CN118330205A - Quantitative detection kit for anti-RA 33 antibody and application thereof - Google Patents
Quantitative detection kit for anti-RA 33 antibody and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of medical examination, and particularly discloses an anti-RA 33 antibody quantitative detection kit and application thereof. The kit comprises a detection card and a diluent; the detection card consists of a bottom plate, and a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially lapped and stuck on the bottom plate along the sample chromatographic direction; the binding pad is provided with a mouse anti-human IgG monoclonal antibody and a chicken IgY antibody which are respectively marked by fluorescent microspheres; the detection pad is coated with RA33 antigen and rabbit anti-chicken IgY polyclonal antibody at different positions to form a detection line and a quality control line respectively; the RA33 antigen is any one of amino acid sequences shown as SEQ ID NO. 1-5. The kit can realize rapid and single quantitative detection by using the fluorescent immunochromatography reagent, and provides great convenience for clinical use.
Description
Technical Field
The application relates to the technical field of medical detection, in particular to an anti-RA 33 antibody quantitative detection kit and application thereof.
Background
The anti-RA 33 antibody is an autoantibody in serum of a patient suffering from rheumatoid arthritis, and the antibody is a nuclear antigen antibody. The target antigen (RA 33 antigen) is a heterogeneous ribonucleoprotein (hnRNP) core protein A2, which can be involved in the formation of nucleoprotein spliceosomes. Since autoantibodies produced by patients with Rheumatoid Arthritis (RA) at the beginning of the course are highly restricted and only for hnRNP, anti-RA 33 antibodies are present in the serum of early RA patients. Thus, detection of anti-RA 33 antibodies is of great value for early diagnosis of RA.
The anti-RA 33 antibody has higher specificity for diagnosing RA, and the specificity can reach 92 percent. The positive rate for RA diagnosis is 20-40%, and the diagnosis sensitivity is 43%. In addition to being positive in RA patients, anti-RA 33 antibodies also have a certain positive rate in non-RA chronic connective tissue diseases such as Systemic Lupus Erythematosus (SLE) and Mixed Connective Tissue Diseases (MCTD).
Anti-RA 33 antibodies are effective serological indicators for diagnosing RA, which are of great value for early RA patients, especially for atypical RA (RF negative) patients. At present, a kit for quantitative detection of an anti-RA 33 antibody is not mentioned in the related art, so that development of an anti-RA 33 antibody detection kit is required.
Disclosure of Invention
The application provides an anti-RA 33 antibody quantitative detection kit and application thereof.
The kit provided by the application adopts a fluorescence immunochromatography method, a nitrocellulose membrane detection line (T line) of a detection card is coated with RA33 antigen, and a quality control line (C line) is coated with rabbit anti-chicken IgY polyclonal antibody. During detection, the sample to be detected and the diluent are mixed uniformly and then accurately added into the sample adding hole of the detection card, and the liquid is chromatographed upwards under the capillary effect. The anti-RA 33 antibody in the sample to be tested is combined with the mouse anti-human IgG monoclonal antibody marked by the fluorescent microsphere in the chromatography process, a solid phase RA33 antigen-anti-RA 33 antibody-mouse anti-human IgG monoclonal antibody-fluorescent microsphere particle complex is formed at the T line, and the fluorescent microsphere marked chicken IgY antibody and the rabbit anti-chicken IgY polyclonal antibody coated at the C line form a complex. The fluorescent microsphere particles emit fluorescent signals under the excitation light, and the ratio (T/C) of the T line signals to the C line signals is in direct proportion to the concentration value of the anti-RA 33 antibody in the sample to be detected. And (3) calculating the concentration of the anti-RA 33 antibody in the sample to be detected through standard curve fitting of the fluorescence immunoassay analyzer, and directly reading the concentration value of the anti-RA 33 antibody in the sample to be detected from a matched analyzer screen.
In a first aspect, the application provides an anti-RA 33 antibody quantitative detection kit, which adopts the following technical scheme:
An anti-RA 33 antibody quantitative detection kit, which comprises a detection card and a diluent; the detection card consists of a bottom plate, and a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially lapped and stuck on the bottom plate along the sample chromatographic direction;
The binding pad is provided with a mouse anti-human IgG monoclonal antibody and a chicken IgY antibody which are respectively marked by fluorescent microspheres;
The detection pad is coated with RA33 antigen and rabbit anti-chicken IgY polyclonal antibody at different positions to form a detection line and a quality control line respectively; the RA33 antigen is any one of amino acid sequences shown as SEQ ID NO. 1-5.
In the kit base, the detection card consists of a bottom plate, and a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially lapped and stuck on the bottom plate along the sample chromatographic direction. The binding pad and the detection pad of the detection card are respectively provided with an antibody which can be specifically bound with the substance to be detected. In the chromatographic process, the liquid containing the substance to be detected flows from the sample pad to the binding pad, and the antibody with fluorescent microsphere marks on the binding pad can be combined with the substance to be detected and continuously flows to the detection pad along with the substance to be detected, so that the substance to be detected also carries fluorescent marks; the antibody on the detection pad is fixed at a specific position in a coating mode to form a detection line, and when a substance to be detected flows through the detection line, the substance to be detected is combined with the antibody on the detection line and is fixed on the detection line. After chromatography is finished, when the light source irradiates the detection card, the detection line excites a fluorescent signal, the fluorescent signal is received by the photoelectric converter and then converted into an electric signal, and the intensity of the electric signal reflects the concentration of the substance to be detected.
In the application, two fluorescent microsphere labeled antibodies which can be respectively combined with the mouse anti-human IgG monoclonal antibody and the chicken IgY antibody in a specific way are arranged on a combining pad of the detection card; and the detection line and the quality control line are respectively formed on the detection pad by coating RA33 antigen and rabbit anti-chicken IgY polyclonal antibody at different positions.
In the present application, RA33 antigen coated on the detection line is further screened for improved detection sensitivity.
In a specific embodiment, the RA33 antigen is any one of the amino acid sequences as depicted in SEQ ID NO. 1-3.
In a specific embodiment, the RA33 antigen is the amino acid sequence shown as SEQ ID No. 1.
In a specific embodiment, the RA33 antigen is the amino acid sequence shown as SEQ ID No. 2.
In a specific embodiment, the RA33 antigen is the amino acid sequence shown as SEQ ID No. 3.
In a specific embodiment, the RA33 antigen is the amino acid sequence shown as SEQ ID No. 4.
In a specific embodiment, the RA33 antigen is the amino acid sequence shown as SEQ ID No. 5.
Further, when the quantitative detection kit for the anti-RA 33 antibody is used, the sample to be detected is diluted by 10-25 times.
In a specific embodiment, the anti-RA 33 antibody quantitative detection kit is used by diluting the test sample 10-fold, 15-fold, 20-fold, 25-fold.
In some specific embodiments, the anti-RA 33 antibody quantitative detection kit is used by diluting the sample to be tested 10-15-fold, 10-20-fold, 10-25-fold, 15-20-fold, 15-25-fold, 20-25-fold.
Further, the diluent comprises the following components: alkyl sulfate, protamine and small molecule amides.
The alkyl sulfates used in the dilutions of the present application are a typical class of anionic surfactants. When protamine or small molecule amides are used alone, the diluent has no cleavage effect. When protamine and alkyl sulfate are used simultaneously or when small molecular amide compounds and alkyl sulfate are used simultaneously, the cracking effect of the diluent still cannot meet the detection requirement of the kit. When protamine, small molecular amide compounds and alkyl sulfate are used simultaneously, the cell lysis efficiency of the diluent can be fully exerted, and even if the dilution factor of a blood sample is very low, the diluent can efficiently and completely lyse cells in the blood, so that hemoglobin is fully released, and the interference of uncleaved cells as components on an immunochromatography result is avoided.
Further, the diluent comprises the following components in parts by weight: every 1000 parts of the diluent comprises 2.5-5 parts of the alkyl sulfate, 4-8 parts of the protamine and 10-20 parts of the small-molecule amide compound.
Further, the alkyl sulfate is an eight to eighteen alkyl sulfate having a alkyl number.
Further, the protamine is selected from one or more of herring, salmon, and rainbow trout protamine.
In a specific embodiment, the protamine is herring protamine.
In a specific embodiment, the protamine is salmon protamine.
In a specific embodiment, the protamine is rainbow trout protamine.
Further, the small molecule amide compound is selected from one or more of acetamide, glutamine and asparagine.
In a specific embodiment, the small molecule amide is acetamide.
In a specific embodiment, the small molecule amide compound is glutamine.
In a specific embodiment, the small molecule amide compound is asparagine.
In a second aspect, the application provides the use of an anti-RA 33 antibody quantitative detection kit as described above in the early screening of RA patients.
In summary, the technical scheme of the application has the following beneficial effects:
1. The kit provided by the application detects the anti-RA 33 antibody by utilizing the indirect method principle, and has the advantages of high sensitivity, strong specificity and good repeatability.
2. The kit provided by the application can realize rapid and single quantitative detection by using the fluorescent immunochromatography reagent, and provides great convenience for clinical use; the device is suitable for various instrument models, has wide application scenes, and is beneficial to application and popularization of primary hospitals; the preparation method is simple, the technology is mature, and batch production can be realized.
Detailed Description
The application provides an anti-RA 33 antibody quantitative detection kit, which consists of a detection card and diluent. The method comprises the following steps:
(1) Detection card
The detection card consists of a bottom plate, and a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially lapped and stuck on the bottom plate along the sample chromatographic direction.
The surface of the binding pad is sprayed with a mouse anti-human IgG monoclonal antibody and a chicken IgY antibody which are respectively marked by fluorescent microspheres. In the application, the fluorescent microsphere marked mouse anti-human IgG monoclonal antibody and chicken IgY antibody are respectively utilized, the concentration is more than or equal to 2mg/ml, and the purity (SDS-PAGE) is more than or equal to 90 percent.
The detection pad is provided with a detection line (T line) and a quality control line (C line) in parallel, and RA33 antigen and rabbit anti-chicken IgY polyclonal antibody are respectively coated.
In the application, the RA33 antigen is any one of amino acid sequences shown as SEQ ID NO. 1-5.
(2) Dilution liquid
Before detecting a sample to be detected, the kit provided by the application needs to uniformly mix the sample to be detected with the diluent according to a certain proportion, and cells in the sample to be detected can be detected by taking a certain volume after being completely cracked under the action of the diluent.
The diluent may be a phosphate buffer of 0.25% Triton 100.
The diluent can also be the diluent designed by the application. The diluent comprises alkyl sulfate, protamine and small molecule amide compounds.
Further, the diluent comprises the following components in parts by weight: every 1000 parts of the diluent comprises 2.5-5 parts of the alkyl sulfate, 4-8 parts of the protamine and 10-20 parts of the small molecular amide compound.
Further, the alkyl sulfate is alkyl sulfate with eight to eighteen alkyl groups.
Further, the protamine is selected from one or more of herring, salmon, and rainbow trout protamine.
Further, the small molecule amide compound is selected from one or more of acetamide, glutamine and asparagine.
In some embodiments, the diluent further comprises a buffer component to stabilize the pH of the diluent at 7.0 to 8.0.
Further, the buffer component may be carbonate, phosphate or borate at a concentration of 10-50 mM.
(3) Sample dilution factor
When the quantitative detection kit for the anti-RA 33 antibody is used, a sample to be detected is diluted by 10-25 times.
(4) Kit detection step
Taking a fresh venous whole blood sample, adding a certain volume of sample into a certain volume of diluent, reversing and uniformly mixing, and reacting for a period of time to enable cells in the sample to be fully cracked by the diluent, taking a certain volume of diluted sample, adding the diluted sample into a sample adding hole, reading fluorescent signals of a C line and a T line by a fluorescent immunoassay analyzer after the chromatographic reaction is finished, and obtaining the actual content of the anti-RA 33 antibody in the sample to be detected according to a standard curve.
The present application will be described in further detail with reference to preparation examples, comparative examples, and test experiments, which should not be construed as limiting the scope of the application as claimed.
Preparation example 1 of test card
The detection card of the preparation example consists of a test strip and a card shell. The test strip is 0.4cm wide and consists of a bottom plate, and a sample pad, a combining pad, a detection pad and a water absorption pad which are sequentially lapped and stuck on the bottom plate along the sample chromatographic direction. And a sample adding hole and an observation window are sequentially arranged right above the sample pad and the detection pad on the cover plate of the clamping shell.
The surface of the binding pad is sprayed with a fluorescent microsphere marked mouse anti-human IgG monoclonal antibody and a fluorescent microsphere marked chicken IgY antibody.
And a quality control line (C line) and a detection line (T line) are sequentially arranged on the detection pad in parallel along the sample chromatography direction. Wherein the C line is coated with rabbit anti-chicken IgY polyclonal antibody, and the T line is coated with RA33 antigen. The RA33 antigen is an amino acid sequence shown as SEQ ID NO. 1.
The preparation method of the detection card comprises the following steps:
1. Coating a detection pad antibody: the concentrations of rabbit anti-chicken IgY polyclonal antibody and RA33 antigen were adjusted to 1mg/mL using 20mM phosphate buffer at pH 7.2 containing 5% sucrose, and coated on detection pads made of nitrocellulose membrane at intervals of 0.5cm in order of 1. Mu.L/cm using a quantitative film spraying apparatus to form C line and T line, and oven-dried at 37deg.C overnight.
2. Fluorescent microsphere marked chicken IgY antibody and fluorescent microsphere marked mouse anti-human IgG monoclonal antibody are prepared and sprayed on the binding pad. Taking a fluorescent microsphere-labeled mouse anti-human IgG monoclonal antibody as an example, the antibody labeling step is described as follows:
(1) Pretreatment of antibodies: the mouse anti-human IgG monoclonal antibody was dialyzed overnight at 4℃with 20mM phosphate buffer, pH 7.2, and the concentration of the dialyzed mouse anti-human IgG monoclonal antibody was adjusted to 1mg/mL.
(2) Activating fluorescent microspheres: washing the microspheres by using 10mM MES buffer with pH of 6.0, shaking and mixing uniformly, sequentially adding 100mg/mL of carbodiimide and 100mg/mL of N-hydroxysuccinimide, activating for 30min in a dark place, fully washing the microspheres by using the MES buffer, and re-dissolving until the concentration of the microspheres is 0.2mg/mL.
(3) Fluorescent microsphere labeling of antibodies: the pretreated mouse anti-human IgG monoclonal antibody was mixed with the activated fluorescent microsphere suspension in equal volume, reacted at room temperature for 2 hours, added with 50mM Tris-HCl blocking solution containing 0.5% BSA and 0.1% glycine and pH 8.0, blocked for 1 hour, washed with 50mM Tris-HCl preserving solution containing 0.5% BSA and 0.05% Tween-20 and pH 8.0 and redissolved to an antibody concentration of 2mg/mL.
The labeling method of the chicken IgY antibody is the same as that of the chicken IgY antibody. After mixing the fluorescent microsphere-labeled antibodies according to a certain proportion, spraying the mixture on a glass fiber membrane (a bonding pad) in an amount of 1.5 mu L/cm by using a quantitative film spraying instrument, and drying the mixture at 37 ℃ overnight in the absence of light.
3. And (3) sequentially overlapping and pasting a sample pad, a bonding pad, a detection pad and a water absorption pad on the bottom plate, cutting into a size with the width of 0.4cm, and assembling into a card shell to obtain the detection card.
Preparation examples 2 to 6 of test card
The test cards of preparation examples 2 to 6 differ from the test card of preparation example 1 in that: the amino acid sequence of the RA33 antigen was identical to that of test card preparation 1. The method comprises the following steps:
Test card preparation example 1: the RA33 antigen is an amino acid sequence shown as SEQ ID NO. 1.
Test card preparation example 2: the RA33 antigen is an amino acid sequence shown as SEQ ID NO. 2.
Test card preparation example 3: the RA33 antigen is an amino acid sequence shown as SEQ ID NO. 3.
Test card preparation 4: the RA33 antigen is an amino acid sequence shown as SEQ ID NO. 4.
Test card preparation 5: the RA33 antigen is an amino acid sequence shown as SEQ ID NO. 5.
Test card preparation 6: the RA33 antigen is an amino acid sequence shown as SEQ ID NO. 6.
Detection card performance detection test-anti-RA 33 antibody quality control detection test
Taking anti-RA 33 antibody quality control products with different concentrations, respectively diluting the quality control products by 20mM phosphate buffer solution with pH of 8.0 for 20 times, taking 100 mu L of diluted quality control products, adding the quality control products into a sample adding hole, and after chromatography for 15min, reading fluorescent signals of a C line and a T line by a fluorescent immunoassay analyzer, and calculating a T/C value. Experimental data and statistical analysis are shown in table 1.
TABLE 1 detection results of anti-RA 33 antibody quality control
As shown in Table 1, the higher the detection sensitivity was when the RA33 antigen in the kit was the amino acid sequences as shown in SEQ ID NO. 1-5. In particular, when the RA33 antigen in the kit is the amino acid sequence shown as SEQ ID NO.1-3, the detection sensitivity is higher. When the RA33 antigen in the kit is an amino acid sequence shown as SEQ ID NO.1, the detection sensitivity is highest.
Preparation of dilution
Preparation examples 1 to 3
Preparation examples 1-3 each provided a diluent. The above preparation differs in that: the types of protamine in the dilutions are shown in table 3.
The preparation method of the diluent specifically comprises the following steps:
(1) 4.107g of potassium dihydrogen phosphate was dissolved in 800mL of ultrapure water to prepare a buffer solution;
(2) Adding alkyl sulfate, protamine and small molecular amide compounds into the buffer solution prepared in the step (1), fully dissolving, fixing the volume to 1000mL, and adjusting the pH to 7.5 to prepare the diluent. Wherein the alkyl sulfate is specifically sodium dodecyl sulfate; the small molecular amide compound is specifically asparagine. The amounts of the components added are shown in Table 1.
PREPARATION EXAMPLES 4 to 5
Preparation examples 4 to 5 each provided a diluent which was different from preparation example 3 in that: the types of small molecule amides are different and are shown in table 2.
TABLE 2 dilution preparation example addition of the Components
Kit examples
Examples 1 to 5
Examples 1-5 provide a kit, respectively. The kit comprises a detection card and a diluent. Wherein the test card is the test card provided in the test card preparation example 1; the dilutions were those provided in preparation examples 1-5, respectively, and are shown in Table 3.
The detection steps of the kit are specifically as follows:
① Standard curve fitting of anti-RA 33 antibody:
Taking quality control products with the anti-RA 33 antibody content of 10AU/mL, 20AU/mL, 40AU/mL, 60AU/mL, 100AU/mL, 150AU/mL and 200AU/mL respectively, diluting by 2 times, taking 100 mu L of diluted quality control product, adding the quality control product into a sample adding hole of a detection card, chromatographic separation for 15min, reading fluorescent signals of a C line and a T line by a fluorescent immunoassay analyzer, and establishing a standard curve of the theoretical concentration of the anti-RA 33 antibody quality control product and the T/C value of the signal.
② And (3) detecting an actual sample:
Taking a fresh venous whole blood sample, diluting the sample by 20 times, reversing and uniformly mixing the sample, taking 100 mu L of the diluted sample after the reaction is performed for 1min, adding the sample into a sample adding hole, performing chromatography for 15min, reading fluorescent signals of a C line and a T line by a fluorescent immunoassay analyzer, and obtaining the actual content of the anti-RA 33 antibody in the sample according to a standard curve.
Example 6
Example 6 provides a kit. The above kit differs from example 1 in that: the dilution in the kit was 0.25% Triton 100 phosphate buffer. The remainder were identical to example 1.
Example 7
Example 7 provides a kit. The above kit differs from example 1 in that: the dilution in the kit was phosphate buffer of 0.1% SDS. The remainder were identical to example 1.
Example 8
Example 8 provides a kit. The above kit differs from example 1 in that: the diluent in the kit is phosphate buffer solution of 0.1% EDTA-Na 2. The remainder were identical to example 1.
TABLE 3 sources of dilutions and assay results in the kits provided in examples 1-6
Test for detecting performance of kit
The kit provided in the above example was used to quantitatively detect anti-RA 33 antibodies in 5 fresh venous whole blood samples, respectively.
The detection results of the HPLC method and the immunofluorescence quantitative chromatography method are respectively used as reference values of the concentration of the anti-RA 33 antibody in the sample to be detected, and the accuracy of the detection results is measured by using the average relative error between the detection results of the embodiment and the reference values. The detection results are shown in Table 3.
As can be seen from Table 3, the diluent provided by the application contains alkyl sulfate, protamine and small molecule amide compounds. Further, examples 1 to 3 are different in the kind of protamine to be added. The detection result shows that the diluent added with the herring protamine, the salmon protamine and the rainbow trout protamine can have a good dilution effect, the detection result is accurate, wherein the embodiment 3 added with the rainbow trout protamine has the best effect, and the detection result is the most accurate. Further, examples 3 to 5 are different in the kinds of addition of the small molecule amide compound. The detection result shows that the diluting solution added with acetamide, glutamine and asparagine has better dilution effect, and the detection result is more accurate, wherein the cracking effect of the embodiment 5 added with glutamine is the best, and the detection result is the most accurate.
Examples 5-8 differ in the diluent. The detection result shows that compared with the diluent in the related technology, the diluent provided by the application can further effectively improve the detection accuracy of the kit for resisting the RA33 antibody.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (10)
1. A quantitative detection kit for an anti-RA 33 antibody is characterized in that,
The kit comprises a detection card and a diluent; the detection card consists of a bottom plate, and a sample pad, a combination pad, a detection pad and a water absorption pad which are sequentially lapped and stuck on the bottom plate along the sample chromatographic direction;
The binding pad is provided with a mouse anti-human IgG monoclonal antibody and a chicken IgY antibody which are respectively marked by fluorescent microspheres;
The detection pad is coated with RA33 antigen and rabbit anti-chicken IgY polyclonal antibody at different positions to form a detection line and a quality control line respectively; the RA33 antigen is any one of amino acid sequences shown as SEQ ID NO. 1-5.
2. The kit for quantitative detection of an anti-RA 33 antibody according to claim 1, wherein the RA33 antigen is any one of the amino acid sequences shown in SEQ ID NO. 1-3.
3. The kit for quantitative detection of an anti-RA 33 antibody according to claim 1, wherein the RA33 antigen comprises an amino acid sequence as shown in SEQ ID No. 1.
4. The quantitative detection kit for an anti-RA 33 antibody according to claim 1, wherein the anti-RA 33 antibody quantitative detection kit is used to dilute a sample to be detected 10 to 25 times.
5. The quantitative detection kit for anti-RA 33 antibodies according to claim 1, wherein the diluent comprises the following components: alkyl sulfate, protamine and small molecule amides.
6. The quantitative detection kit for the anti-RA 33 antibody according to claim 5, wherein the dilution comprises the following components in parts by weight: every 1000 parts of the diluent comprises 2.5-5 parts of the alkyl sulfate, 4-8 parts of the protamine and 10-20 parts of the small-molecule amide compound.
7. The kit for quantitative detection of an anti-RA 33 antibody according to claim 5, wherein the alkyl sulfate is an alkyl sulfate having an alkyl group number of eight to eighteen.
8. The kit for quantitative detection of an anti-RA 33 antibody of claim 5, wherein the protamine is selected from one or more of herring, salmon, and rainbow trout.
9. The kit for quantitative detection of an anti-RA 33 antibody according to claim 5, wherein the small molecule amide compound is selected from one or more of acetamide, glutamine and asparagine.
10. Use of an anti-RA 33 antibody quantitative detection kit according to any one of claims 1-9 in early screening of RA patients.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118330205A true CN118330205A (en) | 2024-07-12 |
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