Biological sample diluent and application of detection method thereof in immunodetection kit

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to application of a biological sample diluent and a detection method thereof in an immunodetection kit. The biological sample diluent is prepared from 6.055 g / L of tris (hydroxymethyl) aminomethane, 35 g / L of sodium chloride, 0.6 mL / L of triton X-100, 0.6 g / L of a surfactant S-9, 1 g / L of bovine serum albumin and 0.2 mL / L of a liquid biological preservative. The surfactant S-9 and the triton X-100 in the biological sample diluent have a synergistic effect under a specific concentration, so that the foaming phenomenon is effectively improved, meanwhile, the biological sample diluent also has high elution and dissolution rate on target protein, and the accuracy of immunodetection is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to the application of a biological sample diluent and a detection method thereof in an immunoassay kit. Background Art

[0002] The IVD product is a total IgE and eosinophil cationic protein (ECP) combined detection kit (colloidal gold method), which uses the colloidal gold immunochromatography double antibody sandwich method to qualitatively detect total IgE and eosinophil cationic protein (ECP) in human nasal swabs. The product uses nasal swabs to collect human nasal secretions, elutes the collected components carried on the nasal swabs through sample diluent, dissolves them in the diluent, and conducts qualitative detection on the diluent.

[0003] In this product, the main uses of the sample diluent are: (1) to elute the sample collected from the nasal swab so that the sample is fully separated from the swab and dissolved in the diluent; (2) to provide a stable buffer environment to ensure the stability of the detection process, uniform chromatography, and accuracy of the detection; (3) to protect the target protein so that its structure and properties are stable during the elution and dissolution process.

[0004] The original diluent of the product is composed of 6 components: tris(hydroxymethyl)aminomethane (Tris Base), sodium chloride (NaCl), Triton X-100 (Triton X-100), surfactant S-9 (Tetronic1307), bovine serum albumin (BSA), and liquid biological preservative (ProClin 300). Among them, Tris Base and NaCl are buffer salt components, providing pH buffering effect; TritonX-100 and surfactant S-9 are surfactant components, providing the function of promoting elution and dissolution; BSA and ProClin 300 are protein protective agents, providing protein stabilization and protection. The above 6 components are dissolved in purified water in a specific ratio. The content of each component is shown in the formula of the original diluent in Table 1.

[0005] The original diluent is prone to foaming during use ( Figure 1-Figure 2 ), a lot of foam is generated during use, so when it is dripped into the sample well of the test card, there are bubbles and a long retention time, which seriously prevents the sample from entering the conjugate pad and extending upward, causing the risk of inaccurate sample addition and abnormal test results. Figure 3As shown in the foaming of the original diluent before and after shaking, 1mL of the existing diluent was added to the sampling tube, and the intelligent rotary vibration mixer (model MIX-S) was used for vibration mode F8, SPEED 50 for 30 seconds. After standing for 30 seconds, obvious foam appeared in the tube, and the foam thickness was about 14.5mm. This foaming situation will have a certain impact on the subsequent sample addition and detection process, making the sample addition amount and test results inaccurate, resulting in result deviation.

[0006] In view of the above problems, it is urgent to provide an optimized formula of the diluent to reduce foaming and reduce the risk of detection. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a biological sample diluent, which has less foaming and can effectively elute and dissolve the target protein, thereby improving the accuracy and sensitivity of detection.

[0008] On the one hand, the present invention provides a biological sample diluent, comprising the following components: 6.055 g / L of tris(hydroxymethyl)aminomethane, 35 g / L of sodium chloride, 0.6 mL / L of Triton X-100, 0.6 g / L of surfactant S-9, 1 g / L of bovine serum albumin and 0.2 mL / L of a biological preservative.

[0009] Specifically, the biological preservative can be selected from one or more of ProClin 300, benzoic acid, sorbic acid, methylparaben, ethylparaben, propylparaben, butylparaben, sodium dehydroacetate, sodium diacetate and nisin.

[0010] More specifically, the biological preservative can be selected from one of ProClin 300, benzoic acid, sorbic acid, methylparaben, ethylparaben, propylparaben, butylparaben, sodium dehydroacetate, sodium diacetate and nisin.

[0011] Preferably, the biological preservative may be ProClin 300.

[0012] Specifically, the biological sample includes, but is not limited to, nasal swab, oral swab, throat swab, cervical swab, male swab, blood, urine or feces.

[0013] Preferably, the biological sample may be a nasal swab, an oral swab, a throat swab, a cervical swab or a male swab.

[0014] Further preferably, the biological sample may be a nasal swab, an oral swab or a throat swab.

[0015] Still further preferably, the biological sample may be a nasal swab.

[0016] In yet another aspect, the present invention provides use of the aforementioned biological sample diluent in the preparation of an immunoassay kit.

[0017] Specifically, the immunoassay is to detect biological samples by using a colloidal gold immunochromatography double antibody sandwich method.

[0018] More specifically, the detection is the detection of immunoglobulin E and / or eosinophil cationic protein in a biological sample.

[0019] Preferably, the detection is the detection of immunoglobulin E and eosinophil cationic protein in a biological sample.

[0020] In yet another aspect, the present invention provides a kit, comprising the aforementioned biological sample diluent.

[0021] Specifically, the kit further includes, but is not limited to: one or more of an ELISA plate, a standard substance, an ELISA antibody, a washing solution or a stop solution.

[0022] In yet another aspect, the present invention provides a detection method, which comprises the aforementioned biological sample diluent or kit.

[0023] The technical effects achieved by the present invention are: (1) The technical solution provided by the present invention can effectively improve the foaming phenomenon.

[0024] (2) The technical solution provided by the present invention has no effect on the elution and solubility of the target protein.

[0025] (3) The technical solution provided by the present invention can meet the technical requirements of a dual-target combined detection kit.

[0026] (4) The technical solution provided by the present invention greatly improves the accuracy of the combined detection kit in the qualitative detection of allergic rhinitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1-Figure 2 The figures show the detection results of the original diluent that is easy to foam during the use of the IgE and ECP combined detection kits.

[0028] Figure 3 This is the foaming situation of the original dilution before and after shaking.

[0029] Figure 4 is the foam thickness of Comparative Example 1.

[0030] Figure 5 is the foam thickness of Example 1 and Comparative Example 2.

[0031] Figure 6It is the foam thickness of different component dilutions, where T represents "Triton X-100", S represents "Surfactant S-9", and B represents "BSA". DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0033] Example 1 This embodiment provides a new diluent formula. Compared with the original diluent formula, Triton X-100 (i.e., Triton X-100) and S-9 (i.e., surfactant S-9) are both adjusted to 60% (i.e., Triton X-100 is 0.6 mL / L, and S-9 is 0.6 g / L). The formula of Example 1 and the original diluent is shown in Table 1.

[0034] Table 1

[0035] Comparative Example 1 Single Component Adjustment The difference from the original dilution in Example 1 is that the single components of Triton X-100, surfactant S-9, and BSA are adjusted to 80%, 60%, 40%, 20%, and 0% of the corresponding components of the original dilution, respectively. The other components are the same as the original dilution in Example 1.

[0036] Comparative Example 2 Two-component adjustment Based on the original dilution in Example 1, the two surfactants Triton X-100 and S-9 were adjusted to three ratios of 80%, 60%, and 40% respectively (except for Example 1, i.e., a combination of 60% S-9 and 60% Triton X-100). The other components were the same as the original dilution in Example 1.

[0037] Effect Example 1 Take 1 mL of each adjusted dilution and place it in the product dilution tube. Use the intelligent rotary vibration mixer (model MIX-S) F8 vibration mode, SPEED 50 for 30 seconds. After standing for 30 seconds, use a vernier caliper to measure the foam thickness. The final result is the average of 2 repeated experiments.

[0038] (1) Comparative Example 1 The foam thickness of Comparative Example 1 is shown in Tables 2 and Figure 4 .

[0039] Table 2 Foam thickness of comparative example 1

[0040] Note: The values ​​in the table are the film thickness, in millimeters (mm); compared with the original dilution, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001.

[0041] like Figure 4 It can be seen that the foaming phenomenon was improved after the proportions of the three diluent components were adjusted separately. Among them, the foaming effect was improved most significantly after the amount of surfactant S-9 was reduced. Since the surfactant S-9 plays a role in promoting protein elution and dissolution in the diluent, the elution and dissolution ability of the target protein was tested for the adjusted formula.

[0042] (2) Example 1 and Comparative Example 2 The foam thickness of Example 1 and Comparative Example 2 is shown in Table 3: Table 3 Foam thickness of Example 1 and Comparative Example 2

[0043] Note: The values ​​in the table are the film thickness, in millimeters (mm); compared with the original dilution, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001.

[0044] like Figure 5 It can be seen that when the proportion of Triton X-100 and surfactant S-9 is 60%, the foaming effect of the dilution is significantly improved, and the foam thickness is the smallest (8.44mm) among all test combinations. The elution and dissolution ability of this formula dilution will be tested later.

[0045] Figure 6 This is an integrated diagram of the foam thickness adjustment of the single component adjustment of Example 1, Example 1, and the two-component adjustment of the foam thickness of Example 2. It can be clearly observed from the graphical results that when the two-component ratio is adjusted to 60% of the original dilution of Triton X-100 and surfactant S-9, the foam formation of the dilution is significantly improved, and the interference of bubbles on the IVD test results is successfully reduced.

[0046] As shown in Table 2, when Triton X-100 is 60%, the thickness of the original diluted foam is reduced by 1.8 mm; when the surfactant S-9 is 60%, the thickness of the original diluted foam is reduced by 2.92 mm; and when Triton X-100 and surfactant S-9 are both at 60% in Table 3, the thickness of the original diluted foam is reduced by 6.07 mm. This shows that when Triton X-100 and surfactant S-9 are both at 60%, they have a synergistic effect in reducing the thickness of the foam.

[0047] Effect Example 2 According to the results of Effect Example 1, the elution and solubility tests were performed on 20% S-9 and 0% S-9, which had good improvement effects on the foaming phenomenon in Comparative Example 1, Example 1 (i.e., 60% S-9 and 60% Triton X-100), 60% S-9 and 40% Triton X-100, 40% S-9 and 60% Triton X-100, and 40% S-9 and 40% Triton X-100 in Comparative Example 2.

[0048] The ECP protein ELISA quantitative detection kit was selected to test the elution and dissolution ability of different diluents. The information of the ECP protein ELISA quantitative detection kit is as follows: name: Human eosinophil cationic protein (ECP) enzyme-linked immunosorbent assay kit, manufacturer: Wuhan Huamei Bioengineering Co., Ltd., detection range: 1.56ng / mL-100ng / mL, sensitivity 0.39ng / mL; item number: CSB-E11729h, specification 96T / 48T.

[0049] Standard solution: Dissolve the ECP protein to a concentration of 1 μg / mL, take 20 μL and add directly to 1 mL of the original diluent as a reference standard solution. The theoretical concentration of the standard solution is 20 ng / mL.

[0050] The operation process for the remaining groups was as follows: 20 μL of 1 μg / mL ECP solution was aspirated by a pipette and added to the nasal swab head. After the liquid was fully absorbed, the swab head was immersed in each group of diluents (1 mL) and mixed thoroughly.

[0051] The concentration points of the standard curve are: 1.56ng / mL, 3.12ng / mL, 6.256ng / mL, 12.5ng / mL, 25ng / mL, 50ng / mL, and 100ng / mL.

[0052] (1) Comparative Example 1 The experimental groups of Comparative Example 1 were: original dilution group, 20% S-9 group, and 0% S-9 group. Five replicate samples were set for each group.

[0053] The curve fitting equation is:

[0054] Where: A=4.40332; B= -1.29288; C=122.06866; D=0.08976; R 2 =0.99961.

[0055] The experimental results are shown in Table 4: Table 4

[0056] Note: Recovery rate = mean concentration of experimental group / mean concentration of standard solution × 100%.

[0057] As shown in Table 4, after reducing the proportion of surfactant S-9, the elution and dissolution efficiency of the target protein decreased to a certain extent. The recovery rate of the original diluent was 98.5%, the recovery rate of 20% S-9 was 87.6%, and the recovery rate of 0% S-9 was 84.7%. Reducing the proportion of surfactant S-9 alone can improve the foaming phenomenon of the diluent to a certain extent, but its elution ability for the target protein is weakened to a certain extent. Therefore, adjusting a single diluent component cannot take into account both improving the foaming problem and not affecting the elution ability.

[0058] (2) Example 1 and Comparative Example 2 The experimental groups of Example 1 and Comparative Example 2 were: Example 1 (i.e., 60% S-9 and 60% Triton X-100), 60% S-9 and 40% Triton X-100, 40% S-9 and 60% Triton X-100, and 40% S-9 and 40% Triton X-100. Five replicate samples were set for each group.

[0059] The curve fitting equation is:

[0060] Where: A=4.22206; B= -1.25372; C=125.03421; D=0.07735; R 2 =0.99947.

[0061] The experimental results are shown in Table 5: Table 5

[0062] Note: Recovery rate = mean concentration of experimental group / mean concentration of standard solution × 100%.

[0063] The experimental results show that the formula diluent of Example 1 (Triton X-100 and S-9 are both 60%) has no effect on the elution and solubility of the target protein, and the recovery rate is 98.2%, which is no different from the recovery rate of the original diluent of 98.5%.

[0064] Effect Example 3 The IgE protein ELISA quantitative detection kit was used to test the elution and dissolution ability of the selected formula diluent (Triton X-100 and S-9 were both 60%). The human immunoglobulin E (IgE) enzyme-linked immunosorbent assay kit was purchased from Beijing Bioson Biotechnology Co., Ltd., with a detection range of 468.75-30000 pg / mL, item number: BSK1158, and specification 96T / box.

[0065] Standard solution: Dissolve IgE protein to a concentration of 500 ng / mL, draw 20 μL, and add directly to 1 mL of the original diluent as a reference standard solution. The theoretical concentration of the standard solution is 10 ng / mL.

[0066] The operation process for the remaining groups is as follows: 20 μL of 500 ng / mL IgE solution is aspirated through a pipette and added to the nasal swab head. After the liquid is fully absorbed, the swab head is immersed in the diluent of each group (1 mL) and mixed thoroughly.

[0067] The concentration points of the standard curve are: 0.02 ng / mL, 0.05 ng / mL, 0.10 ng / mL, 0.39 ng / mL, 1.56ngU / mL, 6.25 ng / mL, and 12.5 ng / mL.

[0068] The experimental groups were divided into: original dilution group, Triton X-100 and S-9 60% group, and 5 replicate samples were set in each group.

[0069] The curve fitting equation is:

[0070] Where: A=4.34256; B= -1.31900; C=5.00045; D=0.05949; R 2 =0.99970.

[0071] The experimental results are shown in Table 6: Table 6

[0072] Note: Recovery rate = mean concentration of experimental group / mean concentration of standard solution × 100%.

[0073] The experimental results show that the formulated diluent (Triton X-100 and S-9 are both 60%) has no significant effect on the elution and solubility of the target protein, and the recovery rate is 98.9%, which is not significantly different from the recovery rate of the original diluent of 99.4%.

[0074] In summary, the formula of Example 1 significantly improves the foaming phenomenon of the diluent, and the formula has no effect on the elution and solubility of the target proteins ECP and IgE. Therefore, the formula is a diluent formula with better performance effect.

[0075] Effect Example 4 Since the IgE content in nasal secretions is low, a slight change in its concentration may significantly affect the test results of the test kit. In order to simultaneously meet the combined detection of total IgE and ECP of this IVD product, the sensitivity of the colloidal gold test strip, false positive, chromatography return time, and gold-labeled antibody release and chromatography were tested. Four formulas were obtained by changing the diluent composition: Among them, the formula of Triton X-100 (i.e., Triton X-100) and S-9 (i.e., surfactant S-9) is shown in Table 7, and the other components are the same as the original diluent formula.

[0076] Table 7

[0077] Sensitivity and false positive test method: Use different sample diluents to add negative nasal swab samples to mix as reference sample diluents, then use different reference sample diluents to dilute the total IgE reference sample to a final concentration of 1.0 IU / mL, 0.5 IU / mL, 0.25 IU / mL, 0.125 IU / mL, 0.0625 IU / mL, 0 IU / mL (no reference sample added to the diluent). Four groups of samples were tested using colloidal gold test strips to obtain the sensitivity under four formulas. Observe whether the 0 IU / mL sample produces a false positive.

[0078] Chromatography whitening time test method: Observe the time required for the chromatographic background to whiten while testing sensitivity and false positives.

[0079] Release and chromatography of gold-labeled antibodies: During the chromatography process, observe whether the gold-labeled antibodies are evenly chromatographed upward after release. After the chromatography background turns white, remove the test strip test card and observe whether the gold-labeled antibodies on the gold-labeled pad are completely released.

[0080] After preparing the above four different formulations of the diluents to be tested, dilute the 800IU / mL reference substance into 10IU / mL using formulation 1, formulation 2, formulation 3, and formulation 4, respectively, and then dilute 10IU / mL into 1IU / mL, 0.5IU / mL, 0.25IU / mL, 0.125IU / mL, and 0IU / mL (without adding the reference substance to the diluent). Use the 20240501 batch of products for testing. The sample volume for each well is 60uL. Start timing after adding the sample, and make the following condition records respectively. Each condition is tested three times.

[0081] Observe the chromatography of the test paper at 5, 8, 10, 13, 15, 20, 25 and 30 minutes respectively.

[0082] Record the time required for the background plate to turn white after adding each formula.

[0083] The sample detection results of each formula were recorded 15-20 minutes after adding the sample, "+" indicates positive and "-" indicates negative.

[0084] After running the plate for 30 minutes, remove the reagent card cover and observe the release of the gold label pad.

[0085] The results are shown in Table 8-Table 12: Table 8 Results of Formulation 1

[0086] Table 9 Results of Formulation 2

[0087] Table 10 Results of Formulation 3

[0088] Table 11 Results of Formulation 4

[0089] Table 12 Summary of test results

[0090] The sensitivity and false positive of colloidal gold test strips directly affect the detection and accuracy of colloidal gold reagents. The chromatographic background return time affects the time to observe the results in colloidal gold detection. If the chromatographic return time is too long, the time to observe the results will be postponed accordingly, which is inconsistent with the purpose of rapid detection. Incomplete release of gold-labeled antibodies and uneven chromatography will affect the uneven color of colloidal gold detection strips and poor detection repeatability. According to the above experiments, Formula 1: The dilution of 60% S9 and 60% Triton x100 components has achieved unexpected technical results and achieved the technical requirements of the dual-target combined detection kit.

[0091] Effect Example 5 Accuracy The samples were obtained from the clinical trial project of this IVD product approved by the Ethics Committee of Beijing Century Altar Hospital, and the sample collection process complies with the relevant provisions of the "Ethical Review Measures for Biomedical Research of the People's Republic of China". The samples used for testing were taken from 10 patients who were enrolled in the group. The samples were tested immediately after collection. The sampling swabs should be avoided from being contaminated during the sampling process, and the samples should be tested immediately after sampling. If the collected samples are not tested immediately, they can be stored at 2~8℃ for 48 hours or frozen at -20℃ for no more than 72 hours. The frozen samples should be fully thawed and restored to room temperature before testing, and shaken well.

[0092] The 22 specific IgE antibody detection kits involved are from Jiangsu Haooubo Biotechnology Co., Ltd. and Hunan Haooubo Biotechnology Co., Ltd., including: house dust mites, dust mites, artemisia, ragweed, dog epithelium, cat epithelium, Alternaria, eggs, milk, peanuts, soybeans, cod, shrimp, crab, wheat flour, willow, cockroaches, Aspergillus fumigatus (M3), Humulus (W22), cypress (T23), common birch (T3), sycamore (T11), and allergens. Suxie registration number 20202401671, 20202401682, 20202401668, 20202401680, 20202401669, 20202401705, 20202401711, 20202401689, 20202401715, 20202401716, 20202401670, 20202401681, 20202401692, 20202401700, 20202401691, 20202401704, 20202401688, Hunan Medical Equipment Registration No. 20212401291, 20212401294, 20212401290, 20212401258, 20212401293.

[0093] The test method of the IVD product of the present invention is as follows: Nasal secretion sampling method: 1. Use a tissue to blow your nose clean.

[0094] 2. Sampling: (1) Carefully open the outer packaging of the swab and avoid touching the swab head with your hands.

[0095] (2) Tilt your head slightly, hold the tail of the swab in your hand (do not touch the swab head with your hand), and insert the swab head into the nasal cavity 1-1.5 cm.

[0096] (3) Keep the nose close to the nasal cavity and rotate at least 4 times (staying for at least 15 seconds).

[0097] (4) Then repeat the above steps for the other nasal cavity using the same swab.

[0098] (5) Take another swab and repeat the previous sampling operation, finally obtaining two nasal swabs.

[0099] Testing steps: (a) Unscrew the caps of the total IgE diluent and ECP diluent tubes; (b) Immediately place one of the nasal swabs in total IgE diluent and the other in ECP diluent. The swab tip should be swirled in the liquid for at least 30 seconds. (c) Use your hands to squeeze the swab head through the outer wall of the tube for at least 5 times to ensure that the liquid is fully released and eluted from the swab; (d) Break the swab at the break point (in the middle) and leave the swab tip in the diluent; (e) Tighten the caps of the total IgE diluent and ECP diluent tubes; (f) Take out the test card, open the aluminum foil bag along the tear edge, take out the test card and lay it flat; (g) Unscrew the small caps on the tubes of total IgE diluent and ECP diluent, invert them vertically, gently pinch the tubes, and drip 3 drops of each liquid into the corresponding sample wells of the test card; (h) Allow the test card to stand for 15 minutes and observe the displayed results.

[0100] IgE and ECP double positive can be diagnosed as allergic rhinitis. The results are shown in Table 13-1 and Table 13-2.

[0101] Table 13-1

[0102] Table 13-2

[0103] According to the results of Effect Examples 4 and 5, the foaming phenomenon generated by the diluent during the detection process will significantly affect the detection amount, sensitivity, whitening time and chromatographic uniformity of the IVD product sample, thereby adversely affecting the accuracy of the final test results. The diluent containing 60% S9 and 60% Triton X-100 described in Effect Example 5 has almost no bubble generation during the dripping process of IVD products, and its test results are highly consistent with the 22 allergen test data provided by the hospital and the clinical diagnosis results obtained by the doctor through physical examination, and the compliance rate is significantly improved, which greatly improves the accuracy of the combined detection kit in the qualitative detection of allergic rhinitis.

Claims

1. A biological sample diluent, characterized in that: The invention comprises the following components: 6.055 g / L of tris(hydroxymethyl)aminomethane, 35 g / L of sodium chloride, 0.6 mL / L of Triton X-100, 0.6 g / L of surfactant S-9, 1 g / L of bovine serum albumin and 0.2 mL / L of biological preservative.

2. The biological sample diluent according to claim 1, characterized in that: The biological preservative is one or more of ProClin300, benzoic acid, sorbic acid, methylparaben, ethylparaben, propylparaben, butylparaben, sodium dehydroacetate, sodium diacetate and nisin.

3. The biological sample diluent according to claim 1 or 2, characterized in that: The biological preservative is ProClin300.

4. The biological sample diluent according to claim 3, characterized in that: The biological sample is a nasal swab, an oral swab, a throat swab, a cervical swab, a male swab, blood, urine or feces.

5. The biological sample diluent according to claim 4, characterized in that: The biological sample is a nasal swab.

6. Use of the biological sample diluent according to any one of claims 1 to 5 in the preparation of an immunoassay kit.

7. The use according to claim 6, characterized in that: The immunoassay is to detect biological samples by using a colloidal gold immunochromatography double antibody sandwich method.

8. The use according to claim 7, characterized in that: The detection is the detection of immunoglobulin E and / or eosinophil cationic protein in the biological sample.

9. A kit, characterized in that: The kit comprises the biological sample diluent according to any one of claims 1 to 5.

10. A detection method, characterized in that: The detection method comprises using the biological sample diluent according to any one of claims 1 to 5 or the kit according to claim 9.

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