WO2018000901A1

WO2018000901A1 - Respiratory syncytial virus chemiluminescence immunoassay kit and preparation method thereof

Info

Publication number: WO2018000901A1
Application number: PCT/CN2017/080405
Authority: WO
Inventors: 何林
Original assignee: 深圳市亚辉龙生物科技股份有限公司
Priority date: 2016-06-30
Filing date: 2017-04-13
Publication date: 2018-01-04
Also published as: CN105891193A

Abstract

Provided are a respiratory syncytial virus chemiluminescence immunoassay kit and a preparation method thereof. The respiratory syncytial virus chemiluminescence immunoassay kit comprises respiratory syncytial virus recombinant protein-coated carboxylated magnetic particles and anti-human immunoglobulin-labeled chemiluminescent labels. The respiratory syncytial virus chemiluminescence immunoassay kit can take a fully automated chemiluminescence immunoassay as a detection tool to complete the detection of respiratory syncytial virus. Through experiments, the detection sensitivity thereof reaches 1U/L, is increased by at least 10 times relative to a traditional detection method of the respiratory syncytial virus, with higher detection accuracy.

Description

Inventive name: Respiratory syncytial virus chemiluminescence immunoassay kit and preparation method thereof

[0001] The present invention relates to the field of in vitro detection, and in particular to a respiratory syncytial virus chemiluminescence immunoassay kit and a preparation method thereof.

Background technique

[0002] Respiratory syncytial virus (RSV) is a first time in 1956 that was isolated from orangutans with symptoms of flu. It is the most common pathogen causing viral pneumonia in children, because it can form special cell fusion lesions in cell culture, hence the name. The infection of the virus can cause interstitial pneumonia and bronchiolitis.

technical problem

[0003] Currently, there are several methods for detecting respiratory syncytial virus infection:

[0004] First, enzyme-linked immunosorbent assay

[0005] Enzyme-linked immunosorbent assay (ELISA) is widely used, but this method also has the following shortcomings: [0006] (1) using 12x8 type, 6x8 type, 8x12 type or whole plate type 96-hole special The microplate is used as an antigen coating device and a reaction container. It can only be divided into 12 batches, 6 batches, 8 batches or whole plates in use, and it is impossible to perform independent, single-person detection;

[0007] (2) There are many kinds of reagents used for quantitative determination, and each test reagent should be filled with a reagent bottle, and each time a reagent is used, the liquid suction nozzle needs to be replaced to be separately added to the microplate. In the micropores, not only the types of reagent bottles are numerous, but also the operation of adding reagents is extremely cumbersome;

[0008] (3) The corresponding labeling of the detection information is lacking, and the production batch number and expiration date information of the detection reagent can only be known or known by looking at the identification of the outer packaging box of the kit, and the known information is not in the detection process. Control, with great randomness;

[0009] (4) The detection reagent is in a space for being exposed during the detection process, which easily causes cross-contamination between various reagents and affects the accuracy of the detection result;

[0010] (5) The detection process is mostly manual operation, the addition amount of the reagent or the sample is not very precise, the operation process is extremely cumbersome and complicated, the operation error is easy to occur, and the accuracy and precision of the detection result are poor; [0011] (6) The number of items in the configuration and use of the test kit is X48/96, and if 10 items need to be detected, the reagent configuration and usage number must be 10x48/96. Only one sample needs to be tested for 10 different items. It also requires 10x48/96 parts of reagents, which is not economically reasonable.

[0012] Second, the routine laboratory detection of a virus separation method

[0013] The gold standard for laboratory diagnosis of respiratory syncytial virus infection is the method of virus isolation. It is advisable to use the specimens for 5 days before the onset of the disease. The patient's nasopharyngeal swabs or sputum are used for virus isolation and culture. The virus cannot proliferate in chicken embryos, and can only be cultured in human and monkey cells such as Hep-2, Hela, etc., and cells that are unclear in cell boundaries and fused into multinucleated giant cells are observed after 2-3 weeks of culture. The lesion, the virus is released by budding. However, the method of virus isolation has serious drawbacks. Because they are both laborious and laborious, they usually require a long day to get the final result, which has certain limitations in the clinical treatment of patients.

[0014] Third, genetic diagnosis

[0015] RT-PCR diagnostic methods are faster and more sensitive than traditional viral isolation and antigen diagnostic methods for many RNA viruses. Moreover, RT-PCR can easily diagnose multiple viruses simultaneously, and other diagnostic methods such as virus isolation and immunofluorescence analysis cannot. However, this method also has shortcomings, such as PCR technology, the university nature of DNA amplification leads to a very small amount of pollution, which can cause false positives, thus distorting the result. In addition, the virus as an invader of the exogenous gene must clarify all or part of its nucleotide sequence to design primers or probes for nucleic acid hybridization and PCR detection.

[0016] From the existing detection methods of respiratory syncytial virus, EIA, virus isolation, and RT-PCR diagnostic methods have certain specificity and sensitivity advantages, but require professional technicians and specialized operations. Disadvantages of equipment and specific conditions and fees.

[0017] The direct chemiluminescence of acridinium ester as a marker has a superior advantage over the above methods, mainly in: the reaction does not require a catalyst, as long as the alkaline environment can be carried out, the reaction is rapid, the background luminescence is low, and the signal-to-noise ratio is high. The interference factor is small, the reagent stability is good, the calibration can be two points, the system is simple, the cost of the excitation liquid is low, the acridine ester is easy to be linked with the protein, and the photon yield after the coupling is not reduced, which has become a new development direction of the syncytial virus diagnosis. .

Problem solution Technical solution

[0018] Based on this, it is necessary to provide a respiratory syncytial virus chemiluminescence immunoassay kit with high detection sensitivity and a preparation method thereof.

[0019] A respiratory syncytial virus chemiluminescence immunoassay kit comprising: a respiratory syncytial virus recombinant protein-coated carboxylated magnetic particle and an anti-human immunoglobulin-labeled chemiluminescent label.

[0020] wherein the respiratory syncytial virus recombinant protein coated carboxylated magnetic particles, the ratio of the respiratory syncytial virus recombinant protein to the carboxylated magnetic particles is 1: 25-35 =

[0021] The anti-human immunoglobulin-labeled chemiluminescent label, wherein the ratio of the anti-human immunoglobulin to the chemical luminescent label is 50:1 to 10.

[0022] The carboxylated magnetic particles have a particle diameter of 0.05 μm to 1 μm.

[0023] The chemiluminescent label is luminol, isoluminol, terpyridine or acridinium ester.

[0024] The respiratory syncytial virus chemiluminescence immunoassay kit further comprises a chemiluminescent substrate liquid, and the chemiluminescent substrate liquid comprises sputum and sputum.

[0025] The liquid A is a H ₂ O ₂ solution, and the liquid B is a NaOH solution.

[0026] The respiratory syncytial virus chemiluminescence immunoassay kit further includes respiratory syncytial virus calibration

p

[0027] The respiratory syncytial virus calibrator is a solution of respiratory syncytial virus at concentrations of 1 U/L, 10 U/L, 100 U/L, 500 U/L, 100 00 U/L, and 2000 U/L, respectively.

[0028] A preparation method of the above-mentioned respiratory syncytial virus chemiluminescence immunoassay kit comprises the following steps:

[0029] taking a suspension of carboxylated magnetic particles, magnetically separating the supernatant, resuspending in MES buffer, and then adding an aqueous solution of E DC to activate the surface carboxyl group of the carboxylated magnetic particles, followed by the recombinant protein of respiratory syncytial virus , suspended at room temperature for 2h~10h, magnetically separated to remove the supernatant and resuspended in Tris buffer to obtain carboxylated magnetic particles coated with respiratory syncytial virus recombinant protein;

[0030] taking anti-human immunoglobulin, adding carbonate buffer and then mixing, then adding a chemiluminescent label, and then mixing, and avoiding light at room temperature for 1h~2h, then obtaining anti-human immunoglobulin labeling. Advantages of the invention of chemiluminescent markers Beneficial effect

[0031] The respiratory syncytial virus chemiluminescence immunoassay kit can be used as a detection tool to complete the detection of respiratory syncytial virus, which is a respiratory syncytial virus chemiluminescence immunoassay kit. The detection sensitivity reaches 1U/L, which is at least 10 times more sensitive than the traditional respiratory syncytial virus detection method. The detection sensitivity of this respiratory syncytial virus chemiluminescence immunoassay kit is high.

Brief description of the drawing

DRAWINGS

1 is a flow chart showing a method of preparing a respiratory syncytial virus chemiluminescence immunoassay kit according to an embodiment;

2 is a standard curve of respiratory syncytial virus obtained in Example 3.

Embodiments of the invention

The specific embodiments of the present invention will be described in detail below with reference to the drawings and specific embodiments. In the following description, numerous specific details are set forth in order to provide a However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention, and thus the present invention is not limited by the specific embodiments disclosed below.

[0035] An embodiment of the respiratory syncytial virus chemiluminescence immunoassay kit, comprising: a respiratory syncytial virus recombinant protein-coated carboxylated magnetic particle and an anti-human immunoglobulin-labeled chemiluminescent label.

[0036] Preferably, in the carboxylated magnetic particles coated with the respiratory syncytial virus recombinant protein, the ratio of the respiratory syncytial virus recombinant protein to the carboxylated magnetic particles is 1:25-35=

Preferably, the anti-human immunoglobulin-labeled chemiluminescent label has a ratio of anti-human immunoglobulin to chemical luminescent label of 50:1 to 10.

Preferably, the carboxylated magnetic particles have a particle diameter of 0.05 μm to 1 μm.

[0039] The chemiluminescent label can be luminol, isoluminol, terpyridine or acridinium ester. Among them, the chemiluminescent label is preferably an acridinium ester.

[0040] In other embodiments, the respiratory syncytial virus chemiluminescence immunoassay kit further includes chemistry Luminescent substrate fluid.

[0041] The chemiluminescent substrate liquid includes a liquid A and a liquid B. Solution A can be H ₂ 0 ₂ solution, and B solution can be NaOH solution.

[0042] In the present embodiment, the liquid A is H ₂ 0 ₂ having a concentration of 0.1 mol/L.

Solution, solution B is a NaOH solution with a concentration of 0.25 mol/L.

[0043] In other embodiments, the respiratory syncytial virus chemiluminescent immunoassay kit further comprises a respiratory syncytial virus calibrator.

[0044] Respiratory syncytial virus calibrators are 1U/L, 10U/L, 100U/L, 500U/L, 1000U/

L and 2000 U/L of a solution of respiratory syncytial virus.

[0045] Specifically, the respiratory syncytial virus calibrator can be used to prepare respiratory syncytial virus at a concentration of 1 U/L, 10 U/L, 100 U/L, 500 U/L, 1000 U/L, and 2000 U using standard buffer. /L solution of respiratory syncytial virus.

[0046] The respiratory syncytial virus chemiluminescence immunoassay kit is used for detecting respiratory syncytial virus, and the respiratory syncytial virus calibrator is detected by a fully automatic chemiluminescence immunoassay analyzer, and a standard curve is drawn, which is built in the computer. Software; then test the actual sample, calculate the sample concentration based on the sample luminescence value; Finally, evaluate the performance (sensitivity, linearity, precision, interference) of the respiratory syncytial virus fully automated chemiluminescence immunoassay system.

[0047] The respiratory syncytial virus chemiluminescence immunoassay kit can complete the detection of respiratory syncytial virus by using a fully automatic chemiluminescence immunoassay analyzer as a detection tool, and the respiratory syncytial virus chemiluminescence immunoassay kit is tested. The detection sensitivity reaches 1U/L, which is at least 10 times more sensitive than the traditional respiratory syncytial virus detection method. The detection sensitivity of this respiratory syncytial virus chemiluminescence immunoassay kit is high.

[0048] In addition, the respiratory syncytial virus chemiluminescence immunoassay kit has the following advantages:

[0049] 1. Selecting acridinium ester as a labeling material and applying to a chemiluminescence immunoassay system, the luminescence system is direct chemiluminescence, and the reaction does not require the participation of an enzyme compared with the conventional enzymatic chemiluminescence, and is more economical. Cost

[0050] 2. The chemiluminescence immunoassay system using acridine ester has a wide linear range and can reach 1U/L~1000U/L, while the detection range of the traditional respiratory syncytial virus detection method is 20U/L~1000U/ L; [0051] 3. The acridine ester chemiluminescence immunoassay system has high reproducibility, and the intra-assay and batch-to-batch difference are within 5%, which is difficult to achieve by other chemiluminescence immunoassay systems;

[0052] 4, the chemiluminescence immunoassay system has achieved the quantification of the sample, through the built-in standard curve to the test software

, you only need to test the sample to get the concentration value of the sample directly;

[0053] 5. The chemiluminescence immunoassay system can be fully automated, and the addition of reagents and samples is completed by the instrument, and the operation is simpler and the human error is reduced.

[0054] The preparation method of the above-mentioned respiratory syncytial virus chemiluminescence immunoassay kit shown in FIG. 1 includes the following steps:

[0055] taking a suspension of carboxylated magnetic particles, magnetically separating the supernatant, resuspending in MES buffer, then adding an aqueous solution of E DC to activate the surface carboxyl group of the carboxylated magnetic particles, and then adding the respiratory syncytial virus recombinant protein After being suspended at room temperature for 2 h to 10 h, the supernatant was removed by magnetic separation and resuspended in Tris buffer to obtain carboxylated magnetic particles coated with respiratory syncytial virus recombinant protein.

[0056] The concentration of MES (2-(N-morpholine)ethanesulfonic acid) buffer was 0.02 M and the pH was 5.5.

[0057] The Tris buffer had a concentration of 0.1 M and contained 2% BSA with a pH of 8.0.

[0058] EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) aqueous solution concentration of 10mg/mL~20m g/mL, ratio of EDC to carboxylated magnetic particles 0.05: 0.1-1 =

[0059] Preferably, in the carboxylated magnetic particles coated with the respiratory syncytial virus recombinant protein, the ratio of the recombinant virus of the respiratory syncytial virus to the carboxylated magnetic particles is 1:25-35=

[0061] Take anti-human immunoglobulin, add carbonate buffer and mix well, then add chemiluminescent label and mix well, avoid the light at room temperature for 1h~2h, get anti-human immunoglobulin label Chemiluminescent label

[0062] The carbonate buffer has a concentration of 0.1 M and a pH of 9.0 9.5.

[0063] The impurity removal operation is desalting of the centrifugal desalting column, and the specific operation is as follows: First, the centrifugal desalting column is treated with pure water and TBS buffer (40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0), and finally A solution of the obtained respiratory syncytial virus recombinant protein-coated carboxylated magnetic particles is added, and finally the liquid in the centrifuge tube is collected.

Preferably, the anti-human immunoglobulin-labeled chemiluminescent label, respiratory syncytial virus recombinant protein The ratio to the chemiluminescent label is 50: 1~10

[0065] The chemiluminescent label can be luminol, isoluminol, terpyridine or acridinium ester. Among them, the chemiluminescent label is preferably an acridinium ester.

The above-mentioned respiratory syncytial virus chemiluminescence immunoassay kit can be obtained by combining the obtained respiratory syncytial virus recombinant protein-coated carboxylated magnetic particles and anti-human immunoglobulin-labeled chemiluminescent label.

[0067] This respiratory syncytial virus chemiluminescence immunoassay kit requires a chemiluminescent substrate solution and a respiratory syncytial virus calibrator in the use of sputum.

[0068] The chemiluminescent substrate fluid and the respiratory syncytial virus calibrator can be prepared by themselves.

[0069] The chemiluminescent substrate liquid includes a liquid A and a liquid B. Solution A can be H ₂ 0 ₂ solution, and B solution can be NaOH solution.

[0070] In the present embodiment, the liquid A is H ₂ O ₂ having a concentration of 0.1 mol/L.

Solution, solution B is a NaOH solution with a concentration of 0.25 mol/L.

[0071] Specifically, the respiratory syncytial virus calibrator can be used to prepare respiratory syncytial virus into a respiratory tract having a concentration of 1 U/L 10 U/L 100 U/L 500 U/L 1000 U/L and 2000 U/L, respectively, using a standard buffer. A solution of syncytial virus.

[0072] The preparation method of the respiratory syncytial virus chemiluminescence immunoassay kit is simple and convenient, and the prepared respiratory syncytial virus chemiluminescence immunoassay kit has high detection sensitivity and has good application before application.

[0073]

[0074] The following are specific examples.

Example 1 : Preparation of Respiratory Syncytial Virus Chemiluminescence Immunoassay Kit

(1) Preparation of Carboxylated Magnetic Particles Coated with Recombinant Syneremic Virus Recombinant Protein:

[0077] A suspension containing 50 mg of carboxylated magnetic particles (MagnaBindTM, Cat. No. 21353) having a particle size of 0.05 μm to 1 μm was taken, and the supernatant was magnetically separated, resuspended in 0.02 M pH 5.5 MES buffer, and added to 1 mL of new Configure 10mg/mL EDC aqueous solution, activate the carboxyl group surface, add 4mg respiratory syncytial virus recombinant protein (biorbyt, no. orb48780), suspend for 6h at room temperature, magnetic separation, remove the supernatant, with 2<3⁄4B SA Resuspend respiratory syncytial virus recombinant protein by resuspending 0.1 M Tris buffer with pH 8.0 to 1 mg/mL The coated carboxylated magnetic particles were stored at 4 ° C for 5 mL per bottle.

(2) Preparation of anti-human immunoglobulin-labeled acridinium ester:

[0079] 50 μί of 25 mg/mL anti-human immunoglobulin was added, and the concentration of 150 μί was 0.1 M, and the pH was 9.0~.

Mix 9.5 carbonate buffer, mix, then add 1.5μL concentration of 5mg/mL acridinium ester solution, avoid the light reaction at room temperature, take it out after 1.5h, desalinate with 2mL zeba centrifugal desalting column, desalting The process is first treated with purified water and TBS buffer, and finally the obtained anti-human immunoglobulin-labeled acridinium ester solution is added, and the liquid in the centrifuge tube is collected to the preservation tube to obtain anti-human immunoglobulin-labeled acridinium ester. , 5mL per bottle is stored at 4 ° C for use.

(3) Preparation of a respiratory syncytial virus calibrator:

Respiratory syncytial virus was formulated to a concentration of 0 U/L, 10 U/L, 100 U/L, 500 U/L using standard buffer (40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0). 1000U/L and 2000U/L, 0.5 mL per bottle is lyophilized and stored at 4 °C.

[0082]

Example 2: Respiratory syncytial virus chemiluminescence immunoassay method

[0084] A fully automatic chemiluminescence immunoassay analyzer (YHLO, item number iFlash3000) is used as a detection tool, and the methodological mode is indirect immunization, that is, the instrument sequentially adds 50 samples, 50 of the respiratory syncytial virus recombinant protein coated carboxylation Magnetic particles and 50 respiratory syncytial virus treatment solution, after reacting for 20 min, add 50 anti-human immunoglobulin acridinium ester, react for 20 min, magnetic separation, the instrument will send the reaction mixture into the dark room, and then add The luminescent substrate A (H ₂ 0 ₂ ) and the B solution (NaOH) were subjected to a luminescence reaction, and finally the luminescence value was recorded.

[0085]

Example 3: Performance Evaluation of Respiratory Syncytial Virus Chemiluminescence Immunoassay Kit

[0087] The respiratory syncytial virus calibrator was tested by the method of Example 2, and a standard curve was drawn as shown in FIG.

[0088] Next, the actual sample is next tested, and the sample concentration is calculated based on the sample luminescence value.

[0089] Sensitivity detection:

[0090] Referring to the CLSI EP17-A document recommended experimental protocol, the sensitivity of the respiratory syncytial virus chemiluminescence immunoassay kit was calculated, and the obtained sensitivity was 1 U/L. [0091] Linear detection:

Linear analysis of concentrations of 1 U/L, 10 U/L, 100 U/L, 500 U/L, 1000 U/L, and 2000 U/L standards, calculating a linear correlation coefficient, r = 0.9996, in addition, the kit pair The linear range of detection of respiratory syncytial virus samples ranged from 1 U/L to 1000 U/L.

[0093] Precision measurement:

[0094] Take two respiratory syncytial virus samples at a concentration of 50 U/L and 500 U/L, and each sample is made up of 3 parallels each. The test is performed in three batches of kits, and the difference between the batch and the batch of the kit is calculated. The results showed that the difference between the batch and batch of the kit was less than 5%.

[0095] Interfering experiments:

[0096] Taking the mixed serum and adding the interference substance respectively includes: combining bilirubin, free bilirubin, hemoglobin, ascorbic acid, glyceride, and adding the ratio according to 1:20, separately measuring the mixed serum and adding various interferents and mixing The measured value of the serum is calculated as the deviation between the two, with an acceptable range of ±10%. The results show that the interference is up to the NCCLS document standard and can be used for accurate assessment of respiratory syncytial virus status in clinical laboratories.

[0097]

Example 4 Comparative Experiment of Respiratory Syncytial Virus Chemiluminescence Immunoassay Kit

[0099] The respiratory syncytial virus samples at concentrations of 0, 50 U/L were detected by chemiluminescence detection method and conventional enzyme-linked immunosorbent assay, respectively. The sensitivity of the two methods was as shown in the following table:

[0101]

[0102]

As can be seen from the above table, the sensitivity of the chemiluminescence detection method is more than 10 times higher than that of the enzyme-linked immunosorbent assay.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is not to be construed as limiting the scope of the invention. It should be noted that various modifications and improvements can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the invention should be subject to the appended claims.

Claims

Claim

[Claim 1] A respiratory syncytial virus chemiluminescence immunoassay kit, comprising: a respiratory syncytial virus recombinant protein-coated carboxylated magnetic particle and an anti-human immunoglobulin-labeled chemiluminescent label .

[Claim 2] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 1, wherein the respiratory syncytial virus-recombined carboxylated magnetic particles, the respiratory syncytium The ratio of the viral recombinant protein to the carboxylated magnetic particles is 1:25-35.

[Claim 3] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 1, wherein the anti-human immunoglobulin-labeled chemiluminescent label, the respiratory syncytial virus recombinant protein The ratio to the chemiluminescent label is 50: 1 to 10.

The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 1, wherein the carboxylated magnetic fine particles have a particle diameter of 0.05 μm to 1 μm.

[Claim 5] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 1, wherein the chemiluminescent label is luminol, isoluminol, terpyridine or acridinium ester .

[Claim 6] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 1, further comprising a chemiluminescent substrate liquid, the chemiluminescent substrate liquid comprising a sputum solution and a B solution.

[Claim 7] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 6, wherein the liquid A is an H ₂ 0 ₂ solution, and the liquid B is a NaOH solution.

[Claim 8] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 1, further comprising a respiratory syncytial virus calibrator.

[Claim 9] The respiratory syncytial virus chemiluminescence immunoassay kit according to claim 8, wherein the respiratory syncytial virus calibrator has a concentration of 1 U/L, 10 U/L, 100 U/ L, 500 U/L, 1000 U/L and 2000 U/L solutions of respiratory syncytial virus.

[Claim 10] The method for preparing a respiratory syncytial virus chemiluminescence immunoassay kit according to any one of claims 1 to 9, comprising the steps of: Taking a suspension of carboxylated magnetic particles, magnetically separating the supernatant, resuspending in MES buffer, then adding EDC aqueous solution to activate the surface carboxyl group of the carboxylated magnetic particles, and then adding the respiratory syncytial virus recombinant protein, mixing at room temperature After hanging for 2h~10h, the supernatant was removed by magnetic separation and resuspended in Tris buffer to obtain carboxylated magnetic particles coated with respiratory syncytial virus recombinant protein;

Take anti-human immunoglobulin, add carbonate buffer and mix well, then add chemiluminescent label, mix well, remove the light at room temperature for 1h~2h, and get the anti-human immunoglobulin-labeled chemiluminescent label. Things.