A sample diluent for indirect immunoassay

Abstract

The invention discloses a sample diluent for indirect immunoassay, and relates to the technical field of immunoassay. The sample diluent of the invention comprises a zwitterionic surfactant, serum and structural analogs of human IgG / IgM. The weight ratio of the zwitterionic surfactant is 1-10 g / kg, the weight ratio of the serum is 100-500 g / kg, and the weight ratio of the immunoglobulin is 1-10 mg / kg. By adding a specific amount of zwitterionic surfactant and structural analogs of human IgG / IgM, an inhibitory and competitive binding effect is formed in the entire reaction system, and the nonspecific adsorption of IgG / IgM in the sample to the solid phase carrier is reduced, thereby reducing the background and reducing false positives.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay, and in particular to a sample diluent used in indirect immunoassay. Background Art

[0002] Indirect immunoassay is to couple specific recombinant or natural antigens to solid phase carriers (common solid phase carriers include magnetic particles, 96-well plates, etc.). The antigen can recognize and bind to the project-specific IgG / IgM in human serum / plasma samples. In addition, anti-human antibodies labeled with tracers (such as alkaline phosphatase, acridinium ester) can react with human IgG / IgM and combine with the above-mentioned immune reactants to form immune reaction complexes. After adding the substrate, a light signal can be generated. The signal value reflects the concentration level of the analyte in the sample. Indirect immunoassay is mainly used for the detection of infectious disease antibody projects, such as anti-hepatitis C antibodies, anti-syphilis antibodies, etc.

[0003] The characteristic of the indirect method is that the anti-human antibody (anti-human IgG / IgM) that marks the tracer cannot specifically recognize the antibody of a certain disease, but recognizes all human IgG / IgM. This will result in that when the sample is tested, human IgG / IgM with extremely high concentration in the sample and non-specific adsorption to the surface of the solid phase carrier will also generate a signal value, resulting in an increase in background and a decrease in sensitivity. In severe cases, it will affect the misjudgment of positive and negative, resulting in a large number of false positive results.

[0004] To solve this problem, the conventional practice is to pre-dilute the sample to reduce the IgG / IgM concentration therein and thereby reduce nonspecific adsorption.

[0005] Commonly used sample diluents consist of a buffer system and protein and can only play a dilution role.

[0006] Chinese patent CN201611095552.0 discloses a spot gold percolation kit for detecting urine microalbumin and its application, including: sample diluent, washing solution, colloidal gold labeled human serum albumin antibody, urine microalbumin chromatograph and calibration card. The sample diluent includes preservative Triton-100, phosphate buffer solution and Tris buffer solvent. The sample diluent of the invention includes preservatives and buffer system, and cannot inhibit nonspecific adsorption in the sample.

[0007] Chinese patent CN202210416061.0 discloses a universal sample diluent for immunochromatographic detection and a preparation method thereof. The universal sample diluent for immunochromatographic detection includes the following components in mass / volume ratio: 6%-12% casein, 2%-20% surfactant, 1%-20% preservative, 5%-20% trehalose, 0.5%-1% sodium chloride, 1%-10% blocking agent, and the sample diluent also includes 0.04mol / L-0.06mol / L HEPES buffer solution. The sample diluent of this invention can be used for different detection items, has the advantages of strong versatility and high detection sensitivity, and the preparation method is simple and convenient, and can be used for large-scale industrial production. Although casein can competitively bind to the surface of a solid phase carrier to a certain extent, its structure is quite different from that of human IgG / IgM and its competitiveness is weak.

[0008] Therefore, in the indirect immunoassay process, a sample diluent is urgently needed that can significantly reduce or eliminate nonspecific adsorption to the solid / liquid surface, further achieving the effect of reducing background, reducing false positives, and improving sensitivity. Summary of the invention

[0009] The purpose of the present invention is to provide a sample diluent for indirect immunoassay. The sample diluent of the present invention, on the basis of sample dilution, further reduces the nonspecific adsorption of IgG / IgM in the sample to the solid phase carrier by adding a specific amount of zwitterionic surfactant and structural analogs of human IgG / IgM, thereby reducing the background and false positives.

[0010] To achieve the above-mentioned purpose of the invention, the technical solution of the present invention is as follows:

[0011] In one aspect, the present invention provides a sample diluent, comprising a zwitterionic surfactant, serum and a structural analogue of human IgG / IgM.

[0012] Specifically, the zwitterionic surfactant is betaine or its analogs.

[0013] More specifically, the components of the betaine or its analogs include cardiac glycosides and other steroid components.

[0014] More specifically, the betaine or its analogue is selected from one or more of betaine, lauryl betaine, tetradecyl dimethyl betaine, octadecyl dimethyl betaine, dodecyl dihydroxyethyl betaine, and octadecyl dihydroxyethyl betaine.

[0015] More specifically, the betaine has a chemical name of 1-carboxy-N,N,N-trimethylaminoethyl lactone.

[0016] More specifically, the lauryl betaine has a chemical name of N-carboxymethyl-N,N-dimethyl-dodecyl ammonium inner salt.

[0017] More specifically, the chemical name of the tetradecyl dimethyl betaine is N-carboxymethyl-N,N-dimethyl-1-tetradecyl ammonium inner salt.

[0018] Specifically, the molecular formula of the octadecyl dimethyl betaine is C 18 H 37 N(CH3)2CH2COO.

[0019] More specifically, the molecular formula of the dodecyl dihydroxyethyl betaine is C 18 H 37 NO4-C 20 H 41 NO4.

[0020] Specifically, the molecular formula of the octadecyl dihydroxyethyl betaine is C 25 H 51 NO4.

[0021] In some embodiments, the betaine or its analogue is purchased from commercial sources; in other embodiments, the betaine or its analogue can be synthesized according to the structure. It should be noted that for those skilled in the art, betaine or its analogue is a substance with a known structure, and the method of obtaining it can be the current conventional method in the art or the possible method of obtaining it in the future, and the method of obtaining it should not be used as a limiting limitation of the present invention, that is, no matter how the betaine or its analogue is obtained, it should be within the protection scope of the present invention.

[0022] Specifically, the weight ratio of betaine or its analogue is 1-10 g / kg; preferably, the weight ratio of betaine or its analogue is 1-5 g / kg; further preferably, the weight ratio of betaine or its analogue is 1-2 g / kg; further preferably, the weight ratio of betaine or its analogue is 2 g / kg.

[0023] In some embodiments, the weight ratio of betaine or its analogue is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and any non-integer therebetween.

[0024] More specifically, the betaine or its analogue is lauryl betaine.

[0025] In some specific embodiments, the weight ratio of lauryl betaine is 2 g / kg.

[0026] Specifically, the serum is selected from at least one of fetal bovine serum, newborn calf serum, goat serum, rabbit serum, chicken serum, pig serum, and donor horse serum.

[0027] More specifically, the serum refers to a pure natural culture medium containing a variety of nutrients required for cell growth and reproduction, such as proteins, polypeptides, etc.

[0028] More specifically, fetal bovine serum is the serum of fetal bovine obtained by sampling blood through cardiac puncture when slaughtering pregnant cows.

[0029] More specifically, the newborn calf serum refers to venous blood collected from newborn calves 12-24 hours after birth.

[0030] More specifically, the goat serum, rabbit serum, chicken serum, pig serum and donor horse serum are derived from non-immune goat, rabbit, chicken, pig and donor horse hosts, respectively, and are processed by collection, separation and microporous filtration.

[0031] Specifically, the weight ratio of the serum is 100-500 g / kg; preferably, the weight ratio of the serum is 150-400 g / kg; further preferably, the weight ratio of the serum is 200-300 g / kg; most preferably, the weight ratio of the serum is 200 g / kg.

[0032] More specifically, the serum is goat serum.

[0033] Specifically, the structural analogue of human IgG / IgM refers to an immunoglobulin that exists in a monomeric form and has a shape similar to a "Y" structure.

[0034] More specifically, the immunoglobulin is selected from at least one of IgG, IgA, IgM, IgD, and IgE.

[0035] More specifically, the weight ratio of the immunoglobulin is 1-10 mg / kg; preferably, the weight ratio of the immunoglobulin is 2-8 mg / kg; further preferably, the weight ratio of the immunoglobulin is 4-6 mg / kg; most preferably, the weight ratio of the immunoglobulin is 5 mg / kg.

[0036] More specifically, immunoglobulin refers to a protein present in plasma and having antibody activity.

[0037] More specifically, the IgG, IgA, IgM, IgD, and IgE represent immunoglobulin G, immunoglobulin A, immunoglobulin M, immunoglobulin D, and immunoglobulin E, respectively.

[0038] More specifically, the immunoglobulin is IgG.

[0039] Preferably, the immunoglobulin is selected from at least one of bovine IgG and mouse IgG.

[0040] More preferably, the immunoglobulin is bovine IgG.

[0041] Preferably, the sample diluent further comprises at least one of a buffer, sodium chloride (NaCl), protein, a preservative, and a non-ionic surfactant.

[0042] Further preferably, the buffer is selected from at least one of tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazineethanesulfonic acid, acetic acid-sodium acetate, 3-(N-morpholinyl)-2-hydroxypropanesulfonic acid, and 2-morpholinoethanesulfonic acid.

[0043] Still further preferably, the buffer is Tris.

[0044] Preferably, the weight ratio of the buffer is 1-10 g / kg; further preferably, the weight ratio of the buffer is 2-6 g / kg; further preferably, the weight ratio of the buffer is 3-5 g / kg; most preferably, the weight ratio of the buffer is 3 g / kg.

[0045] Preferably, the weight ratio of NaCl is 1-10 g / kg; further preferably, the weight ratio of sodium chloride is 5-10 g / kg.

[0046] Preferably, the protein is selected from at least one of casein, BSA, enzymatically hydrolyzed gelatin and gelatin.

[0047] Specifically, the casein is a phosphorus-calcium binding protein, and the CAS number of casein is 9000-71-9.

[0048] Specifically, the BSA is a globulin in bovine serum, with a molecular weight of 66.446 kDa and an isoelectric point of 4.7.

[0049] Specifically, the enzymatically hydrolyzed gelatin is a product of collagen hydrolyzed by enzymes.

[0050] Specifically, the gelatin is a product of partial hydrolysis of collagen, and the CAS number of the gelatin is 9000-70-8.

[0051] More preferably, the protein is casein.

[0052] Preferably, the weight ratio of casein is 1-10 g / kg;

[0053] Further preferably, the weight ratio of casein is 1-5 g / kg;

[0054] Most preferably, the weight ratio of casein is 5 g / kg.

[0055] Preferably, the preservative is selected from at least one of Proclin 300, Bronidox, sodium azide and methylisothiazolinone (MIT).

[0056] Specifically, the CAS of Bronidox is 30007-47-7.

[0057] Specifically, the molecular formula of the sodium azide is NaN3, and the CAS number is 26628-22-8.

[0058] Specifically, the chemical name of the methylisothiazolinone is 2-methyl-4-isothiazolin-3-one, the molecular formula is C4H5NOS, and the CAS number is 2682-20-4.

[0059] Further preferably, the preservative is Proclin300.

[0060] More preferably, the weight ratio of the preservative is 1-10 g / kg; more preferably, the weight ratio of the preservative is 1-5 g / kg; most preferably, the weight ratio of the preservative is 1 g / kg.

[0061] Preferably, the nonionic surfactant is selected from at least one of Tween-20, poloxamer, Tween-40 and polyethylene glycol.

[0062] Specifically, the chemical name of the Tween-20 is polyoxyethylene sorbitan monolaurate, and the molecular formula is C 58 H 114 O 26 , molecular weight is 1227.5, CAS number is 9005-64-5.

[0063] Specifically, the chemical name of the poloxamer is α-hydrogen-ω-hydroxy polyoxyethylene polyoxypropylene polyoxyethylene block copolymer, the molecular formula is HO·(C2H4O)m·(C3H6O)n·H, the molecular weight is 102.13, and the CAS number is 9003-11-6.

[0064] Specifically, the chemical name of Tween-40 is polyoxyethylene sorbitan monopalmitate, and its molecular formula is C 44 H 54 N7O8P, molecular weight is 839.915, CAS number is 9005-66-7.

[0065] Specifically, the molecular formula of the polyethylene glycol is HO(CH2CH2O)nH, and the CAS number is 25322-68-3.

[0066] Further preferably, the nonionic surfactant is Tween-20;

[0067] More preferably, the weight ratio of the nonionic surfactant is 1-10 g / kg; more preferably, the weight ratio of the nonionic surfactant is 1-5 g / kg; more preferably, the weight ratio of the nonionic surfactant is 1-3 g / kg; most preferably, the weight ratio of the nonionic surfactant is 1 g / kg.

[0068] Preferably, the present invention provides a method for preparing a sample diluent, comprising the following steps:

[0069] (1) Add buffer, NaCl and water, mix well, dissolve, and adjust the pH to 7.5-8;

[0070] (2) Add protein, serum, immunoglobulin, betaine, preservative and surfactant, fully dissolve, adjust pH to 7.5-8, and add water to the total mass of the buffer solution.

[0071] Preferably, when the buffer in step (1) is Tris, the pH value is adjusted with 6 mol / L HCl.

[0072] Preferably, the pH in step (1) and step (2) is 7.5-7.8.

[0073] In another aspect, the present invention provides use of the sample diluent in indirect immunoassay.

[0074] The beneficial effects of the present invention are:

[0075] (1) Lauryl betaine is a zwitterionic surfactant that has a significant effect on reducing or eliminating nonspecific adsorption on solid / liquid surfaces.

[0076] (2) Goat serum and bovine IgG provide proteins that compete with human IgG / IgM for binding to the surface of the solid phase carrier. Lauryl betaine, goat serum, and bovine IgG are added to the sample diluent in a certain ratio to form an inhibitory and competitive binding effect in the entire reaction system, thereby reducing background, false positives, and improving sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 It is a bar graph of the ratios of positive samples to negative samples of sample dilutions 1-6 in Example 1 of the present invention. DETAILED DESCRIPTION

[0078] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of protection claimed in this application, and those skilled in the art can make various changes and modifications to the invention of this application based on the disclosed content, and they should also fall within the scope of protection claimed in this application.

[0079] The present invention is further described below by way of specific examples. Unless otherwise specified, the various biological reagents used in the examples of the present invention are obtained through conventional commercial channels.

[0080] The manufacturer and product number of the raw material components:

[0081] MS160 / Streptavidin was purchased from JSR, catalog number 1811R-01;

[0082] TPO antigen was purchased from Diarect, catalog number 12101;

[0083] Anti-human IgG was purchased from Abcam, catalog number ab98616;

[0084] Acridinium ester was purchased from Helison Company with the catalog number HS-11015005;

[0085] Tris was purchased from Sigma, catalog number 252859-500G;

[0086] Casein sodium salt was purchased from Sigma, catalog number C8654;

[0087] Goat serum was purchased from Sijiqing Company, catalog number 50012-8615;

[0088] Bovine IgG was purchased from Sigma, catalog number I5506;

[0089] Betaine was purchased from Aladdin Company with the product number L134724;

[0090] Proclin300 was purchased from Sigma, catalog number 48914-U;

[0091] Tween-20 was purchased from Bioscientific with the catalog number A600560-0500.

[0092] Example 1

[0093] Reagent preparation:

[0094] (1) Magnetic particle suspension

[0095] MS160 / Streptavidin and biotin-labeled TPO antigen were mixed according to 1 mg magnetic particles and 1 μg biotin-labeled TPO antigen, and the mixture was shaken at room temperature for 1 hour. The mixture was washed 2-3 times with magnetic particle buffer and the volume was fixed to 0.25 mg / mL for later use.

[0096] (2) AE component reagents

[0097] Anti-human IgG and acridinium ester were labeled, and the anti-human IgG and acridinium ester labels were diluted with AE labeling buffer to a concentration of 0.1 μg / mL for later use.

[0098] The method for preparing the AE marker buffer comprises the following steps:

[0099] a) Measure the amount of various raw materials according to the prepared volume of reaction buffer.

[0100] b) Add 80% of the required amount of purified water to the container, then add Tris to adjust the pH to 7.2±0.1; then add BSA, Tween-20 and Proclin300, and stir for more than 30 minutes until there are no solid particles.

[0101] c) Add purified water to make up the total volume of the buffer.

[0102] d) Filter with a 0.45 μm filter membrane; label the product (name, batch number, preparer, preparation date, expiration date, preparation quantity), and store at 2-8°C until use.

[0103] The amounts of each raw material are as follows:

[0104]

[0105]

[0106] (3) Preparation of sample diluent

[0107] The components of the sample diluent 1 and the conventional sample diluent 2 of the present application are as described in Table 1 below:

[0108] Table 1

[0109]

[0110] Sample diluent 3

[0111] The difference from sample diluent 1 is that goat serum is not added, and 200 g of purified water equal to the goat serum is added, and the rest is the same.

[0112] Sample diluent 4

[0113] The difference from sample diluent 1 is that no bovine IgG is added, and purified water equal to 5 mg of bovine IgG is added, and the rest is the same.

[0114] Sample diluent 5

[0115] The difference from the sample dilution 1 is that no lauryl betaine is added, and 2 g of purified water equal to the amount of lauryl betaine is added, and the rest is the same.

[0116] Sample diluent 6

[0117] The difference from sample diluent 1 is that the added amount of goat serum is 50 g, the added amount of bovine IgG is 0.5 mg, and the added amount of lauryl betaine is 0.5 g, and the rest are the same.

[0118] (4) Sample dilution

[0119] Use the sample diluent prepared above to dilute the negative sample, weak, medium, and strong positive samples 50 times, respectively. The sample volume is 10 μl and the sample diluent is 490 μL.

[0120] Detection of luminescent signal values ​​of different sample dilutions

[0121] Testing steps:

[0122] Add 10 μL of sample and 50 μL of sample diluent to 50 μL of magnetic particle suspension, place at 37°C for 15 minutes, and then wash 4 times with cleaning solution. Then add 100 μL of AE component reagent, react at 37°C for 7 minutes, and then wash 4 times with cleaning solution. Then add 100 μL of pre-excitation solution (mainly hydrogen peroxide) and 100 μL of excitation solution (mainly sodium hydroxide), and measure the luminescence signal value. The results are shown in Table 2 below.

[0123] Table 2

[0124]

[0125] As can be seen from Table 2, the background signal values ​​of sample diluents 2, 3, 4, 5, and 6 are all higher than the background value of sample diluent 1, while the detection signal values ​​of weak, medium, and strong positive samples have no obvious changes; taking sample diluent 1 as the control, the background signal value of the sample diluent 2 with the absence of goat serum, bovine IgG, and lauryl betaine is the highest, and after the absence of single components such as goat serum (sample diluent 3), bovine IgG (sample diluent 4), and lauryl betaine (sample diluent 5), the background value also increases, but the increase is not as large as that of sample diluent 2. When all three are added, but the addition amount is reduced (sample diluent 6), the background signal value also increases.

[0126] P / N values ​​of different sample dilutions

[0127] The P / N values ​​of sample dilutions 1-6 are shown in Table 3.

[0128] Table 3

[0129] Weak positive / negative Medium Yang / Negative Strong positive / negative Sample diluent 1 8.50 33.76 82.15 Sample diluent 2 4.28 17.01 40.70 Sample diluent 3 5.65 23.28 54.62 Sample diluent 4 5.74 21.74 53.71 Sample diluent 5 5.31 22.82 55.57 Sample diluent 6 6.57 25.87 63.80

[0130] As can be seen from Table 3, with sample diluent 1 as the control, the P / N of the sample diluent 2 lacking goat serum, bovine IgG, and lauryl betaine is the lowest. When a single component such as goat serum (sample diluent 3), bovine IgG (sample diluent 4), and lauryl betaine (sample diluent 5) is missing, the P / N is lower than that of sample diluent 1, but higher than that of sample diluent 2. When all three are added but the added amount is reduced (sample diluent 6), the P / N is larger, but lower than that of sample diluent 1.

[0131] The above shows that when the sample diluent described in the present invention is used for testing, the background value is lower, the sensitivity is significantly improved, and the probability of false positive samples can be reduced.

[0132] The bar graph of P / N values ​​is shown in Figure 1 .

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A sample diluent, characterized in that: The sample diluent includes a zwitterionic surfactant, serum and a structural analog of human IgG / IgM; The zwitterionic surfactant includes betaine or its derivatives; The structural analogue of human IgG / IgM is immunoglobulin; The weight ratio of betaine or its derivative is 2g / kg; The weight ratio of the immunoglobulin is 5 mg / kg; The weight ratio of the serum is 200 g / kg; The betaine or its derivative is lauryl betaine; The serum is goat serum; The immunoglobulin is IgG.

2. The sample diluent according to claim 1, characterized in that: The immunoglobulin is bovine IgG.

3. The sample diluent according to any one of claims 1 to 2, characterized in that: The sample diluent further comprises at least one of a buffer, sodium chloride, protein, a preservative, and a non-ionic surfactant.

4. Use of the sample diluent according to any one of claims 1 to 3 in indirect immunoassay.

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