Kit for detecting chlamydia pneumoniae antigen and preparation method thereof

Abstract

The invention discloses a kit for detecting a chlamydia pneumoniae antigen and a preparation method of the kit, and particularly relates to the technical field of biological detection. The kit comprises: antigen detection test paper; the antigen detection test paper comprises an immune microsphere pad and an immune nitrocellulose membrane; the immune microsphere pad is coated with a chlamydia pneumoniae monoclonal antibody 1 marked by colored latex microspheres; a detection line coated with a chlamydia pneumoniae monoclonal antibody 2 and a quality control line coated with a goat anti-mouse IgG polyclonal antibody are arranged on the immune nitrocellulose membrane; the chlamydia pneumoniae monoclonal antibody 1 and the chlamydia pneumoniae monoclonal antibody 2 are obtained by screening and culturing artificial antigens. The antigen detection is based on the immunoreaction between the specific antibody and the virus protein in the sample, and the sample at the infected part can be directly detected.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a kit for detecting Chlamydia pneumoniae antigen and a preparation method thereof. Background Art

[0002] Chlamydia pneumoniae is one of the common pathogens that cause respiratory tract infections. The onset of infection with Chlamydia pneumoniae is relatively slow, and the most prominent symptoms are in the elderly and infants with relatively low immunity. The main clinical manifestations are cough, fever, sore throat, sputum, etc. The symptoms are relatively mild, but patients with Chlamydia pneumoniae infection who have underlying diseases may develop atypical pneumonia and other systemic complications such as nerves and circulation, which have a more prominent impact on patients and should be taken seriously. Mycoplasma pneumoniae colonizes in the respiratory tract through adhesion organelles, leading to diseases such as community-acquired pneumonia. Adhesion organelles are composed of surface structures and internal structures. The surface structure is responsible for the binding of Mycoplasma pneumoniae to the surface of host cells, including P1 adhesin, P30 adhesin, P40, and P90. The internal structure is divided into a translucent area and a core structure, and its functions include the formation and maintenance of organelles and the generation and transmission of force.

[0003] At present, it is difficult to promote the application of laboratory pathogen culture tests in the laboratory testing of some hospitals for the diagnosis of Chlamydia pneumoniae. The PCR test has strict requirements for specimen collection, which makes its quality control relatively difficult. The commonly used serum antibody test has a lag in pathogen detection. Summary of the invention

[0004] To this end, the present invention provides a kit for detecting Chlamydia pneumoniae antigen and a preparation method thereof, so as to solve the problems of difficult quality control of existing laboratory tests and lag in pathogen detection. The purpose of the present invention is to provide a kit for detecting Chlamydia pneumoniae antigen with low cost and relatively low detection requirements.

[0005] The present invention is a kit for directly detecting Chlamydia pneumoniae antigens, which has good characteristics, high sensitivity, simple operation, does not require any equipment or instruments, and can directly read the results with the naked eye. The present invention uses colored latex microspheres as a labeled tracer, which have a higher sensitivity than colloidal gold, and are combined with antibodies by chemical bond coupling. The colored latex microspheres are more firmly combined with antibodies, and the product is more stable.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] According to one aspect of the present invention, a kit for detecting Chlamydia pneumoniae antigen is provided, the kit comprising: an antigen detection test paper;

[0008] The antigen detection test paper comprises an immune microsphere pad and an immune nitrocellulose membrane; the immune microsphere pad is coated with a Chlamydia pneumoniae monoclonal antibody 1 marked with colored latex microspheres;

[0009] The immune nitrocellulose membrane is provided with a detection line coated with a Chlamydia pneumoniae monoclonal antibody 2 and a quality control line coated with a goat anti-mouse IgG polyclonal antibody;

[0010] The Chlamydia pneumoniae monoclonal antibody 1 and the Chlamydia pneumoniae monoclonal antibody 2 are both obtained by artificial antigen screening and cultivation;

[0011] The antigen detection test paper also includes a liner and absorbent paper;

[0012] The immune microsphere pad, immune nitrocellulose membrane and absorbent paper are pasted on the lining sheet.

[0013] Furthermore, the colored latex microspheres are red latex microspheres with a diameter of 200-300 nm.

[0014] In some embodiments, it is more preferred that the colored latex microspheres are red colored latex microspheres with a diameter of about 300 nm.

[0015] Furthermore, the detection kit also includes a sample extract, which uses 0.05M Tris-HCl (PH=8.0) as a basic solution and contains S9 and NP-40; the concentration of S9 in the sample extract is 2%, and the concentration of NP-40 in the sample extract is 1%.

[0016] According to another aspect of the present invention, a method for preparing the above-mentioned Chlamydia pneumoniae antigen detection kit is provided, characterized in that it comprises the following steps:

[0017] Step 1, preparation of immune microsphere pad: labeling Chlamydia pneumoniae monoclonal antibody 1 with colored latex microspheres to prepare immune microsphere solution, and coating the solution on a release pad to prepare an immune microsphere pad;

[0018] Step 2, preparation of immunonitrocellulose membrane: using Chlamydia pneumoniae monoclonal antibody 2 goat anti-mouse IgG polyclonal antibody to prepare detection line solution and quality control line solution, respectively, and coating them on immunonitrocellulose membrane;

[0019] Step 3, preparation of antigen test paper: sticking the immune microsphere pad, immune nitrocellulose membrane and absorbent paper on the lining to prepare a semi-finished plate of test paper; cutting the semi-finished plate of test paper to prepare strip-shaped antigen test paper.

[0020] Furthermore, in the step 1, during the preparation of the immune microsphere pad, the spray volume of the immune microsphere solution on the release pad is 5-7 μL / cm, and the immune microsphere pad obtained after spraying is dried at 38-42° C. for 4-16 hours; in the step 2, during the preparation of the immune nitrocellulose membrane, the test line solution and the quality control line solution are 1.0-2.0 mg / ml, and the prepared immune nitrocellulose membrane is dried at 38-42° C. for 4-16 hours.

[0021] Furthermore, the specific method of labeling Chlamydia pneumoniae monoclonal antibody 1 with colored latex microspheres includes:

[0022] Step 1, pretreatment of colored latex microspheres: washing the colored latex microsphere solution with a labeled buffer solution MES to obtain pretreated colored latex microspheres;

[0023] Step 2, EDC activation: adding labeling buffer MES, EDC and NHS solution to the pre-treated colored latex microspheres obtained in step 1 for mixed reaction to obtain a mixed solution;

[0024] Step 3, latex labeling: centrifuge the mixed solution after the reaction, take the precipitate and add MES buffer to re-dissolve it, and ultrasonicate it evenly, add Chlamydia pneumoniae monoclonal antibody 1, and react under ultrasonic conditions;

[0025] Step 4, blocking: continue to add aminoethanol solution, react under ultrasonic conditions, block; centrifuge, discard the supernatant, and precipitate for later use;

[0026] Step 5, centrifugal purification: re-dissolve the precipitate with TBS buffer and homogenize by ultrasonication to obtain the immune microsphere solution.

[0027] Furthermore, the mass ratio of the amount of EDC to the colored latex microspheres is 1:10.

[0028] Furthermore, the mass ratio of the Chlamydia pneumoniae monoclonal antibody 1 to the colored latex microspheres is 1:20.

[0029] Furthermore, the reaction time of latex labeling in step three is 1 hour.

[0030] Furthermore, in step 4, the latex sealing time is 1 hour.

[0031] In some embodiments, more preferably, the dosage ratio of the activator EDC is as follows: when the concentration of the colored latex microsphere solution is 1% and the marking volume is 1 ml, the concentration of the EDC solution is 20 mg / ml and the dosage is 50 μL; that is, the mass ratio of the dosage of EDC to the colored latex microspheres is 1:10.

[0032] The dosage ratio of latex-labeled Chlamydia pneumoniae monoclonal antibody 1 is as follows: when the concentration of the colored latex microsphere solution is 1% and the labeled volume is 1 ml, the preferred dosage of Chlamydia pneumoniae monoclonal antibody 1 is 0.5 mg / ml, that is, the mass ratio of Chlamydia pneumoniae monoclonal antibody 1 to colored latex microspheres is 1:20.

[0033] The conventional latex labeling reaction time is 3-5 hours, but the reaction under ultrasonic conditions can shorten the reaction time and complete the coupling of antibodies and colored latex microspheres in 1 hour.

[0034] Antigen / antibody testing uses the principle of antigen-antibody immune reaction to detect antibodies produced by the human immune response induced by viral antigens and viral proteins in the sample.

[0035] Antibodies require that after the virus infects the human body, the antigenic determinant cluster contacts the lymphocytes and then induces the lymphocytes to produce specific antibodies. Different individuals have different immune responses to invading antigens in different intensities and reaction times, which will have a great impact on the sensitivity of antibody detection.

[0036] Advantages of the present invention:

[0037] The antibody detection of the present invention mainly detects antibodies through immune reaction between specific antigen recombinant proteins and potential Chlamydia pneumoniae antigen antibodies in blood samples. The antigen detection of the present invention is based on the immune reaction between specific antibodies and viral proteins in samples, and can directly detect samples of infected sites.

[0038] The present invention uses latex particles as markers and tracers, and uses antigen-antibody reaction and lateral chromatography to detect and analyze the target. In actual operation, it has the advantages of convenience and speed: the detection personnel do not need professional detection knowledge, and the detection experiment does not require professional equipment configuration, and can be widely carried out in grassroots community hospitals and health service centers.

[0039] The reason why the present invention uses latex particles as the labeling tracer is that the latex particles have uniform particle size, bright color, and can be coupled with functional groups on the surface, so they can be labeled in a covalent manner. The labeled antibody binding is stable and not easy to fall off, which is an ideal naked eye visible labeling tracer. In addition, the latex particle manufacturing process is mature, and there are many high-quality latex particle suppliers on the market, such as Thermo Fisher Scientific, Merck, and Suzhou Weidu. The company purchases high-quality latex particles and directly performs production labeling operations. The uniformity of the tracer is good, the coefficient of variation (CV) of the product is relatively easy to control, and the difference between batches is also small. DETAILED DESCRIPTION

[0040] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The Chlamydia pneumoniae monoclonal antibodies 1 and 2 used in the embodiments of the present invention were obtained by screening by our company:

[0042] The present invention selects an immunogen with good specificity. The P1 protein is one of the main adhesion proteins of Mycoplasma pneumoniae, contains 4884bp, has a GC content of 53.5%, a relative molecular weight of 170kda, and is composed of 1627 amino acid residues. Detecting the P1 adhesin of Mycoplasma pneumoniae can be used as a method for detecting Mycoplasma pneumoniae. The present invention further designs an immunogen for the antigenic epitopes of the amino acid positions T13-R50 (sequence as shown in SEQ ID NO.1) and V1381-S1406 (sequence as shown in SEQ ID NO.2) on the P1 adhesin protein group to prepare subsequent monoclonal antibodies, and it is found that the immunogens designed for the two targets have better specificity for the prepared monoclonal antibodies.

[0043] The specific method is as follows: the screening is to use the antigen to coat the 96-well plate at 100μg / well, then add the corresponding monoclonal antibody sample and shake at 37℃ for 30min, add sheep anti-mouse enzyme-labeled antibody (100μg / well, 100μL / well) at 37℃ for 30min after elution, add 1% BSA 200μL per well for blocking, elute after half an hour of blocking, add 50μL of stop solution after adding the color developing solution to terminate the experiment, and select the cell lines corresponding to the wells with OD values ​​above 1.0.

[0044] Cell strains that can be paired with Mycoplasma pneumoniae pathogen cultures were selected, and then cell strains with higher OD values ​​were cloned, and finally Chlamydia pneumoniae antigen (monoclonal antibody 1 (antibody strain 1693)) Chlamydia pneumoniae antigen (monoclonal antibody 2 (antibody strain 1694)) were selected.

[0045] Finally, it was determined that monoclonal antibody 1 and monoclonal antibody 2 could complete sandwich pairing detection of the target.

[0046] Verification: By testing 20 positive throat swab samples (confirmed by PCR) and 10 negative throat swab samples (confirmed by PCR), the positive detection rate is 85%. All negative test results are negative. The total compliance rate is 90%. The results can basically meet certain clinical use needs.

[0047] Example 1

[0048] This embodiment provides a screening of a labeled tracer:

[0049] In this embodiment, colloidal gold and red colored latex microspheres are used as markers for comparison, wherein the colloidal gold uses colloidal gold of about 40 nm (chloroauric acid: trisodium citrate = 1:1.1), and the red colored latex microspheres use red colored latex microspheres of about 300 nm in diameter.

[0050] 1. Colloidal gold labeling process:

[0051] Take 5ml of colloidal gold and add 40μL of 0.1MK 2 CO 3 Adjust the colloidal gold pH to 7.0; add 100 μg of Chlamydia pneumoniae antigen (monoclonal antibody 1), let stand for 45 minutes; centrifuge at 8000 r / min for 15 minutes and discard the supernatant; re-dissolve the precipitate obtained by centrifugation with 5 ml of gold label working solution, and apply the re-dissolved solution to the glass fiber membrane SB08. After drying overnight, it is used in combination with the nitrocellulose membrane coated with Chlamydia pneumoniae antigen monoclonal antibody 2 to form a reagent strip A.

[0052] 2. Latex marking process:

[0053] Take 1mL of 1% colored latex microspheres and centrifuge at 13000rpm for 20 minutes, discard the supernatant; add 1mL MES to the precipitate to dissolve and ultrasonicate. With 50μL of 20mg / mL EDC and 35.5μL of 10mg / ml NHS, shake and react for 1 hour. Centrifuge at 13000rpm for 20 minutes, discard the supernatant; add MES to the precipitate to dissolve in 1mL MES, and ultrasonicate evenly; add 0.5mg of Chlamydia pneumoniae antigen monoclonal antibody 1 to reach the batch volume and react overnight; add 15μL of 1M aminoethanol, react for 30 minutes, and block; centrifuge at 13000rpm for 20 minutes, discard the supernatant; re-dissolve the precipitate particles with TBS to 10mL. After ultrasonic mixing, apply the re-dissolved solution to the glass fiber membrane SB08. After drying overnight, it is used with the nitrocellulose membrane coated with Chlamydia pneumoniae antigen monoclonal antibody 2 to form reagent strip B.

[0054] 3. Marking comparison:

[0055] The Chlamydia pneumoniae antigen recombinant protein with a product standard concentration of 2.2 mg / ml was serially diluted with the sample extract to obtain the results shown in Table 1.

[0056] Table 1: Results of the selection experiments of labeled tracers

[0057]

[0058] From the data in the table, it can be seen that the sensitivity of colloidal gold markers and latex markers differs by about 10 times, so colored latex microspheres are selected as markers for this reagent.

[0059] Example 2

[0060] This embodiment provides a method for preparing a kit:

[0061] 1) Use colored latex microspheres to label Chlamydia pneumoniae antigen monoclonal antibody 1 to prepare an immune microsphere solution (colored latex microspheres-Chlamydia pneumoniae antigen (monoclonal antibody 1 complex). 2) Dilute the immune microsphere solution (colored latex microspheres-Chlamydia pneumoniae monoclonal antibody 1 complex) appropriately and coat it on the release pad to prepare an immune microsphere pad. 3) Use Chlamydia pneumoniae monoclonal antibody 2 to prepare a test line (T line) solution, and use goat anti-mouse IgG to prepare a quality control line (C line) solution. 4) Spray C and T lines on a nitrocellulose membrane to prepare an immune nitrocellulose membrane. 5) Dry the immune microsphere pad and the immune nitrocellulose membrane. 6) Use the immune microsphere pad to coat the colored latex microspheres-Chlamydia pneumoniae antigen (monoclonal antibody 1 complex), the immune nitrocellulose membrane, the Chlamydia pneumoniae antigen (monoclonal antibody 2, goat anti-mouse IgG), and the absorbent paper to stick on the plastic liner to form a semi-finished plate of Chlamydia pneumoniae antigen detection paper. 7) a. Paste the handle paper, MAX glue, etc. onto the semi-finished plate, cut the semi-finished plate of Chlamydia pneumoniae antigen (test paper) into reagent strips, and put them into an aluminum foil bag together with a desiccant, seal and package to obtain a single-portion Chlamydia pneumoniae antigen test paper (strip type). b. Cut the semi-finished plate of Chlamydia pneumoniae antigen test paper into reagent strips, put them into a plastic card slot, assemble them into a card, and put them into an aluminum foil bag together with a desiccant, seal and package to obtain a single-portion Chlamydia pneumoniae antigen test paper (card type). 8) Package several single-portion Chlamydia pneumoniae antigen test papers, sample extracts, and instructions together to obtain a finished product of the Chlamydia pneumoniae antigen test kit (latex method).

[0062] Example 3

[0063] This embodiment provides a screening of process parameters for preparing a kit:

[0064] 1. Preparation process of immune microsphere solution

[0065] The latex labeling procedure was carried out in accordance with relevant literature and manufacturer's instructions, using the EDC activation labeling principle. The basic steps are as follows:

[0066] (1) Latex pretreatment: Take a measure of the colored latex microsphere solution diluted to 1%, add the labeling buffer MES to the batch volume and wash, centrifuge at 13000rpm for 20 minutes, and discard the supernatant; add MES to the precipitate to the batch volume for re-dissolution, and ultrasonically homogenize, centrifuge at 13000rpm for 20 minutes, and discard the supernatant; (2) EDC activation: Add a certain volume of labeling buffer MES to the precipitate, mix with a certain volume of 20mg / ml EDC (0.05ml / ml, the ratio of EDC addition volume to the addition volume of 1% colored latex microsphere solution, to avoid the effect of volume change on the system) and 10mg / ml NHS (0.075ml / ml, the ratio of NHS addition volume to the addition volume of 1% colored latex microsphere solution, to avoid the effect of volume change on the system) to the batch volume, and react for 20min. Centrifuge at 13000rpm for 20 minutes, discard the supernatant; add MES to the precipitate to a certain volume, re-dissolve it in the batch volume, and mix it evenly by ultrasonication; (3) Latex labeling: take a certain amount of Chlamydia pneumoniae monoclonal antibody 1 and add it to the batch volume, and react for 1 hour under ultrasonic conditions; (4) Blocking: add 1M aminoethanol (0.03ml / ml, the ratio of the volume of aminoethanol added to the volume of the 1% colored latex microsphere solution added to avoid the impact of volume changes on the system), react for 1 hour under ultrasonic conditions, and block; (5) Centrifugal purification: centrifuge at 13000rpm for 20 minutes, discard the supernatant; re-dissolve the precipitate particles with TBS to the batch volume. Mix it evenly by ultrasonication; (6) Repeat the centrifugation operation again: centrifuge at 13000rpm for 20 minutes. Discard the supernatant; re-dissolve the precipitate particles with TBS to the batch volume. Mix it evenly by ultrasonication and set aside. The labeling work is completed. The immune microsphere solution (Chlamydia pneumoniae monoclonal antibody 1-colored latex microsphere complex) is obtained. Several important parameters in the labeling process are: EDC amount, labeling antibody concentration and reaction time. The present invention studies each of them and obtains the optimal process.

[0067] 1.1 Determination of EDC quantity:

[0068] As an activator, if the concentration of EDC is too low, the labeling efficiency will be reduced and the product will not reach the expected sensitivity; if the concentration is too high, it may cause agglutination or increase non-specific reaction after latex labeling. Therefore, the present invention optimizes the concentration of EDC while fixing the amount of microspheres and the volume of EDC addition (the test comparison is carried out when the concentration of the colored latex microsphere solution is 1% and the volume is 1mL). The experimental results are shown in Table 2.

[0069] Table 2: EDC concentration determination test results

[0070]

[0071]

[0072] Conclusion: Under the same conditions, with the increase of EDC concentration, the sensitivity showed an upward trend, but false positive reactions occurred above 2 mg / ml. The EDC concentration of 1 mg / ml showed good sensitivity and specificity, so the EDC usage was determined to be 1% of the colored latex microsphere solution 1mL added with EDC to make its concentration 1 mg / ml, and the mass ratio of microspheres and EDC for the best reaction condition was 1:10.

[0073] 1.2 Determination of the amount of labeled antibody

[0074] The amount of labeled antibody has a certain influence on the product performance. Too low concentration will lead to low sensitivity, and too high concentration will easily increase non-specific reactions. Therefore, the present invention optimizes the amount of labeled antibody (with a colored latex microsphere solution concentration of 1%, a volume of 1mL, and an EDC concentration of 1mg / ml for test comparison). The experimental results are shown in Table 3.

[0075] Table 3: Results of the test to determine the amount of labeled antibodies

[0076]

[0077] Conclusion: Under the same other conditions, as the amount of labeled antibody increases, the sensitivity increases, and a weak false positive reaction occurs at 1.0 mg / ml. When the labeled amount is 0.5 mg / ml and 0.75 mg / ml, the performance meets the requirements. Considering the saving of antibodies, the final amount of labeled antibody is determined to be 0.5 mg / ml, that is, 1 mg of Chlamydia pneumoniae monoclonal antibody 1 is added to 1 mL of 1% colored latex microsphere solution, and the mass ratio of Chlamydia pneumoniae monoclonal antibody 1 to colored latex microspheres is 10:1.

[0078] 1.3 Determination of marking time

[0079] The reaction time affects the binding efficiency of the antibody and latex. The shorter the time, the lower the labeling efficiency. The longer the time, the more complete the labeling. Therefore, the present invention confirms the minimum required reaction time (tested with the above-determined preferred colored latex microspheres and EDC, Chlamydia pneumoniae monoclonal antibody 1 dosage). The experimental results are shown in Table 4.

[0080] Table 4: Reaction time determination test results

[0081]

[0082] Conclusion: When other conditions are fixed, different reaction times have a significant impact on the performance after labeling. From the table above, we can see that the best labeling state is achieved when the reaction time is 1 hour. Therefore, 1 hour is determined to be the optimal time required for the reaction.

[0083] 2. Preparation process of immune microsphere pad

[0084] 2.1 Determination of the optimal spray volume of immunomicrosphere dilution solution

[0085] The release pad has strong protein adsorption capacity and hydrophilicity. By spraying an appropriate concentration of immune microsphere solution on the pad and drying it, a solid phase protein can be obtained. In the immunochromatographic test, too high or too low a spray volume will affect the sensitivity, specificity and uniformity of the product. If the spray volume is too high, it may cause some non-specific reactions and waste antibodies; if the spray volume is too low, it may reduce the sensitivity of the product; during the spraying process of the release pad, if the spray volume is too high, it is easy to exceed the effective adsorption capacity of the release pad, resulting in too dark background in the detection process, unclear plate running, unclean release and other results that are difficult to judge. There are two purposes for determining the spray volume: one is to ensure that the immune reaction is fully carried out; the other is to save antibody protein as much as possible to avoid non-specific reactions and excessive waste.

[0086] Test method: The immune microsphere dilution solution (colored latex microsphere-Chlamydia pneumoniae antigen (monoclonal antibody 1 complex) was sprayed at a gradient rate of 1.0 μL / cm, 2.0 μL / cm, 2.5 μL / cm, 3 μL / cm, and 5 μL / cm. The line was drawn using the parameter of 1 μl / cm, and after drying, the test strip was assembled for performance testing to determine the optimal spray rate. The test results are shown in Table 5.

[0087] Table 5: Test results for determining the optimal spray volume of immune microsphere pad

[0088]

[0089]

[0090] Note: “+” indicates weak color, “++” indicates obvious color, “+++” indicates very dark color, and “—” indicates invisible color.

[0091] The test results show that when the spray volume is 1.0μL / cm and 2.5μL / cm, the line color is weak and uneven, and the immune response is insufficient; when the spray volume is 5μL / cm and 6μL / cm, the color is easy to judge, and the strip color is uniform and consistent, and the immune response is sufficient. Based on the principle of no waste and reducing nonspecific reactions, the spray volume of the process is determined to be 5μL / cm.

[0092] 2.2 Determination of the optimal drying conditions for immunomicrosphere pads

[0093] The drying conditions after spraying have an impact on the performance of the immunomicrosphere pad. The test found that the immunomicrosphere pad after spraying can achieve good results if it is placed in a 38-42℃ oven for more than 4 hours.

[0094] Test method: Place the sprayed immunomicrosphere pad in a 38-42°C oven and dry for 2 hours, 3 hours, 4 hours, 8 hours, 16 hours and 18 hours. After drying, assemble the test strips for property observation and performance testing. The best drying time is determined by whether the properties are uniform and whether the performance test is qualified. The test results are shown in Table 6 below:

[0095] Table 6: Test results of optimal drying time of immune microsphere pad

[0096]

[0097] The test results show that when the drying time is less than 4 hours, the background of the immunomicrosphere pad is red during detection, resulting in false positive results; when the drying time is greater than 16 hours, false negative results appear in the test results; when the drying time is 4 to 16 hours, the background of the immunomicrosphere pad is clean and the performance tests are qualified.

[0098] Therefore, the optimal drying time of the immunomicrosphere pad was determined to be 4 to 16 hours.

[0099] 3. Preparation process of immunonitrocellulose membrane

[0100] 3.1 Determination of the optimal concentration of antibody protein coating on nitrocellulose membrane

[0101] There are two protein-coated bands on the immunonitrocellulose membrane, namely the quality control line (C line) and the test line (T line). The C line is coated with goat anti-mouse IgG antibody; the T line is coated with Chlamydia pneumoniae antigen (monoclonal antibody 2). There are two purposes for determining the coating concentration of the C / T line: one is to ensure that the immune response is fully carried out; the other is to save antibody protein as much as possible to avoid nonspecific reactions and excessive waste.

[0102] Test method: Dilute goat anti-mouse IgG antibody and Chlamydia pneumoniae antigen (monoclonal antibody 2) to 1.0 mg / ml, 1.5 mg / ml, 2.0 mg / ml, and 2.5 mg / ml, respectively. Use the parameter of 1 μl / cm for streaking, assemble the test strips after drying for performance testing, and observe the color depth and uniformity of the C / T line to determine the appropriate concentration of the coating. The test results are shown in Table 7.

[0103] Table 7: Test results for determining the optimal concentration of antibody protein coating on nitrocellulose membrane

[0104]

[0105] Note: “+” indicates weak color, “++” indicates obvious color, “+++” indicates very dark color, and “—” indicates invisible color.

[0106] The test results show that when the coating concentration is 0.5mg / ml, the color of the C / T line is relatively weak and uneven, which affects the interpretation of the results; when the coating concentration is 0.75mg / ml, the color of the C line is obvious, but the color of the T line is weak, and the immune response is insufficient; when the coating concentration is 1.0mg / ml and 2.0mg / ml, the color of the C line and T line is easy to judge, and the color of the strip is uniform and consistent, and the immune response is sufficient. Based on the principle of no waste and reducing nonspecific reactions, the protein coating concentration of the C line and T line in the streaking process is determined to be 2.0mg / ml.

[0107] 3.2 Determination of the optimal drying conditions for immunonitrocellulose membranes

[0108] The drying conditions after streaking have an impact on the performance of the immunonitrocellulose membrane. The experiment found that the immunonitrocellulose membrane after streaking can achieve good results by drying it in an oven at 38-42℃ for more than 4 hours.

[0109] Test method: Place the nitrocellulose membrane with the lines drawn in a 38-42°C oven and dry for 2 hours, 3 hours, 4 hours, 8 hours, 16 hours and 18 hours respectively. After drying, assemble the test strips for property observation and performance testing. The optimal drying time is determined by whether the C / T line is uniform and whether the performance test is qualified. The test results are shown in Table 8.

[0110] Table 8: Results of the test on the optimal drying time of immunonitrocellulose membrane

[0111]

[0112] The test results show that when the drying time is less than 4 hours, the C / T line has dragging during detection and the edges are not very neat and are relatively scattered; when the drying time is more than 16 hours, there are false negatives in the test results; when the drying time is 4 to 16 hours, the C / T line is neat and has no dragging, and the performance tests are qualified.

[0113] Therefore, the optimal drying time for immunonitrocellulose membrane was determined to be 4 to 16 hours.

[0114] 4. Sample Extract Preparation Process

[0115] 4.1 Materials

[0116] 0.05M Tris-HCl (PH=8.0), S9, NP-40.

[0117] 4.2 Preparation method

[0118] Using 0.05M Tris-HCl (PH=8.0) as the base solution, the concentrations of S9 and NP-40 were screened, so as to screen out the best formula for sample extracts to ensure the suitability of the reagent card reaction system. Prepare S9 extracts with concentrations of 0.5%, 1%, 2%, and 3% respectively; prepare NP-40 extracts with concentrations of 0.1%, 0.25%, 0.5%, 0.75%, and 1%. Take the minimum detection limit reference S1 and negative throat swab samples for testing, add samples to test the extracts of the two formulas, and record the test results and phenomena.

[0119] 4.3 Results

[0120] 4.3.1 Experimental results of different concentrations of Triton-100

[0121] 1) The test results of the minimum detection limit reference material P1 are shown in Table 9.

[0122] Table 9

[0123]

[0124]

[0125] 2) The test results of negative throat swab samples are shown in Table 10.

[0126] Table 10

[0127]

[0128] The test results show that the best concentration of S9 is 2%.

[0129] 4.3.2 Results

[0130] 1) The experimental results of different concentrations of NP-40 are shown in Table 11.

[0131] Table 11

[0132]

[0133] 2) The test results of negative throat swab samples are shown in Table 12.

[0134] Table 12

[0135]

[0136] The test results show that the best concentration of NP-40 is 0.5%.

[0137] 4.3.3 Verification of two optimal component combinations

[0138] The extract was prepared with 0.15M Nacl, 2% S9, and 0.5% NP-40 as a combination, and the lowest detection limit reference S1 and 10 negative throat swab samples were tested in parallel for 5 times for verification. The results are as follows:

[0139] 1) The test results of the minimum detection limit reference material S1 are shown in Table 13.

[0140] Table 13

[0141]

[0142] 2) Negative swab sample test results are shown in Table 14.

[0143] Table 14

[0144]

[0145] The test results show that the best combination of extracting solution is when the concentration of Triton-100 is 1% and the concentration of NP-40 is 0.5%.

[0146] Example 4

[0147] This embodiment provides Chlamydia pneumoniae antigen detection.

[0148] Methods for determining the effectiveness of the system

[0149] 1. After the production process and reaction system are determined, the finished product is prepared for performance testing to verify the effectiveness of the production process and reaction system.

[0150] 2. Test method: According to the determined production process, prepare the immune microsphere pad and immune nitrocellulose membrane, assemble and package them. Then perform performance testing according to the reaction conditions of the determined reaction system and record the test results. The test results are shown in Table 15 below:

[0151] Table 15: Results of validation of production process and reaction system effectiveness

[0152]

[0153]

[0154] 3. Test results: The minimum detection limit, positive compliance rate, negative compliance rate and repeatability of the test all meet the requirements.

[0155] 4. Conclusion: The above-mentioned production process and reaction system can meet the expected use purpose of this product.

[0156] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0157]

Claims

1. A kit for detecting Chlamydia pneumoniae antigen, characterized in that: The kit comprises: an antigen detection test paper; The antigen detection test paper comprises an immune microsphere pad and an immune nitrocellulose membrane; the immune microsphere pad is coated with a Chlamydia pneumoniae monoclonal antibody 1 marked with colored latex microspheres; The immune nitrocellulose membrane is provided with a detection line coated with a Chlamydia pneumoniae monoclonal antibody 2 and a quality control line coated with a goat anti-mouse IgG polyclonal antibody; The Chlamydia pneumoniae monoclonal antibody 1 and the Chlamydia pneumoniae monoclonal antibody 2 are both obtained by artificial antigen screening and cultivation; The colored latex microspheres are red latex microspheres with a diameter of 200-300 nm.

2. The kit according to claim 1, characterized in that The antigen detection test paper also includes a lining sheet and absorbent paper; the immune microsphere pad, immune nitrocellulose membrane and absorbent paper are pasted on the lining sheet.

3. The kit according to claim 1, characterized in that The detection kit also includes a sample extract, which uses 0.05M Tris-HCl (PH=8.0) as a basic solution and contains S9 and NP-40; the concentration of S9 in the sample extract is 2%, and the concentration of NP-40 in the sample extract is 1%.

4. The method for preparing a kit for detecting Chlamydia pneumoniae antigen according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, preparation of immune microsphere pad: labeling Chlamydia pneumoniae monoclonal antibody 1 with colored latex microspheres to prepare immune microsphere solution, and coating the solution on a release pad to prepare an immune microsphere pad; Step 2, preparation of immunonitrocellulose membrane: using Chlamydia pneumoniae monoclonal antibody 2 goat anti-mouse IgG polyclonal antibody to prepare detection line solution and quality control line solution, respectively, and coating them on immunonitrocellulose membrane; Step 3, preparation of antigen test paper: sticking the immune microsphere pad, immune nitrocellulose membrane and absorbent paper on the lining to prepare a semi-finished plate of test paper; cutting the semi-finished plate of test paper to prepare strip-shaped antigen test paper.

5. The preparation method according to claim 4, characterized in that: The specific method of labeling Chlamydia pneumoniae monoclonal antibody 1 with colored latex microspheres comprises: Step 1, pretreatment of colored latex microspheres: washing the colored latex microsphere solution with a labeled buffer solution MES to obtain pretreated colored latex microspheres; Step 2, EDC activation: adding labeling buffer MES, EDC and NHS solution to the pre-treated colored latex microspheres obtained in step 1 for mixed reaction to obtain a mixed solution; Step 3, latex labeling: centrifuge the mixed solution after the reaction, take the precipitate and add MES buffer to re-dissolve it, and ultrasonicate it evenly, add Chlamydia pneumoniae monoclonal antibody 1, and react under ultrasonic conditions; Step 4, blocking: continue to add aminoethanol solution, react under ultrasonic conditions, block; centrifuge, discard the supernatant, and precipitate for later use; Step 5, centrifugal purification: re-dissolve the precipitate with TBS buffer and homogenize by ultrasonication to obtain the immune microsphere solution.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the amount of EDC to the colored latex microspheres is 1:

10.

7. The preparation method according to claim 6, characterized in that: The mass ratio of the Chlamydia pneumoniae monoclonal antibody 1 to the colored latex microspheres is 1:

20.

8. The preparation method according to claim 7, characterized in that: The reaction time of latex labeling in step 3 is 1 hour.

9. The preparation method according to claim 8, characterized in that: The latex sealing time in the step 4 is 1 hour.

10. The preparation method according to claim 4, characterized in that: In the step 1, during the preparation of the immune microsphere pad, the spray volume of the immune microsphere solution on the release pad is 5-7 μL / cm, and the immune microsphere pad obtained after spraying is dried at 38-42° C. for 4-16 hours; In the step 2, during the preparation of the immunonitrocellulose membrane, the concentration of the test line solution and the quality control line solution is 1.0-2.0 mg / ml, and the prepared immunonitrocellulose membrane is dried at 38-42° C. for 4-16 hours.

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