CN105974111A - Joint detection method for rheumatoid factors (IgM type) and other antigen-specific IgM antibodies - Google Patents

Abstract

The invention provides a joint detection test strip for rheumatoid factor (IgM type) and other antigen-specific IgM antibodies. plain film and water-absorbent pad, the nitrocellulose membrane is respectively coated with a first detection line, an adsorption line, one or more detection lines and a quality control line; the first detection line is denatured rabbit/human IgG, The adsorption line is an antibody against human IgG, the quality control line is human IgM, and the one or more detection lines are respectively selected from the following one or more specific antigens: Mycoplasma pneumoniae specific antigen, influenza B specific antigen Sexual antigens, Toxoplasma gondii pathogen-specific antigens, autoimmune antigens and allergens. The present invention also provides a kit including the test strip, a preparation method of the test strip, a detection method using the test strip or the kit, and an application of the test strip or the kit.

Description

Combined detection of a rheumatoid factor (IgM type) and other antigen-specific IgM antibodies Methods

technical field

The invention relates to a test strip. Specifically, the present invention relates to a joint detection test strip for rheumatoid factor (IgM type) and other antigen-specific IgM antibodies, and a test kit including the test strip, a method for preparing the test strip, and using the test strip The method for detecting the test strip or kit, and the application of the test strip or kit.

Background technique

Rheumatoid factor (RF) is an autoantibody that targets denatured immunoglobulin G (IgG) and has no species specificity. Rheumatoid arthritis (RA) patients and about 50% of healthy people have RF-producing B cell clones, which can be synthesized and produced in large quantities under the direct action of certain pathological factors such as denatured IgG or Epstein-Barr virus. RF has poor binding ability to natural IgG, but it is easy to react with IgG or polymerized IgG in immune complexes. There are five types of RF: IgG, IgA, IgM, IgD, and IgE. The method for detecting RF is mainly to measure IgM RF. Detection of RF is of great significance to the diagnosis, classification and curative effect observation of rheumatoid arthritis (RA).

Immunoglobulin M (IgM) is a very important antibody in humans or animals. When the body is infected with pathogens or other antigens, in the body, pathogen or other antigen-specific IgM antibodies first appear, and then pathogen or other antigen-specific IgG antibodies appear. Therefore, by detecting the presence or absence of antigen-specific IgM in the body, it is possible to diagnose the immune response state of humans or animals recently infected with pathogens or other antigens. If specific pathogen IgM or specific IgM caused by other types of antigens are detected in the serum, it can prove that the body has recent pathogen infection or the occurrence of autoimmune diseases caused by other antigens, which can be used as a test for pathogen infection and immune diseases, etc. Diagnosis and medication basis can give patients better treatment as soon as possible. Accurate and rapid diagnostic methods can avoid unnecessary use of antibiotics or other drugs, and can provide timely and accurate treatment options, so the establishment of pathogen or other antigen-specific IgM detection methods is of great significance.

From the perspective of detecting the principle of immune response, the detection of antigen-specific IgM usually uses capture method and indirect method. The capture method generally improves the specificity of detection, but because anti-IgM antibodies cannot recognize specific and non-specific IgM, it often causes missed detection, resulting in low detection sensitivity; and if the principle of indirect method is used for detection, it is generally considered that detection Before specific IgM antibody, if the IgG antibody in the patient sample is not removed, it is easy to cause rheumatoid factor (IgM type) to react with specific IgG and specific antigen complex, resulting in false positive of IgM, and also cause specific IgG and Specific IgM competes for antigen binding sites resulting in IgM false negatives.

At present, it is a mature technology to detect rheumatoid factor or antigen-specific IgM in samples by enzyme-linked immunosorbent assay kit. Testing products are basically divided into three categories:

One is the traditional microwell plate product, but its detection process takes a long time (usually several hours), and the reagents and samples used are also large, which cannot meet the needs of a small number of samples or clinical timely detection. The principle is a simple capture method or an indirect method, both of which have certain deficiencies.

One is fluorescent label detection glass slide products, which reduce the amount of reagents used, but the detection time is still long, and the results of most of these products need to be observed and interpreted with the help of expensive fluorescent microscopes. This intuitive judgment requires technicians Extensive experience and time-consuming, so it is not suitable for on-site rapid detection.

The other type is immunochromatography products, mostly colloidal gold test strips, which are relatively fast and simple, but the application of existing products has certain limitations. For example, most products only use the principle of capture method to detect IgM antibodies, resulting in a missed detection rate. It is relatively high, and it cannot meet the simultaneous detection of multi-indicators and multi-samples. In addition, there are few products that use the principle of indirect method for combined detection, but if the product does not process and eliminate the interference of specific IgG and rheumatoid factor, false positives and false negatives are prone to occur, resulting in inaccurate results.

Immunochromatography technology is a rapid on-site detection technology, easy to operate, such as colloidal gold test strip products, the results can be read by naked eyes within 15 minutes, and its basic structure is that the chromatography test strip is coated with polyvinyl chloride (PVC) ) one end of the back plate is attached with a sample pad, a composite pad, and a nitrocellulose membrane sequentially overlapping each other, and an absorbent pad is attached at the other end. The complex pad is a glass cellulose membrane coated with an antibody-colloidal gold complex (or other microparticle complexes). There have been many literature reports on the use of colloidal gold test strips to detect IgM antibodies, some of which did not deal with the interference of IgG and rheumatoid factors, which may easily cause inaccurate results; some simply increased the ability to absorb human IgG and rheumatoid factors An adsorption line or an adsorption treatment step does not increase the detection line of rheumatoid factor, and the detection of rheumatoid factor cannot be realized.

However, with the growth of market demand, hospitals and patients urgently need products that can be combined and quickly tested, which has become the development trend of today's testing products. Therefore, based on the interference and principle analysis of rheumatoid factor on the detection of antigen-specific IgM antibody, it is very meaningful to carry out the research and development of the combination of the two and rapid detection products.

Contents of the invention

Therefore, based on the defects of the above-mentioned prior art, the purpose of the present invention is to provide a joint detection test strip for rheumatoid factor (IgM type) and other antigen-specific IgM antibodies, and a test kit comprising the test strip. The preparation method of the strip, the detection method using the test strip or the kit, and the application of the test strip or the kit.

For above-mentioned purpose of the invention, the present invention is achieved through the following technical solutions:

The invention provides a joint detection test strip for rheumatoid factor (IgM type) and other antigen-specific IgM antibodies, wherein the test strip includes a sample pad, a conjugate pad, A nitrocellulose membrane and an absorbent pad, the nitrocellulose membrane is coated with a first detection line (hereinafter abbreviated as T1 line), an adsorption line (hereinafter abbreviated as A line), one or more detection lines (hereinafter may be referred to as T2 line, T3 line, ...) and quality control line (hereinafter may be referred to as C line for short); the first detection line is denatured rabbit/human IgG, and the adsorption line is anti-human IgG antibody, the quality control line is human IgM, and the detection line is selected from one or more of the following specific antigens: Mycoplasma pneumoniae specific antigens, influenza type B specific antigens, Toxoplasma gondii and other pathogen specific antigens, Autoimmune antigens and allergens, etc. Preferably, the specific antigen is a multi-epitope antigen obtained after the cultured pathogen is lysed and/or inactivated and purified. Wherein, when pathogen antigens are involved in coating the detection line, the pathogen antigens may be multi-epitope pathogen antigens extracted from natural and purified, and the multi-epitope pathogen antigens have higher sensitivity than recombinant antigens.

Preferably, according to the aforementioned test strip, wherein the sample pad is a glass fiber membrane. And/or the absorbent pad is absorbent paper.

More preferably, according to the aforementioned test strip, wherein the conjugate pad is a glass fiber membrane coated with an anti-human IgM antibody-colloidal gold conjugate. The immunogen of the anti-human IgM antibody is preferably an Fc5μ fragment of human IgM; and/or the anti-human IgM antibody is preferably a goat anti-human IgM IgG antibody, more preferably the F(ab' of the goat-derived IgG antibody )2 fragments. Wherein, the conjugate pad can also be a glass fiber membrane coated with anti-human IgM antibody-other colored or particulate conjugates. Wherein, the anti-human IgM antibody-colloidal gold conjugate has high specificity and will not cross-react with the IgG antibody adsorbed by the adsorption line to develop color. The high specificity of the anti-human IgM antibody-colloidal gold conjugate can also be manifested as: the anti-human IgM antibody can be the F(ab')2 fragment of the IgG antibody after the Fc fragment is removed, and the anti-human IgM antibody binds After the serum sample-specific IgM forms a complex, it will not be combined with rheumatoid factor (binding to the Fc segment of denatured IgG) to cause a false negative of the rheumatoid factor detection line, thereby improving the specificity of the rheumatoid factor detection line in the present invention. sex.

Still preferably, according to the aforementioned test strip, wherein, the width of the test strip is an appropriate width. Preferably it is 3 to 5 mm. More preferably, it is 4 mm.

Still preferably, according to the aforementioned test strip, wherein, the distance between adjacent lines of the first detection line, adsorption line, detection line and quality control line is an appropriate width. Preferably it is 3-8 mm. More preferably, it is 3 mm. The overlap between the sample pad and the conjugate pad is 1-2 mm, preferably 1 mm. The overlap between the conjugate pad and the nitrocellulose membrane is 1-2 mm, preferably 1 mm. And/or the overlap between the nitrocellulose membrane and the absorbent pad is 1-2 mm, preferably 1 mm.

The present invention also provides the preparation method of above-mentioned test strip, wherein, described preparation method comprises:

(1) Prepare colloidal gold solution by reducing chloroauric acid with sodium citrate: add 1000mL triple distilled water to the beaker, then add 10mL of 1% by mass sodium citrate solution, heat to boiling, then add 10mL of 1% by mass % chloroauric acid solution, after boiling for 15min to cooling, obtains the colloidal gold solution;

(2) Prepare the binding pad: take 20mL of the colloidal gold solution prepared in step (1) and put it in a beaker, add 150μL of 0.1M K ₂ CO ₃ solution to adjust the pH of the solution to 7.6, stir well, and then add the centrifuged anti-human IgM antibody, stirred for 20min, then added 2mL of 10% by mass bovine serum albumin solution, and centrifuged for 40Min at a speed of 10000rpm, then discarded the supernatant, then added 1% by mass of bovine serum albumin solution to continue centrifugation, Discard the supernatant, use the recovery solution to recover the precipitate, coat it on a glass fiber membrane with a size of 200 square centimeters, freeze it at -45°C, and use it as a conjugate pad, wherein the component of the recovery solution is 150mM NaCl, 1 mass % bovine serum albumin solution, 0.5 mass % sucrose and 0.5 mass % sodium caseinate, the prepared conjugate pad can be stored in the dark at room temperature for subsequent use after lyophilization;

(3) Assemble the test strip: attach the sample pad, the conjugate pad prepared in step (2), the nitrocellulose membrane and the water-absorbing pad to the polyvinyl chloride base plate in order to obtain the assembled test strip;

(4) Coating with nitrocellulose membrane: Coat the denatured rabbit/human IgG, anti-human IgG antibody, antigen-specific IgM antibody antigen and human IgM on the assembled test paper of step (3) in turn by a film-drawing instrument On the nitrocellulose membrane of the strip, that is the test strip. The test strip can also be further cut, packaged, sealed and stored to prepare corresponding products on demand, for example, the PVC rubber sheet and the attached material of the test strip are cut into test strips with a width of 4mm;

Preferably, the anti-human IgM antibody in step (2) is previously treated by the following method: pepsin is used to digest the anti-human IgM IgG antibody, and the F(ab')2 fragment of the anti-human IgM antibody is obtained by affinity purification.

Among them, the glassware used can be soaked overnight with the lotion in advance, and then rinsed.

Preferably, according to the preparation method of the aforementioned test strip, wherein the denatured rabbit/human IgG, anti-human IgG antibody, antigen-specific IgM antibody antigen and human IgM concentrations in step (4) are independently 0.3 mg/mL.

The present invention also provides a combined detection kit for rheumatoid factor (IgM type) and other antigen-specific IgM antibodies, wherein the kit includes the above-mentioned test strip or the test strip prepared by the above-mentioned method. Preferably, the kit further includes an aluminum foil bag and a desiccant sealed in the aluminum foil bag. and/or dropper.

The present invention also provides a detection method for rheumatoid factor (IgM type) and other antigen-specific IgM, the detection method is based on the above-mentioned test strip, the test strip prepared by the above-mentioned method or the above-mentioned kit, wherein , the detection method includes:

(1) Put the test strip flat on the detection table, add 100 μl of sample to the sample pad dropwise, the sample is whole blood or serum, observe the first detection line, detection line and quality control line after 10 to 15 minutes The color development result after 30 minutes is deemed invalid;

(2) Judging the situation of rheumatoid factor (IgM type) and other antigen-specific IgM antibodies in the sample according to the standard:

When the first detection line, detection line and quality control line all show red bands, the result is judged as; rheumatoid factor (IgM type) positive and other antigen-specific IgM antibodies positive,

When the first test line does not show a red band, and both the test line and the quality control line show a red band, the result is judged as; rheumatoid factor (IgM type) negative and other antigen-specific IgM antibodies positive,

When both the first test line and the quality control line show red bands, but the test line does not show red bands, the result is judged as; rheumatoid factor (IgM type) positive and other antigen-specific IgM antibodies negative,

When the first test line and the test line do not show a red band, and the quality control line shows a red band, the result is judged as; rheumatoid factor (IgM type) negative and other antigen-specific IgM antibodies negative,

When the quality control line does not show a red band, the result is judged as; the test is invalid.

Specifically, the following table can also be used to judge the situation of rheumatoid factor (IgM type) and other antigen-specific IgM antibodies in the sample:

Where "+" indicates that the red band is displayed, "-" indicates that the red band is not displayed, and "/" indicates that the red band is displayed or not:

The present invention also provides the application of the above-mentioned test strip, the test strip prepared by the above-mentioned method, or the above-mentioned kit in the preparation of one or more products for detecting and/or diagnosing the following: rheumatoid immune disease , preferably, the rheumatoid immune disease is rheumatoid arthritis; pathogen recent infection, preferably, the pathogen is Mycoplasma pneumoniae and/or influenza B virus and other pathogens; prenatal and postnatal care, preferably, the Prenatal and postnatal care is the detection of Toxoplasma gondii; autoimmune system diseases, preferably, the autoimmune system diseases are systemic lupus erythematosus (SLE) and/or Sjogren's disease (SS) and the like.

Specifically, the present invention relates to a method capable of jointly detecting rheumatoid factor (IgM) and other antigen-specific IgM antibodies. In this method, the sample passes through the rheumatoid factor (IgM) detection area (i.e. T1 line), IgG adsorption area (i.e. A line), and other antigen-specific IgM detection areas (i.e. T2 line, T3 line, ...). It also provides a preparation method for applying the method in colloidal gold test strips for rapid detection based on immunochromatography.

The specific content is as follows:

Paste the sample pad, the conjugate pad, the nitrocellulose membrane, and the absorbent paper on one end of the polyvinyl chloride (PVC) rubber sheet in sequence, and then cut the PVC rubber sheet and the attached material into suitable (4mm) Width test strips.

Wherein, the sample pad is a glass fiber membrane, and the conjugate pad is a glass fiber membrane covered with an anti-human IgM antibody-colloidal gold conjugate or other colored or particle conjugates, preferably a colloidal gold conjugate; the nitrocellulose The film is coated with at least three lines, which are: T1 line is denatured rabbit/human IgG, used to detect human rheumatism factor (IgM); A line is anti-human IgG antibody, used to absorb IgG antibodies in samples; T2 line (T3, T4...) lines are specific antigens that cause antibody responses to different pathogens or other types; C lines are human IgM (as shown in Figure 1).

Among them, the T1 line is the human rheumatism factor (IgM) detection line, and the T2 (T3, T4...) line is the purified natural multi-epitope specific antigen optimized for different pathogen-specific antigens or other diseases that can cause human IgM antibody responses. Sexual antigens, such as Mycoplasma pneumoniae antigens caused by infection with pathogens, dsDNA antigens for detection of autoimmune diseases such as systemic lupus erythematosus, eugenics such as Toxoplasma gondii antigens, etc., or any specificity or Multiple specific antigens. According to the requirements of the detection project, the detection of specific IgM antibodies to various antigens can be realized and the sensitivity can be improved.

The present invention also relates to an analysis method capable of jointly detecting rheumatoid factor (IgM) and other specific IgM antibodies, including colloidal gold test strip technology in rheumatoid immune diseases, recent infection of pathogens and autoimmune system diseases or other detection purposes The specific content of the application in the detection of specific IgM antibody is as follows:

After the sample to be tested is added to the sample loading area (i.e., the sample pad), the combination of IgM antibodies and anti-human IgM-colloidal gold in the sample moves toward one end of the absorbent paper through chromatography/capillary action. If the tested sample contains rheumatoid factor (IgM type), when the sample moves to the T1 line, that is, the denatured IgG coating line, the combination of rheumatoid factor (IgM type) and anti-human IgM-colloidal gold is captured, A purple-red band is displayed on the T1 line, and the rheumatoid factor (IgM type) no longer moves backward to cause interference to the specific IgM detection area. The sample continues to flow, and the IgG antibody in the sample is combined with the A line, that is, the anti-human IgG antibody, which is used to adsorb the IgG antibody in the sample, and the specific IgG antibody is captured on the A line, and no longer reacts with the same antigen. Specific IgM competes for the reaction site, enhancing the sensitivity and specificity of specific IgM detection. The sample continues to flow, containing IgM antibodies against one or more specific antigens coated once, when the sample passes through the T2 (T3, T4...) line, that is, different pathogens or other types of specific antigen-coated lines that cause antibody responses When the specific IgM antibody in the sample and the conjugate of anti-human IgM antibody-colloidal gold are captured, one or more purple-red bands are displayed on the T2 (T3, T4...) line through the principle of the indirect method. The detection of region-specific IgM is no longer interfered by false positives or false negatives caused by rheumatoid factors and IgG that may exist in the sample. When the sample continues to move to the C line, the anti-human IgM antibody-colloidal gold conjugate carried by the sample binds to the human IgM antibody at the C line, and a purple-red band is displayed on the C line.

Preferably, the sample to be tested is whole blood or serum.

If both T1 and T2 (T3, T4...) lines show red bands, it means that the sample contains both rheumatoid factor (IgM) and a corresponding specific IgM; if T1 line does not show red bands, but T2 (T3, T4...) lines show red bands, indicating that they contain IgM corresponding to a certain specificity; if T1 lines show red bands, but T2 (T3, T4...) lines do not show red bands, they contain Rheumatoid factor (IgM); if T1 and T2 (T3, T4...) lines are not colored, it means that rheumatoid factor (IgM) and certain specific IgM are negative; regardless of T1, T2 (T3, T4... ...) line, as long as there is no red band at line C, the test strip is judged to be invalid.

If the specific IgM is positive, it indicates that the body has recently been infected with a certain pathogen, or has an abnormal autoimmune antibody reaction; if RF (IgM type) is positive, it indicates that the body may have abnormal factors that lead to the production of rheumatoids, prompting There is a risk of rheumatoid immune disease; if both RF (IgM type) and specific IgM are positive, it indicates that the body has recently been infected with a certain pathogen, and there are abnormal factors that may lead to the generation of rheumatoid; if RF ( IgM type) and specific IgM are both negative, indicating that they have not been infected with a certain pathogen, and the risk of abnormal factors produced by the wet class is low; regardless of the situation at T1 and T2 (T3, T4...), if the C line If no color develops, the test strip is considered invalid.

The test strip provided by the present invention is based on the analysis method that this sample sequentially passes through the rheumatoid factor (IgM) detection area, IgG adsorption area, and other antigen-specific IgM detection areas, changing the traditional separate detection of specific IgM antibody or rheumatoid factor (IgM type) method, to overcome the deficiency that rheumatoid factor is easy to interfere with the detection of specific IgM antibody in routine detection, and the combined detection can rule out whether there is rheumatoid factor (IgM type) in the same sample. The presence of rheumatoid factors will cause false positive interference in the subsequent specific IgM antibody detection. For example, if the test result of a sample for rheumatoid factor (IgM type) is negative, it can be ruled out that the sample does not have rheumatoid factor ( IgM type) interferes with the subsequent specific IgM antibody detection; if a sample detects rheumatoid factor (IgM type) and the test result is positive, it shows that our detection method first clarifies that this is a case of rheumatoid factor (IgM type) Type) samples that interfere with the detection of specific IgM antibodies, but because our design has increased the detection of rheumatoid factors (IgM type) in the sample, it avoids interference with the detection of specific IgM antibodies later The test strip adds rheumatoid factor before the specific antigen IgM antibody detection line

(IgM) detection line and adsorption IgG line, not only can simultaneously detect multiple specific IgM antibodies in one detection and remove the interference of IgG, but also realize the simultaneous detection of rheumatoid factor (IgM type), and subtract the The false-positive interference caused by rheumatoid factor and IgG antibody to the detection of specific IgM antibody by the indirect method fully reflects the advantages of the sensitivity and specificity of the detection of specific IgM by the principle of the indirect method in this detection scheme.

Based on immunochromatography technology, the test strip highlights the characteristics of rapid detection of multi-index colloidal gold immunochromatography technology, low price, quick and intuitive reading results, etc. It does not require special equipment or professional operation , the overall coincidence rate of the test results is high, which can greatly shorten the test time and reduce the test cost, and achieve the purpose of on-site testing.

In addition, this test strip improves the deficiency of the detection of specific IgM antibodies by the principle of capture method commonly used in previous immunochromatography techniques, because the detection area using the principle of capture method cannot recognize specific IgM and non-specific IgM, which may cause Only non-specific IgM is captured, and specific IgM is missed, which reduces the sensitivity; secondly, it improves the previous immunochromatography technology that uses the principle of indirect method to coat recombinant antigens, which leads to a single epitope and reduces the sensitivity. This method encapsulates the multi-epitope antigen after the subsequent treatment and purification of the optimized natural cultured pathogen, which improves the sensitivity; it also improves the principle of the indirect method used in the previous immunochromatography technology, but does not remove the detection of the influence of IgG and rheumatoid factor method, resulting in false positives and false negatives in the detection of specific IgM antibodies, or some indirect methods have IgG adsorption treatment, but the rheumatoid factor has not been realized

Simultaneous detection of (IgM) cannot clarify and rule out whether there is really rheumatoid factor in a certain sample, which causes the interpretation of IgM detection interference. In other words, the test strip provided by the present invention can not only avoid the interference of IgG antibody and rheumatoid factor in the sample in one detection, but also improve the sensitivity by using the indirect method, and realize simultaneous detection and clearing of specific IgM and rheumatoid factor. Factor (IgM) negative and positive results, so as to obtain more relevant information, has important clinical significance.

Description of drawings

Below, describe embodiment of the present invention in detail in conjunction with accompanying drawing, wherein:

Fig. 1 shows a schematic diagram of the detection method of rheumatoid factor (IgM type) and other antigen-specific IgM antibodies;

Fig. 2 shows the schematic diagram of the combined detection test strip for rheumatoid factor (IgM type) and other antigen-specific IgM antibodies;

Fig. 3 shows the mark used in Fig. 3～5 and described test strip structure;

Fig. 4 has shown the effective detection result of described test strip;

Fig. 5 shows the detection result that described test strip is invalid;

Explanation of reference signs:

1. Sample loading area; 2. Anti-human IgM antibody-particle conjugate area; 3. Nitrocellulose membrane; 4. RF (IgM type) detection area; 5. Adsorption IgG antibody area; 6. Specific IgM antibody detection area ;7. Quality inspection area; 8. Absorbent pad;

9, sample pad; 10, conjugate pad (can be the conjugate pad that is covered with anti-human IgM antibody-colloidal gold conjugate); 11, T1 line (promptly detect rheumatoid factor (IgM type) district); 12, A 13. T2 line (i.e. specific IgM antibody detection area); 14. C line (i.e. quality control area); 15. Absorbent pad (can be absorbent paper);

16. Denatured IgG (can be denatured rabbit/human IgG); 17. Anti-human IgG antibody; 18. Rheumatoid factor (can be rheumatoid factor (IgM type)); 19. Specific IgM; 20. Human IgM; 21. Antigen of specific IgM; 22. Gold-labeled antibody conjugate (can be anti-human IgM antibody-colloidal gold conjugate); 23. Nanoparticles (can be colloidal gold); 24. Anti-human IgM antibody; 25. Human IgG;

26. The test result is RF (IgM) positive and specific antibody IgM positive; 27. The test result is RF (IgM) negative and specific antibody IgM positive; 28. The test result is RF (IgM) positive and specific antibody IgM negative ;29. The test result was negative for RF (IgM) and negative for specific antibody IgM;

30-33 are the four situations where the test results are invalid.

detailed description

The present invention will be further illustrated by specific examples below, but it should be understood that these examples are only used for more detailed description, and should not be construed as limiting the present invention in any form.

In the following examples, many materials and operating methods used in the examples are well known in the art, except for the specifically indicated test materials, conditions and operating methods. Accordingly, it will be apparent to those skilled in the art that, in the context and context, the materials employed and methods of operation used in the present invention are those known in the art, unless otherwise specified.

The reagents and instruments used in the following examples are as follows:

Reagent:

Denatured rabbit/human IgG: Faipeng Biological Co., Ltd.;

Anti-human IgM antibody, anti-human IgG antibody: Beijing Boaosen Biotechnology Co., Ltd.;

Human IgM: sigma;

Nitrocellulose membrane: Merck Millipore;

PVC rubber sheet, absorbent paper, glass fiber membrane: Shanghai Jinbiao Biotechnology Co., Ltd.;

Mycoplasma pneumoniae IgM antibody detection kit (ELISA): Shenzhen Anqun Bioengineering Co., Ltd.;

Rheumatoid factor IgM antibody detection kit (colloidal gold method): Weifang Kanghua Biotechnology Co., Ltd.

instrument:

Three-dimensional planar film drawing instrument: Shanghai Jinbiao Biotechnology Co., Ltd.;

Freeze dryer: Beijing Boyikang Experimental Instrument Co., Ltd.;

Strip cutting machine: Shanghai Gold Standard Biotechnology Co., Ltd.

Example 1

This example is used to illustrate the verification method and results of the highly specific anti-human IgM antibody selected in the present invention.

Method 1 includes: Purified human IgG with a concentration of 1ug/ml, 100ul/well coated in a polystyrene microwell plate at 4 overnight; use 3% bovine serum albumin (BSA), 200ul/well, 37 degrees for 2 hours Blocking; then add anti-human IgM-HRP, 100ul/hole, 37 degrees for 30 minutes, after washing, add TMB chromogenic solution, and carry out microplate reader detection OD _450nm-630nm after 10 minutes in the dark; and do coating human IgM Positive control wells and blank control wells; all multi-wells are parallel, and the average value is calculated. The test results are shown in Table 1.

Method 2 includes: the test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbent pad that are attached sequentially on a polyvinyl chloride rubber sheet; the sample pad is a glass fiber membrane; the conjugate pad is a A glass fiber membrane of an anti-human IgM antibody-colloidal gold conjugate; the nitrocellulose membrane is respectively coated with a detection line and a quality control line; the detection line is human IgG, and the quality control line is human IgM; the The absorbent pad is absorbent paper. Add 100 μl of PBS buffer solution dropwise to the sample pad, observe the color development results of the test line and quality control line after 10 to 15 minutes, and the color development results after 30 minutes are considered invalid. The test results are shown in Table 2.

From the detection results of method 1 and method 2 in this example, it can be seen that the anti-human IgM antibody used in the present invention has high specificity and does not cross-react with human IgG.

Table 1

Table 2

Example 2

This example is used to illustrate the joint detection test strip and kit of rheumatoid factor (IgM type) and other antigen-specific IgM antibodies and the preparation method thereof of the present invention.

As shown in Figure 2, the test strip of the present embodiment comprises a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbent pad attached successively on a polyvinyl chloride rubber sheet; the sample pad is a glass fiber membrane; The conjugate pad is a glass fiber membrane with an anti-human IgM antibody-colloidal gold conjugate; the nitrocellulose membrane is respectively coated with a first detection line, an adsorption line, a detection line and a quality control line; The first detection line is denatured rabbit/human IgG, the adsorption line is an anti-human IgG antibody, the quality control line is human IgM, and the detection line is the purified Mycoplasma pneumoniae-specific natural multi-epitope antigen; The absorbent pad is absorbent paper.

Wherein, the distance between adjacent lines of the first detection line, the adsorption line, the detection line and the quality control line is 3-8 mm. The overlap between the sample pad and the conjugate pad is 1-2 mm, the overlap between the conjugate pad and the nitrocellulose membrane is 1-2 mm, and the overlap between the nitrocellulose membrane and the water-absorbing pad is 1-2 mm. The width of the test strip is a suitable width. In this embodiment, the distance between adjacent lines of the first detection line, adsorption line, detection line and quality control line is 3 mm. The overlap between the sample pad and the conjugate pad is 1 mm, the overlap between the conjugate pad and the nitrocellulose membrane is 1 mm, the overlap between the nitrocellulose membrane and the absorbent pad is 1 mm, and the width of the test strip is 4mm.

The preparation method of above-mentioned test strip is as follows:

(1) Prepare colloidal gold solution by reducing chloroauric acid with sodium citrate: add 1000mL triple distilled water to the beaker, then add 10mL of 1% by mass sodium citrate solution, heat to boiling, then add 10mL of 1% by mass % chloroauric acid solution, after boiling for 15min to cooling, obtains the colloidal gold solution;

(2) Prepare the binding pad: take 20mL of the colloidal gold solution prepared in step (1) and put it in a beaker, add 150μL of 0.1M K ₂ CO ₃ solution to adjust the pH of the solution to 7.6, stir well, and then add the centrifuged anti-human IgM antibody, stirred for 20min, then added 2mL of 10% by mass bovine serum albumin solution, centrifuged for 40Min at a speed of 10000rpm, discarded the supernatant, then added 1% by mass of bovine serum albumin solution to continue centrifugation, discarded Remove the supernatant, use the recovery solution to recover the precipitate, coat it on a glass fiber membrane with a size of 200 square centimeters, freeze at -45°C, use it as a conjugate pad, and store it in the dark at room temperature after freeze-drying Standby, the composition of the recovery solution is 150mM NaCl, 1 mass% bovine serum albumin solution, 0.5 mass% sucrose and 0.5 mass% sodium caseinate;

(3) Assembling the test strip: attach the sample pad, the conjugate pad prepared in step (2), the nitrocellulose membrane, and the water-absorbent pad to the polyvinyl chloride base plate in sequence, with the adjacent parts overlapping by 1 mm. Get assembled test strips;

(4) Coating nitrocellulose membrane: use a three-dimensional planar filming instrument to coat denatured rabbit/human IgG, anti-human IgG antibody, Mycoplasma pneumoniae specific antigen and human IgM on the assembled test paper of step (3) in sequence On the nitrocellulose membrane of the strip, the setting parameters of the scratching instrument are: the marking interval is 3mm, and the marking concentration is 0.3mg/mL;

(5) Cut the assembled test paper processed in step (4) into test strips with a width of 4 mm. This embodiment can further provide a kit, which includes an aluminum foil bag, a desiccant sealed in the aluminum foil bag, a dropper, and the test strip prepared above.

The preparation method of the kit is as follows: the test strips prepared above are packed in an aluminum foil bag, and then a desiccant and a dropper are packed in, sealed and stored at room temperature.

Example 3

This embodiment is used to illustrate the application of the test strip or kit prepared in Example 2 in the detection of RF (IgM type) and Mycoplasma pneumoniae samples.

The steps of using the test strip are as follows:

(1) Collect serum samples;

(2) Put the test strip flat on the detection table, add 100 μl of the serum sample in step (1) dropwise to the sample pad, observe the color development results of the first detection line, detection line and quality control line after 10 to 15 minutes, The color development result after 30 minutes is considered invalid;

(3) Judgment of results: Judgment results according to Table 3, where "+" means that the red band is displayed, "-" means that the red band is not displayed, and "/" means that the red band is displayed or not:

table 3

The test strips prepared in Example 2 were used to detect clinically confirmed positive and negative sera, and the results are shown in Table 4.

Table 4 Example 2 detection results

Example 4

This example is used to illustrate the joint detection test strip and kit of rheumatoid factor (IgM type) and other antigen-specific IgM antibodies and the preparation method thereof of the present invention.

As shown in Figure 2, the test strip of the present embodiment includes sample pads, conjugate pads, nitrocellulose membranes and water-absorbing pads attached sequentially on a polyvinyl chloride rubber sheet; the sample pads are glass fiber membranes; The conjugate pad is a glass fiber membrane with an anti-human IgM antibody-colloidal gold conjugate; the nitrocellulose membrane is respectively coated with a first detection line, an adsorption line, two detection lines and a quality control line; The first detection line is denatured rabbit/human IgG, the adsorption line is an anti-human IgG antibody, the quality control line is human IgM, and the detection lines are respectively purified Mycoplasma pneumoniae-specific natural multi-epitope Antigen and purified influenza type B specific natural multi-epitope antigen; the absorbent pad is absorbent paper.

Wherein, the distance between adjacent lines of the first detection line, adsorption line, detection line and quality control line is 3 mm. The overlap between the sample pad and the conjugate pad is 1 mm, the overlap between the conjugate pad and the nitrocellulose membrane is 1 mm, and the overlap between the nitrocellulose membrane and the absorbent pad is 1 mm. The width of the test strip is 4mm.

The preparation method of above-mentioned test strip is as follows:

(1) Prepare colloidal gold solution by reducing chloroauric acid with sodium citrate: add 1000mL triple distilled water to the beaker, then add 10mL of 1% by mass sodium citrate solution, heat to boiling, then add 10mL of 1% by mass % chloroauric acid solution, after boiling for 15min to cooling, obtains the colloidal gold solution;

(2) Prepare the binding pad: take 20mL of the colloidal gold solution prepared in step (1) and put it in a beaker, add 150μL of 0.1M K ₂ CO ₃ solution to adjust the pH of the solution to 7.6, stir well, and then add the centrifuged anti-human IgM antibody, stirred for 20min, then added 2mL of 10% by mass bovine serum albumin solution, centrifuged for 40Min at a speed of 10000rpm, discarded the supernatant, then added 1% by mass of bovine serum albumin solution to continue centrifugation, discarded Remove the supernatant, use the recovery solution to recover the precipitate, coat it on a glass fiber membrane with a size of 200 square centimeters, freeze at -45°C, use it as a conjugate pad, and store it in the dark at room temperature after freeze-drying Standby, the composition of the recovery solution is 150mM NaCl, 1 mass% bovine serum albumin solution, 0.5 mass% sucrose and 0.5 mass% sodium caseinate;

(3) Assembling the test strip: attach the sample pad, the conjugate pad prepared in step (2), the nitrocellulose membrane, and the water-absorbent pad to the polyvinyl chloride base plate in sequence, with the adjacent parts overlapping by 1 mm. Get assembled test strips;

(4) Coating nitrocellulose membrane: using a three-dimensional planar filming instrument to coat denatured rabbit/human IgG, anti-human IgG antibody, Mycoplasma pneumoniae-specific antigen, influenza B-specific antigen and human IgM respectively in the step (3) On the nitrocellulose membrane of the assembled test strip, wherein the setting parameters of the film-drawing instrument are: the marking interval is 3 mm, and the marking concentration is 0.3 mg/mL;

(5) Cut the assembled test paper processed in step (4) into test strips with a width of 4 mm. This embodiment can further provide a kit, which includes an aluminum foil bag, a desiccant sealed in the aluminum foil bag, a dropper, and the test strip prepared above.

The preparation method of the kit is as follows: the test strips prepared above are packed in an aluminum foil bag, and then a desiccant and a dropper are packed in, sealed and stored at room temperature.

Example 5

This embodiment is used to illustrate the application of the test strip or kit prepared in Example 4 in the detection of RF (IgM type), Mycoplasma pneumoniae, and influenza B type samples.

The steps of using the test strip are as follows:

(1) Collect serum samples;

(2) Put the test strip flat on the detection table, add 100 μl of the serum sample in step (1) dropwise to the sample pad, observe the color development results of the first detection line, detection line and quality control line after 10 to 15 minutes, The color development result after 30 minutes is considered invalid;

(3) Judgment of results: judge the results according to Table 3.

The test strip prepared in Example 4 is used to detect the clinically confirmed positive serum and negative serum, wherein the RF (IgM type) and Mycoplasma pneumoniae IgM antibody positive samples are the same as in the implementation case 2, and the specificity of influenza B type is newly added 100 cases of IgM-positive samples and 150 cases of three-index IgM-negative samples were tested.

The results are shown in Table 5.

Table 5 Example 5 detection results

Test example 1

This test example is used to illustrate the detection comparison test between the test strip prepared in Example 2 and commercially available detection products.

The tested samples were 100 cases of clinically confirmed positive samples of rheumatoid factor (IgM type), 100 cases of clinically confirmed positive samples of Mycoplasma pneumoniae and 150 cases of co-negative samples.

The test strip prepared in Example 2 is detected by the method in Example 3.

Mycoplasma pneumoniae IgM antibody detection kit (enzyme-linked immunoassay) and rheumatoid factor IgM antibody detection kit (colloidal gold method) were detected using the methods used in their instructions.

The test results are shown in Table 6. As can be seen from the test results, compared with the Mycoplasma pneumoniae IgM antibody detection kit (enzyme-linked immunoassay), the test strip prepared in Example 2 has a higher coincidence rate to the tested sample; Compared with the kit (colloidal gold method), the test strip prepared in Example 2 has a stable and more accurate detection result for rheumatoid factor.

Table 6

Although the invention has been described to a certain extent herein, it will be apparent that appropriate changes can be made in various conditions by those skilled in the art without departing from the spirit and scope of the invention. It is to be understood that the invention is not to be limited to the general and specific examples of the embodiments described, but rather to the scope of the claims and to include equivalents of each element described.

Claims (10)

1. a rheumatoid factor (IgM type) and other antigen-specific IgM antibody combined detection test papers, it is characterised in that Described test strips is included in sample pad, conjugate pad, nitrocellulose filter and the adsorptive pads attached successively on polyvinyl chloride plastic sheet, The first detection line, adsorption line, one or more detection line and nature controlling line it is coated with the most respectively on described nitrocellulose filter；Institute Stating the first detection line is degeneration rabbit/human IgG, and described adsorption line is the antibody of anti-human igg, described nature controlling line behaviour IgM, described inspection Survey line is respectively selected from one or more of specific antigen: mycoplasma pneumoniae specific antigen, influenza Type B specific antigen, Toxoplasmosis pathogen-specific antigen, autoimmunity antigen and anaphylactogen；Preferably, described specific antigen is for cultivating cause of disease Body is after cracking and/or inactivation and through the multi-epitope antigen obtained after purification.

Test strips the most according to claim 1, it is characterised in that described sample pad is glass fibre membrane；And/or described suction Water cushion is absorbent paper.

Test strips the most according to claim 1 and 2, it is characterised in that described conjugate pad for be covered with anti-human IgM antibodies- The glass fibre membrane of colloidal gold conjugate；The immunogen of described anti-human IgM antibodies is preferably the Fc5 μ fragment of people IgM；And/or institute State anti-human IgM antibodies and be preferably the IgG antibody of goat-anti people IgM, F (ab') 2 fragment of the IgG antibody of the most described Yang Yuan.

Test strips the most according to any one of claim 1 to 3, it is characterised in that the width of described test strips is suitable Width, preferably 3～5mm, more preferably 4mm.

Test strips the most according to any one of claim 1 to 4, it is characterised in that

Described first detection line, adsorption line, detection line and nature controlling line each adjacent lines be spaced apart convenient width, preferably 3～ 8mm, more preferably 3mm；

Between described sample pad with conjugate pad overlapping 1～2mm, preferably 1mm；

Between described conjugate pad and nitrocellulose filter overlapping 1～2mm, preferably 1mm；And/or

Between described nitrocellulose filter and adsorptive pads overlapping 1～2mm, preferably 1mm.

6. the preparation method of the test strips according to any one of claim 1 to 5, it is characterised in that described preparation method includes:

(1) reduction of sodium citrate gold chloride method is used to prepare colloidal gold solution: to add 1000mL tri-distilled water in beaker, then add Enter the sodium citrate solution of 1 mass % of 10mL, after being heated to boiling, add the chlorauric acid solution of 1 mass % of 10mL, boil To cooling after boiling 15min, obtain described colloidal gold solution；

(2) pad is prepared: take colloidal gold solution prepared by 20mL step (1) and be placed in beaker, add 150 μ L 0.1M's K₂CO₃The pH to 7.6 of solution regulation solution, stirs, is subsequently adding the anti-human IgM antibodies being centrifuged, and stirs 20min, then Adding the bovine serum albumen solution of 2mL 10 mass %, be centrifuged, centrifugation time is 40Min, and rotating speed is 10000rpm, discards Supernatant, the bovine serum albumen solution adding 1 mass % continues centrifugal, abandoning supernatant, uses recovery liquid to enter precipitate Row recovers, and is coated onto above the glass fibre membrane of 200 square centimeters of sizes, after-45 DEG C of freezings, as conjugate pad, institute State the NaCl that composition is 150mM recovering liquid, the bovine serum albumen solution of 1 mass %, the sucrose of 0.5 mass % and 0.5 matter The casein sidium of amount %；

(3) test strips is assembled: conjugate pad, nitrocellulose filter and the adsorptive pads sample pad, step (2) prepared are the most mutual It is attached on polrvinyl chloride base plate overlap joint, obtains assembling test strips；

(4) it is coated nitrocellulose filter: use and draw film instrument by degeneration rabbit/human IgG, the antibody of anti-human igg, antigen-specific IgM On the nitrocellulose filter of the assembling test strips that the antigen of antibody and people IgM are coated on step (3) the most respectively；

Preferably, described in step (2), anti-human IgM antibodies processes the most by the following method: use pepsin digestion anti-human The IgG antibody of IgM, affinity purification processes and obtains described anti-human IgM antibodies.

The preparation method of test strips the most according to claim 6, it is characterised in that degeneration rabbit/people described in step (4) The concentration of IgG, the antibody of anti-human igg, the antigen of antigen-specific IgM antibody and people IgM is each independently 0.3mg/mL.

8. a rheumatoid factor (IgM type) and other antigen-specific IgM antibody combined detection kits, it is characterised in that Described test kit includes: the test strips according to any one of claim 1 to 5 or employing method as described in claim 6 or 7 The test strips of preparation；Preferably, described test kit farther includes:

Aluminium foil bag and be sealed in the desiccant in described aluminium foil bag；And/or

Dropper.

9. rheumatoid factor (IgM type) and a detection method for other antigen-specific IgM antibody, described detection method based on Test strips according to any one of claim 1 to 5, use test strips or right prepared by the method described in claim 6 or 7 Require the test kit described in 8, it is characterised in that described detection method includes:

(1) described test strips being lain on detection table top, drip 100 μ l samples to sample pad, described sample is whole blood or blood Clearly, observing the first detection line, detection line and the colour developing result of nature controlling line after 10～15 minutes, the colour developing result after 30 minutes is considered as Invalid；

(2) rheumatoid factor in described sample (IgM type) and the feelings of other antigen-specific IgM antibody are judged according to following standard Condition:

When the first detection line, detection line and nature controlling line all show red stripes, and result is judged to；Rheumatoid factor (IgM type) sun Property and other antigen-specific IgM antibody positive,

When the first detection line does not shows that red stripes, detection line and nature controlling line all show red stripes, and result is judged to；Rheumatoid The factor (IgM type) is negative and other antigen-specific IgM antibody are positive,

When the first detection line and nature controlling line all show that red stripes, detection line do not show that red stripes, result are judged to；Rheumatoid The factor (IgM type) is positive and other antigen-specific IgM antibody are negative,

When the first detection line and detection line do not show red stripes, and nature controlling line display red stripes, result is judged to；Rheumatoid because of Son (IgM type) is negative and other antigen-specific IgM antibody are negative,

When nature controlling line does not shows that red stripes, result are judged to；It is invalid to detect.

10. test strips according to any one of claim 1 to 5, use reagent paper prepared by the method described in claim 6 or 7 Test kit described in bar or claim 8 is selected from the product of one or more of for detection and/or diagnosis in preparation Application:

Rheumatoid immunological diseases, it is preferable that described rheumatoid immunological diseases are rheumatoid arthritis；

Pathogen recent infection, it is preferable that described pathogen is mycoplasma pneumoniae and/or Type B influenza virus；

Prenatal and postnatal care, it is preferable that described prenatal and postnatal care is detection toxoplasma；

And autoimmune pathologies, it is preferable that described autoimmune pathologies is systemic lupus erythematosus (sle) and/or is dried Disease.