a bottom plate, a water absorption pad, a chromatographic membrane, a conjugate release pad and a sample pad, wherein the water absorption pad, the chromatographic membrane, the conjugate release pad and the sample pad are arranged on the bottom plate in an overlapped mode, a bovine lactoferrin antibody a complex marked by colloidal gold is coated on the conjugate release pad, a detection line and a quality control line are arranged on the chromatographic membrane, an anti-bovine lactoferrin antibody b is coated on the detection line, and a goat anti-mouse IgG is coated on the quality control line.
2. The colloidal gold immunochromatographic test strip according to claim 1, wherein the detection line on the chromatographic membrane is coated with anti-bovine lactoferrin antibody b at a concentration of 0.5-2mg/mL, and the quality control line on the chromatographic membrane is coated with goat anti-mouse IgG at a concentration of 0.5-1 mg/mL.
3. The colloidal gold immunochromatographic test strip according to claim 1, wherein the anti-bovine lactoferrin antibody a and the anti-bovine lactoferrin antibody b are both one of anti-bovine lactoferrin monoclonal antibodies or anti-bovine lactoferrin polyclonal antibodies or anti-bovine lactoferrin recombinant antibodies.
4. The colloidal gold immunochromatographic test strip according to claim 3, wherein the anti-bovine lactoferrin antibody a and the anti-bovine lactoferrin antibody b are both anti-bovine lactoferrin monoclonal antibodies.
5. A preparation method of a colloidal gold-labeled bovine lactoferrin antibody a compound is characterized by comprising the following steps:
10mL of the calcined colloidal gold solution was added to the solution at 0.1M K2CO3Regulating the pH value of the solution to 7.5, dropwise adding 1mL of bovine lactoferrin antibody solution with the concentration of 110 mu g/mL under magnetic stirring, uniformly mixing, and standing for 1 h;
and (3) dropwise adding 1.3mL of boric acid buffer solution containing 10% BSA and 1% PEG into the solution after standing, uniformly mixing, standing for 1h, centrifuging at 13500r/min for 30min, discarding the supernatant, re-dissolving with the boric acid buffer solution, repeatedly centrifuging once, and finally precipitating to a constant volume of 1mL with 1% BSA and 5% sucrose, namely the colloidal gold-labeled bovine lactoferrin antibody a compound concentrated solution.
6. The method for preparing a colloidal gold-labeled bovine lactoferrin antibody-a complex according to claim 5, wherein the colloidal gold is 20 to 60nm colloidal gold particles obtained by firing chloroauric acid under reduction of sodium citrate.
7. The method for preparing a colloidal gold-labeled bovine lactoferrin antibody-a complex according to claim 6, wherein the colloidal gold particles are 20 to 60 nm.
8. The preparation method of the bovine lactoferrin double-antibody sandwich colloidal gold immunochromatographic test strip is characterized by comprising the following steps of:
step 1, preparing a sample pad: the sample pad adopts a glass fiber membrane, the glass fiber membrane is treated by phosphate buffer solution containing 1% BSA, 2% sucrose, 0.1% PEG and 0.05% Tween-20 for 1h, and then the sample pad is prepared by drying;
step 2, preparation of conjugate release pad: the conjugate release pad adopts a glass fiber membrane, the glass fiber membrane is pretreated by phosphate buffer solution containing 1% BSA, 2% sucrose, 0.1% PEG and 0.05% Tween-20 for 1h, and then dried for standby; diluting the concentrate of the colloidal gold-labeled bovine lactoferrin antibody a complex prepared by the method of any one of claims 7, 8 or 9 by 10-20 times, uniformly spraying the diluted concentrate onto a pretreated glass fiber membrane at 3 μ L/cm to obtain a conjugate release pad coated with the colloidal gold-labeled bovine lactoferrin antibody a complex, drying the conjugate release pad at 37 ℃ for 24 hours, and sealing the conjugate release pad for later use;
the conjugate release pad is coated with a bovine lactoferrin monoclonal antibody a marked by colloidal gold, the concentration of the bovine lactoferrin monoclonal antibody a is 0.5-1mg/mL, and the bovine lactoferrin monoclonal antibody a is obtained by spraying and drying a gold spraying and membrane scratching instrument;
step 3, preparing a chromatographic membrane: the chromatographic membrane adopts a nitrocellulose membrane, a detection line and a quality control line are arranged on the chromatographic membrane, the detection line is coated with 1mg/mL of anti-bovine lactoferrin monoclonal antibody b, and the quality control line is coated with 0.5mg/mL of goat anti-mouse anti-IgG;
step 4, assembling the test strip: the sample pad, the conjugate release pad and the chromatographic membrane are sequentially pressed on the PVC base plate, and are overlapped by 2-3 mm at the edge; and pressing the water absorption pad on the other end of the chromatographic membrane, and overlapping the edge of the water absorption pad by 2-3 mm to obtain the assembled quantitative detection cow milk immunoglobulin double-antibody sandwich colloidal gold immunochromatographic test strip.
9. A double-antibody sandwich colloidal gold immunochromatographic kit for quantitatively detecting bovine milk immunoglobulin, which is characterized by comprising the colloidal gold immunochromatographic test strip of any one of claims 1, 2, 3, 4 or 5 and a gold-labeled immunoassay analyzer.
10. A method of detection, the method comprising the steps of:
placing the colloidal gold immunochromatographic test paper horizontally; a series of samples containing bovine lactoferrin concentration gradients are sucked, and 4 drops of each sample with each concentration gradient are respectively dripped onto a sample pad;
the sample is specifically combined with the bovine lactoferrin antibody a complex labeled by the colloidal gold on the conjugate release pad through chromatography;
further passing the bovine lactoferrin in the sample and the bovine lactoferrin antibody compound marked by the colloidal gold through a detection line and a quality control line;
standing for 10-15 min;
the color development intensity of the detection line and the quality control line is measured by a gold-labeled immunoassay analyzer, so that the bovine lactoferrin content in the sample is accurately quantified.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20220146506A1 (en) * | 2020-11-10 | 2022-05-12 | Hangzhou Fudemin Biotechnology Co., Ltd. | Test strip for milk immunofluorescence assay (ifa) and use thereof |
| CN115420893A (en) * | 2022-07-11 | 2022-12-02 | 昂科生物医学技术(苏州)有限公司 | PSEP colloidal gold test paper, kit, preparation method and application thereof |
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