CN111579774A - Card type standard substance for calibration and quality control, and preparation method and application thereof - Google Patents
Card type standard substance for calibration and quality control, and preparation method and application thereof Download PDFInfo
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- CN111579774A CN111579774A CN202010407461.6A CN202010407461A CN111579774A CN 111579774 A CN111579774 A CN 111579774A CN 202010407461 A CN202010407461 A CN 202010407461A CN 111579774 A CN111579774 A CN 111579774A
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Abstract
The card type standard substance provided by the invention comprises a bottom plate, a sample pad, a standard substance curing film, a combination pad, a base film and absorbent paper, wherein the standard substance is sprayed on the standard substance curing film. The preparation method of the card-type standard substance comprises the following steps: accurately determining the value of the standard substance; diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration; spraying a standard substance solution with a certain concentration on a glass cellulose membrane, and drying to obtain a standard substance solidified membrane; and sequentially overlapping the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper on the bottom plate to obtain the card type standard substance. The preparation method of the card type standard substance is also suitable for preparing card type reference substances, card type calibration substances, card type quality control substances and card type reference substances with similar purposes. The card type standard substance is a brand new form of standard substance, and can be directly used for qualitative detection, semi-quantitative detection, quantitative detection and calibration of related instruments.
Description
Technical Field
The invention belongs to the field of immunochromatography rapid detection, and relates to a card-type standard substance for calibration and quality control, and a preparation method and application thereof.
Background
The immunochromatography technology is an immunoassay mode which is based on specific reaction among antigen and antibodies and carries out qualitative or quantitative detection on a certain substance through a marking material in the early 80 s, and the immunochromatography technology represented by a colloidal gold test strip is widely applied to the fields of food safety, medical inspection, clinical diagnosis, environmental monitoring and the like because of the advantages of rapidness, convenience, sensitivity, accuracy, low price, capability of realizing online rapid detection and monitoring of a target object and the like.
However, most immunochromatography techniques can only carry out qualitative or semi-quantitative detection on a target substance, and limit the application range to a certain extent. In recent years, according to the optical properties of nano-label materials, a corresponding immunochromatographic signal reader is matched with an immunochromatographic detection card to carry out quantitative detection on a target object in a sample by an external standard method. The principle of quantitative detection based on the external standard method is as follows: before sample detection, diluting a standard substance of a target object into a known series of concentrations, detecting by using an immunochromatography detection card, digitizing a signal value of a detection line by using a signal reader, drawing a curve according to the relation between the concentration and the signal intensity, taking the curve as a standard curve for quantitative detection by an external standard method, and embedding the curve into the immunochromatography signal reader; when a sample is detected, the immunochromatographic signal reader can convert the signal intensity of the detection line into a concentration value through an embedded standard curve and output the concentration value, so that the content of a target object in the sample is obtained. However, this method of drawing a quantitative standard curve by diluting the standard substance and embedding it in the instrument has the following disadvantages:
(1) standard substances with a series of concentrations need to be prepared again each time a quantitative standard curve is established, and the quantitative result is inaccurate due to experimental errors caused by factors such as operation, environment and the like;
(2) after the quantitative standard curve is embedded, the flexibility is poor, and due to the change of the detection environment, no corresponding standard substance is used for calibration, so that the quantitative detection result is easy to be inaccurate;
(3) the biological activity of the solution standard substance is reduced due to the problems of storage, transportation, use and the like, and the accuracy of a detection result is influenced, so that the solution standard substance is not matched with a corresponding standard substance to a terminal user generally.
Disclosure of Invention
The invention provides a novel card-type standard substance for calibration and quality control and a preparation method thereof aiming at the defects in the existing fast detection technologies such as immunochromatography and the like.
The invention provides a card-type standard substance for calibration and quality control, which comprises a bottom plate, a sample pad, a standard substance curing film, a combination pad, a base film and absorbent paper, wherein the standard substance curing film is sprayed with the standard substance.
Preferably, the preparation method of the standard substance cured film comprises the following steps:
accurately determining the value of the standard substance;
diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration;
spraying a standard substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 mu L/cm, and drying to obtain a standard substance solidified membrane.
Preferably, the drying is carried out for 1 to 10 hours at a temperature of between 25 and 37 ℃ under vacuum.
Preferably, the sample pad, the standard substance curing film, the bonding pad, the base film and the absorbent paper of the card-type standard substance are respectively overlapped on the bottom plate, the sample pad is pressed on the standard substance curing film, the standard substance curing film is pressed on the bonding pad, and the bonding pad and the absorbent paper are respectively pressed on two ends of the base film.
Preferably, the base film comprises a detection line and a quality control line.
Preferably, the binding pad is sprayed with nano-material labeled antigen or antibody capable of specifically binding with standard substance; the detection line is coated with an antigen or an antibody which can specifically bind to a standard substance, and the quality control line is coated with a quality control antibody.
In a second aspect, the present invention provides a method for preparing the card-type standard substance for calibration and quality control of the present invention, comprising the steps of:
accurately determining the value of the standard substance;
diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration;
spraying a standard substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 mu L/cm, and drying to obtain a standard substance solidified membrane;
and sequentially overlapping the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper on a bottom plate, and cutting the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper into test strips with the width of 2-5mm, namely the card type standard substance.
The preparation method can also be used for preparing reference substances, calibration substances, quality control substances and reference substances for calibration and quality control.
A third aspect of the invention provides the use of the present invention as a calibration, quality control cartridge standard, including use in qualitative, semi-quantitative, quantitative assays in solid support-based rapid assay technologies and calibration of related instruments.
Preferably, the application of the standard substance in the form of a card to quantitative detection comprises the following steps:
taking more than or equal to 3 series contents of the card-type standard substances, respectively dripping negative samples on sample pads of more than or equal to 3 series contents of the card-type standard substances, incubating for 1-30min at 20-40 ℃, digitizing signals by a signal reader, and drawing a quantitative standard curve by taking the concentration of the standard substances as abscissa and the intensity of the signals as ordinate;
embedding the standard curve into a signal reader through an editing program, and when a sample is detected, converting the signal reader into a concentration value through the embedded standard curve according to the signal intensity of a detection line and outputting the concentration value, so that the content of a target object in the sample is obtained.
Compared with the prior art, the invention has the beneficial effects that:
(1) the standard substance is fixed on the standard substance curing membrane, so that the card type standard substance is obtained, and is a brand new form of the standard substance;
(2) the card-type standard substance can be directly used for contrast in semi-quantitative detection, establishment and calibration of a standard curve in quantitative detection and calibration of related instruments in a rapid detection technology based on a solid phase carrier, and the effectiveness of quantity value transmission is ensured, so that the accuracy, comparability and traceability of rapid detection data such as immunochromatography and the like are improved;
(3) the card type standard substance of the invention has accurate and stable quality value, can be matched with the card type standard substance with corresponding series content to a terminal user, and the user can use the card type standard substance immediately, thereby being convenient and rapid, and avoiding the operation that the series concentration standard substance needs to be prepared again when a quantitative standard curve is established in the prior art.
Drawings
FIG. 1 is a schematic diagram of a card-type standard substance;
FIG. 2 is a quantitative curve of a novel coronavirus N-protein human IgG monoclonal antibody card-type standard substance of example 1;
FIG. 3 is a quantitative curve of the novel coronavirus S protein human IgG monoclonal antibody card standard substance of example 2;
in fig. 1, a bottom plate, 2, a sample pad, 3, a standard substance curing film, 4, a combination pad, 5, a base film, 6, absorbent paper, 7, a detection line, 8 and a quality control line.
Detailed Description
Hereinafter, embodiments will be described in detail with respect to the card-type standard substance of the present invention and the preparation method and application thereof, however, these embodiments are exemplary and the present disclosure is not limited thereto. And the drawings used herein are for the purpose of illustrating the disclosure better and are not intended to limit the scope of the invention.
In some embodiments of the present invention, the calibration and quality control card-type standard substance, as shown in fig. 1, includes a base plate 1, a sample pad 2, a standard substance cured film 3, a bonding pad 4, a base film 5, absorbent paper 6, a detection line 7, and a quality control line 8.
In a preferred embodiment, the standard substance cured film is sprayed with a standard substance, and the preparation method of the standard substance cured film comprises the following steps:
accurately determining the value of the standard substance;
diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration;
spraying a standard substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 mu L/cm, and drying to obtain a standard substance solidified membrane.
The standard substance corresponds to a target substance in a sample to be detected, and if the target substance in the sample to be detected is a novel coronavirus nucleocapsid protein (N protein) human IgG monoclonal antibody, the standard substance is a novel coronavirus nucleocapsid protein (N protein) human IgG monoclonal antibody standard substance. The standard substance can be prepared by self or purchased commercially, and before use, the standard substance needs to be subjected to value determination to ensure the accuracy of the content of the standard substance. And (3) adopting two or more different principle methods to carry out valuing on the standard substance, and taking the average value of the valuing results of the two or more methods as the final valuing result of the standard substance.
In a preferred embodiment, the drying is carried out under vacuum at 25 ℃ to 37 ℃ for 1 to 10 hours. Under the vacuum condition, the boiling point of water is reduced, so that the water can be removed at a lower temperature, and the activity of the standard substance is kept. The degree of vacuum may further preferably be 50 to 98 kpa.
The sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper of the card type standard substance are respectively lapped on the bottom plate, the sample pad is pressed on the standard substance curing film, the standard substance curing film is pressed on the combination pad, the combination pad and the absorbent paper are respectively pressed at two ends of the base film, the base film comprises a detection line and a quality control line, and the detection line is closer to the combination pad relative to the quality control line.
The base plate, the sample pad, the combination pad, the base film and the absorbent paper are common parts in the field, the base plate is exemplified by polyvinyl chloride (PVC) and polyethylene plastic, the sample pad and the combination pad are exemplified by a polyester cellulose film and a glass cellulose film, the base film is exemplified by a nitrocellulose film, and the absorbent paper is exemplified by a plant fiber filter paper and a cellulose acetate film, which are merely exemplified and not limited.
The binding pad is sprayed with antigen or antibody marked by nano material, the antigen or antibody can be specifically bound with standard substance, and the nano material can be colloidal gold, graphene, quantum dots and the like; the detection line is coated with an antigen or an antibody which is specifically combined with a standard substance, and the quality control line is coated with a quality control antibody.
The antigen or antibody on the binding pad and the detection line and the quality control antibody of the quality control line are configured according to a competition method or a double-antigen sandwich method or a double-antibody sandwich method or an indirect method or other principles in the rapid detection technology such as immunochromatography and the like.
Examples are: if the standard substance is an antibody, the binding pad is an antigen labeled by a nano material specifically bound with the antibody, the detection line is another antigen capable of being bound with the antibody or a secondary antibody capable of being bound with the antibody, and the quality control line is a quality control antibody. If the standard substance is an antigen, the binding pad is a nano material labeled antibody specifically bound with the antigen, the detection line is the same antigen and can be competitively bound with the antibody, or the detection line is an antibody capable of being bound with the standard substance, the antibody is different from the antibody on the binding pad, and the quality control line is a quality control antibody.
In some embodiments of the invention, the method for preparing the calibration, quality control cartridge standard substance comprises the following steps:
firstly, preparing a standard substance cured film, wherein the preparation method of the standard substance cured film comprises the following steps: accurately determining the value of the standard substance; diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration; spraying a standard substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 muL/cm, and drying to obtain a standard substance solidified membrane;
and sequentially overlapping the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper on a bottom plate, and cutting the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper into test strips with the width of 2-5mm, namely the card type standard substance.
Before the bonding pad is lapped, nano material marked antigen or antibody capable of specifically bonding with standard substance is sprayed; before the base film is lapped, an antigen or an antibody which is specifically combined with a standard substance is sprayed to form a detection line, and then a quality control antibody is sprayed to serve as a quality control line.
The content of the standard substance in the prepared card-type standard substance was obtained according to the following formula:
the content of standard substance in the card-type standard substance (ng) ═ concentration of standard substance sprayed (ng/μ L) × spray rate (μ L/cm) × width of test strip (cm).
And (3) putting the prepared card type standard substance into a plastic card shell marked with corresponding standard substance content, drying at low temperature, sealing and storing.
The preparation method of the card type standard substance can also be used for preparing card type reference substances, card type calibration substances, card type quality control substances and card type reference substances.
During the preparation process, the reference substance, the calibrator, the quality control substance and the reference substance are used for replacing the standard substance, and the corresponding card type reference substance, calibrator, quality control substance and reference substance can be obtained.
The preparation method of the control product in the form of card comprises the following steps:
accurately determining the value of the reference substance;
diluting a reference substance with a fixed value by using a buffer solution to prepare a reference substance solution with a certain concentration;
spraying a reference substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 muL/cm, and drying to obtain a reference substance cured membrane;
and sequentially overlapping the sample pad, the reference substance curing film, the combination pad, the base film and the absorbent paper on a bottom plate, and cutting into test strips with the width of 2-5mm, namely the card-type reference substance.
In some embodiments of the invention, the application of the card-type standard substance comprises qualitative detection, semi-quantitative detection, quantitative detection and calibration of related instruments in a solid-phase carrier-based rapid detection technology.
The semi-quantitative detection is directly interpreted by naked eyes, the color development intensity of the card-type standard substance and the color development intensity of the detection sample are compared, then the detection result is interpreted, and the result is obtained simply and quickly.
The quantitative detection utilizes a signal reader to obtain a more accurate detection result, and the application of the card-type standard substance to the quantitative detection specifically comprises the following steps:
taking more than or equal to 3 series contents of the card-type standard substances, respectively dripping negative samples on sample pads of more than or equal to 3 series contents of the card-type standard substances, incubating for 1-30min at 20-40 ℃, digitizing detection line signals by a signal reader, and drawing a quantitative standard curve by taking the standard substance concentration as a horizontal coordinate and the signal intensity as a vertical coordinate;
embedding the standard curve into a signal reader through an editing program, and when a sample is detected, converting the signal reader into a concentration value through the embedded standard curve according to the signal intensity of a sample detection line and outputting the concentration value, so that the content of a target object in the sample is obtained.
In quantitative detection, the card-type standard substances are used for drawing a standard curve, the number of the card-type standard substances is more than or equal to 3, and the standard substance content of each card-type standard substance is different to form a series of contents. The number of the standard substances in the form of a card is preferably 4 to 6.
The preparation of the cassette standard substance with more than or equal to 3 serial contents comprises the following steps:
accurately determining the value of the standard substance;
diluting the standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with more than or equal to 3 series concentrations;
respectively spraying more than or equal to 3 standard substance solutions with series concentrations on more than or equal to 3 glass cellulose membranes, and drying to obtain more than or equal to 3 standard substance solidified membranes;
and sequentially overlapping the sample pad, the standard substance curing membrane, the combination pad, the base membrane and the water absorption pad on the bottom plate, and shearing to obtain more than or equal to 3 test strips with the width of 2-5mm, namely obtaining more than or equal to 3 series of content card-type standard substances.
The negative sample dripped on the sample pad is a detection sample containing no standard substance component.
Hereinafter, the technical solution of the present invention will be further described and illustrated by specific examples. However, these embodiments are exemplary, and the present disclosure is not limited thereto. Unless otherwise specified, the raw materials used in the following specific examples of the present invention are those commonly used in the art, and the methods used in the examples are those conventional in the art.
Example 1
1.1 fixed value of novel coronavirus nucleocapsid protein (N protein) human IgG monoclonal antibody standard substance
Based on the sequence of a novel coronavirus N gene (28274-29533 site) published in NCBI GenBank, a monoclonal antibody aiming at the 30 th-213 th amino acid sequence at the N end of the N Protein is screened and humanized, expressed by 293T cells, and purified by a Protein A column to obtain an N Protein human IgG monoclonal antibody standard substance raw material. The novel coronavirus N protein human IgG monoclonal antibody standard substance is subjected to valuing by adopting an amino acid hydrolysis isotope dilution mass spectrometry and a peptide fragment enzymolysis isotope dilution mass spectrometry, and the average value of the valuing results of the two methods is taken as a final valuing result of 2.64 +/-0.25 g/kg.
1.2 preparation of novel coronavirus N protein human IgG monoclonal antibody cassette standard substance with series content
1.2.1 preparation of cured film of Standard substance
The standard substance solution of the novel coronavirus N protein human IgG monoclonal antibody was diluted to different concentrations with a buffer solution containing a protein protectant (0.01M PBS, pH 7.4, buffer solution containing 3% by mass of bovine serum albumin as a protein protectant): 50. 100, 200 and 400 mu g/mL, then respectively spraying the solution on a glass cellulose membrane at the spraying speed of 2 mu L/cm to obtain a series of standard substance solidified membranes with different contents, drying the standard substance solidified membranes at 30 ℃ for 4 hours under the vacuum degree of 90kpa, sealing, drying and storing.
1.2.2 preparation of gold-labeled antigen
Adjusting the pH value of 1mL of colloidal gold solution to 9.0, uniformly stirring by using a constant speed stirrer, simultaneously dropwise adding 100 mu L of novel coronavirus N protein with the concentration of 50 mu g/mL, reacting for 1 hour, adding a sealing agent (10% bovine serum albumin solution), continuously stirring for 30min, centrifuging for 30min at 4 ℃ to obtain gold-labeled antigen precipitate, adding a resuspension solution, uniformly mixing, and moving to a clean beaker for later use. The resuspension solution is as follows: 0.01M PBS buffer containing 1% bovine serum albumin, 0.2% sucrose, 0.1% Tween-20, 0.05% PEG4000, 0.05% Triton X-100, 0.01% Procline 300.
1.2.3 preparation of conjugate pad, nitrocellulose Membrane detection line and quality control line
Uniformly spraying the gold-labeled antigen solution on a glass cellulose membrane to obtain a bonding pad, drying, and sealing for storage; uniformly spraying the diluted mouse anti-human IgG monoclonal antibody on a nitrocellulose membrane to obtain a detection line; and (3) uniformly spraying the diluted goat anti-mouse IgG monoclonal antibody on a nitrocellulose membrane to obtain a quality control line, drying, and sealing for storage.
1.2.4 Assembly of card Standard substances
The sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are overlapped and fixed on a PVC base plate in sequence, the combination pad and the absorbent paper are pressed at two ends of the nitrocellulose membrane respectively, the standard substance curing membrane is pressed on the combination pad, the sample pad is pressed on the standard substance curing membrane, the sample pad is cut into test strips with the width of 3.5mm, the test strips are arranged in a plastic card shell marked with corresponding standard substance content to prepare novel human IgG monoclonal antibody card type standard substances with serial contents of coronavirus N protein, and the contents of the standard substances are respectively 35 ng, 70 ng, 140 ng and 280ng according to the spraying speed and the width of the test strips.
1.3 application of novel coronavirus N protein human IgG monoclonal antibody card type standard substance 1.3.1 standard curve drawing
10 mu L of negative serum was dropped into the sample pad of the prepared test strip containing the novel coronavirus N protein human IgG monoclonal antibody card-type standard substance in the series of contents, and 80 mu L of sample diluent (0.01M PBS buffer containing 1% bovine serum albumin and 0) was added.01% Procline300 and 0.1% Tween 20), incubating at 37 deg.C for 15min, digitizing the detection line signal with immunochromatography signal reader, and drawing quantitative standard curve with standard substance concentration as abscissa and signal intensity as ordinate, the result is shown in FIG. 2, and correlation coefficient R is20.9938, the kit has good linearity, and can be used for quantitative detection of the novel coronavirus N protein IgG monoclonal antibody in serum.
1.3.2 determination of clinical serum samples
Embedding the standard curve drawn by 1.3.1 into an immunochromatography signal reader;
a sample pad of a colloidal gold immunochromatography detection card (the detection card is different from a card-type standard substance only in that a standard substance solidified membrane is not included, and other standard substances are the same as the card-type standard substance) is added with 10 mu L of clinical serum sample, then 80 mu L of sample diluent is added, after incubation for 15min at 37 ℃, an immunochromatography signal reader is used for obtaining a detection line signal, and according to an embedded quantitative standard curve, the output result is shown in the following table.
TABLE 1 concentration of novel coronavirus N protein IgG monoclonal antibody in clinical serum samples
| Sample numbering | Novel coronavirus N protein IgG monoclonal antibody concentration (mu g/mL) |
| 1 | 0 |
| 2 | 17.7 |
| 3 | 8.1 |
| 4 | 10.3 |
| 5 | 6.4 |
Example 2
2.1 fixed value of novel coronavirus spike glycoprotein (S protein) human IgG monoclonal antibody standard substance
Based on the sequence of a novel coronavirus S gene (21563-25384 site) published in NCBI GenBank, a monoclonal antibody aiming at a receptor binding sequence of an S Protein S1 fragment is screened and humanized, expressed by 293T cells and purified by a Protein A column to obtain a S Protein humanized IgG monoclonal antibody standard substance raw material. The novel coronavirus S protein human IgG monoclonal antibody standard substance is subjected to valuing by adopting an amino acid hydrolysis isotope dilution mass spectrometry and a peptide fragment enzymolysis isotope dilution mass spectrometry, and the average value of the valuing results of the two methods is used as a final valuing result of 1.77 +/-0.14 g/kg.
2.2 preparation of novel coronavirus S protein human IgG monoclonal antibody cassette standard substance with series content
2.1 preparation of cured films of Standard substances
The standard substance solution of the novel coronavirus S protein human IgG monoclonal antibody was diluted to different concentrations with a buffer solution containing a protein protectant (0.01M PBS, pH 7.4 buffer solution, bovine serum albumin containing 3% by mass fraction as a protein protectant): 100. 200, 400 and 800 mu g/mL, then respectively spraying the solution on a glass cellulose membrane at the spraying speed of 2 mu L/cm to obtain a series of standard substance solidified membranes with different contents, drying the standard substance solidified membranes at 32 ℃ for 4 hours under the vacuum degree of 90kpa, sealing, drying and storing.
2.2 preparation of gold-labeled antigen
Adjusting the pH value of 1mL of colloidal gold solution to 8.0, uniformly stirring by using a constant speed stirrer, simultaneously dropwise adding 100 mu L of novel coronavirus S protein with the concentration of 80 mu g/mL, reacting for 1 hour, adding a sealing agent (10% bovine serum albumin solution), continuously stirring for 30min, centrifuging for 30min at 4 ℃ to obtain gold-labeled antigen precipitate, adding a resuspension solution, uniformly mixing, and moving to a clean beaker for later use. The resuspension solution is as follows: 0.01M PBS buffer containing 1% bovine serum albumin, 0.2% sucrose, 0.1% Tween-20, 0.05% PEG4000, 0.05% Triton X-100, 0.01% Procline 300.
2.3 preparation of conjugate pad, nitrocellulose Membrane detection line and quality control line
Uniformly spraying the gold-labeled antigen solution on a glass cellulose membrane to obtain a bonding pad, drying, and sealing for storage; uniformly spraying the diluted mouse anti-human IgG monoclonal antibody on a nitrocellulose membrane to obtain a detection line; and (3) uniformly spraying the diluted goat anti-mouse IgG monoclonal antibody on a nitrocellulose membrane to obtain a quality control line, drying, and sealing for storage.
2.4 assembling of card-type Standard substance
The sample pad, the combination pad, the nitrocellulose membrane and the absorbent paper are overlapped and fixed on a PVC base plate in sequence, the combination pad and the absorbent paper are pressed at two ends of the nitrocellulose membrane respectively, the standard substance curing membrane is pressed on the combination pad, the sample pad is pressed on the standard substance curing membrane, the sample pad is cut into test strips with the width of 3.5mm, the test strips are arranged in a plastic card shell marked with corresponding standard substance content to prepare novel coronavirus S protein human IgG monoclonal antibody card type standard substances with series content, and the standard substance content is converted into 70 ng, 140 ng, 280ng and 560ng respectively according to the spraying speed and the test strip width.
3. Application of novel coronavirus S protein human IgG monoclonal antibody cassette standard substance
3.1 Standard Curve plotting
Dripping 10 μ L of negative serum into the sample pad of the prepared series of novel coronavirus S protein human IgG monoclonal antibody card type standard substance test strip, adding 80 μ L of sample diluent (0.01M PBS buffer solution containing 1% bovine serum albumin, 0.01% Procline300 and 0.1% Tween 20), incubating at 37 deg.C for 15min, digitizing the detection line signal by an immunochromatography signal reader, drawing quantitative standard curve with the standard substance concentration as abscissa and the signal intensity as ordinate, and obtaining the result shown in FIG. 3, wherein the correlation coefficient R is20.9908, the kit has good linearity, and can be used for quantitative detection of the novel coronavirus S protein IgG monoclonal antibody in serum.
3.2 determination of clinical serum samples
Embedding the standard curve drawn by 3.1 into an immunochromatography signal reader;
a sample pad of a colloidal gold immunochromatography detection card (the detection card is different from a card-type standard substance only in that a standard substance solidified membrane is not included, and other standard substances are the same as the card-type standard substance) is added with 10 mu L of clinical serum sample, then 80 mu L of sample diluent (the same as the above) is added, after incubation for 15min at 37 ℃, an immunochromatography signal reader is used for acquiring a detection line signal, and according to an embedded quantitative standard curve, the output result is shown in the following table.
TABLE 2 novel coronavirus S protein IgG monoclonal antibody concentrations in clinical serum samples
| Sample numbering | Novel coronavirus S protein IgG monoclonal antibody concentration (mu g/mL) |
| 1 | 0 |
| 2 | 50.1 |
| 3 | 23.0 |
| 4 | 38.7 |
| 5 | 11.9 |
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Claims (10)
1. The card type standard substance for calibration and quality control is characterized by comprising a bottom plate, a sample pad, a standard substance curing film, a combination pad, a base film and absorbent paper, wherein the standard substance is sprayed on the standard substance curing film.
2. The card-type standard substance according to claim 1, characterized in that the method for preparing the standard substance cured film comprises the steps of:
accurately determining the value of the standard substance;
diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration;
spraying a standard substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 mu L/cm, and drying to obtain a standard substance solidified membrane.
3. The card-type standard substance according to claim 2, wherein the drying is drying under vacuum at 25 ℃ -37 ℃ for 1-10 hours.
4. The card type standard substance according to claim 1, wherein a sample pad, a standard substance cured film, a bonding pad, a base film and a water absorbent paper of the card type standard substance are respectively lapped on the base plate, the sample pad is pressed against the standard substance cured film, the standard substance cured film is pressed against the bonding pad, and the bonding pad and the water absorbent paper are respectively pressed against both ends of the base film.
5. The card type standard substance according to claim 1, characterized in that a detection line and a quality control line are contained on a base film of the card type standard substance.
6. The card-type standard substance according to claim 5, wherein the binding pad is sprayed with a nanomaterial-labeled antigen or antibody that can specifically bind to a standard substance; the detection line is coated with an antigen or an antibody which can specifically bind to a standard substance, and the quality control line is coated with a quality control antibody.
7. A method for preparing the card-type standard substance according to claim 1, comprising the steps of:
accurately determining the value of the standard substance;
diluting a standard substance with a fixed value by using a buffer solution to prepare a standard substance solution with a certain concentration;
spraying a standard substance solution with a certain concentration on a glass cellulose membrane at a spraying speed of 1.0-10.0 muL/cm, and drying to obtain a standard substance solidified membrane;
and sequentially overlapping the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper on a bottom plate, and cutting the sample pad, the standard substance curing film, the combination pad, the base film and the absorbent paper into test strips with the width of 2-5mm, namely the card type standard substance.
8. The method for preparing the card-type standard substance according to claim 7, wherein the method is further used for preparing a card-type reference substance, a card-type calibration substance, a card-type quality control substance and a card-type reference substance for calibration and quality control.
9. Use of the card-type standard substance according to claim 1, comprising qualitative detection, semi-quantitative detection, quantitative detection and calibration of the related instruments for rapid detection technologies based on solid phase carriers.
10. The use of the card-type standard substance according to claim 9, wherein the use of the card-type standard substance for quantitative detection comprises the steps of:
taking more than or equal to 3 series contents of the card-type standard substances, respectively dripping negative samples on sample pads of more than or equal to 3 series contents of the card-type standard substances, incubating for 1-30min at 20-40 ℃, digitizing signals by a signal reader, and drawing a quantitative standard curve by taking the concentration of the standard substances as abscissa and the intensity of the signals as ordinate;
and embedding the standard curve into a signal reader, and when a sample is detected, converting the signal reader into a concentration value through the embedded standard curve according to the signal intensity of the sample and outputting the concentration value, thereby obtaining the content of the target object in the sample.
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| CN202010407461.6A CN111579774A (en) | 2020-05-14 | 2020-05-14 | Card type standard substance for calibration and quality control, and preparation method and application thereof |
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| CN202010407461.6A CN111579774A (en) | 2020-05-14 | 2020-05-14 | Card type standard substance for calibration and quality control, and preparation method and application thereof |
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