IgG antibody, IgM antibody and method

Abstract

The present invention relates to the field of in vitro immunodiagnosis and detection, and in particular to an IgG antibody, an IgM antibody and a method. The present invention provides an IgG antibody that specifically binds to a coxsackie virus, characterized in that the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region of the IgG antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The IgG antibody provided by the present invention not only has good specificity and can specifically identify coxsackie viruses, but also has a high level of sensitivity and stability.

Description

Technical Field

[0001] The present invention relates to the field of in vitro immunodiagnosis and detection, and in particular to an IgG antibody, an IgM antibody and a method. Background Art

[0002] Pathogens are a type of microorganisms or parasites that can cause disease, also known as pathogenic microorganisms, including bacteria, viruses, fungi, parasites, protozoa, etc. Pathogens cause diseases by directly invading the host, releasing toxins, interfering with the normal functions of host cells, and causing immune responses. Bacteria and viruses are the most harmful. Common epidemic pathogens include: bacteria, respiratory viruses and enteric viruses.

[0003] Pathogen detection refers to the process of identifying, detecting and quantifying pathogens through laboratory techniques and methods. Pathogen detection plays an important role in determining the cause of the disease, monitoring the spread of the disease, and assessing the severity of the disease. It can also guide effective treatment and guide the formation of preventive measures. It has now become an important task in clinical diagnosis.

[0004] With the advancement of molecular biological diagnostic technology, the detection methods of pathogens (such as Coxsackie virus) have gone through the stages of pathogen isolation and culture detection, PCR method and immunological detection method. The pathogen isolation and culture detection method requires the pathogen to be isolated and then cultured. The operation is complicated, time-consuming, and there is a certain degree of subjectivity in the judgment of the results. The PCR method detects pathogens by detecting the target DNA or RNA sequence. Although it has great advantages in specificity and price, the amplified product must be interpreted by agarose gel electrophoresis, which has low sensitivity and is time-consuming. The immunological detection method is based on the reaction principle of antigen-antibody specific binding to detect pathogens. Although it has the advantages of simple operation, convenience and speed, it has not yet overcome the shortcomings of insufficient sensitivity and easy to cause false positive and false negative results. Summary of the invention

[0005] In the first aspect, the present invention provides an IgG antibody that specifically binds to Coxsackie virus, characterized in that the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region of the IgG antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0006] In some embodiments, the IgG antibody is coupled to magnetic beads.

[0007] In some embodiments, the IgG antibody is labeled with an acridinium ester.

[0008] In some embodiments, the IgG antibody is conjugated to biotin.

[0009] In a second aspect, the present invention further provides an IgM antibody, characterized in that the IgM antibody is transformed from the IgG antibody of the first aspect, and the IgM antibody comprises a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region.

[0010] In some embodiments, the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0011] In some embodiments, the heavy chain constant region includes a first constant region, a second constant region, a third constant region and a fourth constant region, wherein the first constant region is derived from a mouse IgG antibody, and the amino acid sequence of the first constant region is shown in SEQ ID NO: 13.

[0012] In some embodiments, the second constant region, the third constant region, and the fourth constant region are derived from a murine IgM antibody.

[0013] In some embodiments, the amino acid sequences of the second constant region, the third constant region, and the fourth constant region are shown as SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0014] In some embodiments, the IgM antibody is coupled to magnetic beads.

[0015] In some embodiments, the IgM antibody is labeled with an acridinium ester.

[0016] In some embodiments, the IgM antibody is conjugated to biotin.

[0017] In a third aspect, the present invention also provides an IgG antibody that specifically binds to Coxsackie virus, characterized in that the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region of the IgG antibody are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0018] In some embodiments, the IgG antibody is coupled to magnetic beads.

[0019] In some embodiments, the IgG antibody is labeled with an acridinium ester.

[0020] In some embodiments, the IgG antibody is conjugated to biotin.

[0021] In a fourth aspect, the present invention further provides an IgM antibody, characterized in that the IgM antibody is transformed from the IgG antibody of the third aspect, and the IgM antibody comprises a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region.

[0022] In some embodiments, the heavy chain constant region includes a first constant region, a second constant region, a third constant region and a fourth constant region, wherein the first constant region is derived from a mouse IgG antibody, and the amino acid sequence of the first constant region is shown in SEQ ID NO: 13.

[0023] In some embodiments, the second constant region, the third constant region, and the fourth constant region are derived from a murine IgM antibody.

[0024] In some embodiments, the amino acid sequences of the second constant region, the third constant region, and the fourth constant region are shown as SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0025] In some embodiments, the IgM antibody is coupled to magnetic beads.

[0026] In some embodiments, the magnetic bead coating surface is a carboxyl surface, which can be replaced by an original surface, a modified surface (amino, hydroxyl and sulfate), a pre-activated surface (tosyl, epoxy and chloromethyl groups) and a biologically activated surface (protein A, protein G, streptavidin, biotin).

[0027] In some embodiments, the IgM antibody is labeled with an acridinium ester.

[0028] In some embodiments, the IgM antibody is conjugated to biotin.

[0029] In a fifth aspect, the present invention further provides a detection reagent, characterized in that the detection reagent comprises the IgM antibody described in the second aspect and / or the IgM antibody described in the fourth aspect.

[0030] In some embodiments, the detection reagent comprises the IgM antibody of the second aspect coupled to magnetic beads and / or the IgM antibody of the fourth aspect coupled to magnetic beads.

[0031] In some embodiments, the preparation method of the detection reagent comprises: (a) adding 50 μL of carboxyl magnetic beads to 300 μL of 0.01M PBS, vortexing for 5 min, removing the supernatant by magnetic aspiration, and repeating twice; (b) adding 150 μL of pH 6.0 MES to dissolve, vortexing for 30 s to mix; (c) adding 20 mg / mL EDC and 20 mg / mL sulfo-NHS, vortexing again to mix, and activating at 37°C for 30 min; (d) adding 300 μL of 0.01M PBS, vortexing for 30 s, removing the supernatant by magnetic aspiration, and repeating 3 times; (e) adding 300 μL of 0.5 mg / mL of the IgM antibody described in the second aspect or the fourth aspect, vortexing at 37°C for 2 h, and removing the supernatant by magnetic aspiration; (f) adding 5 mL of blocking solution (1% BSA) to resuspend, vortexing at 37°C for 10 min, removing the supernatant by magnetic aspiration, and repeating twice; (g) Add 5 mL of blocking solution (1% BSA) and resuspend to obtain the detection reagent, which is stored at 4°C for later use.

[0032] In some embodiments, the detection reagent comprises the IgM antibody of the second aspect coupled to magnetic beads and the IgM antibody of the fourth aspect labeled with acridinium ester; or

[0033] The IgM antibody of the fourth aspect coupled to magnetic beads and the IgM antibody of the second aspect labeled with acridinium ester.

[0034] In a sixth aspect, the present invention further provides a detection reagent, characterized in that the detection reagent comprises:

[0035] Magnetic beads-streptavidin conjugate;

[0036] a biotin-modified primary IgM antibody; and

[0037] acridinium ester-labeled secondary IgM antibody;

[0038] The first IgM antibody and the second IgM antibody are selected from the IgM antibodies described in the second aspect or the fourth aspect, and the first IgM antibody and the second IgM antibody are different.

[0039] In some embodiments, the mass ratio of the magnetic bead-streptavidin conjugate to the biotin-modified first IgM antibody is 1:2.

[0040] In some embodiments, the method for preparing the biotin-modified first IgM antibody comprises: mixing the first IgM antibody with biotin, wherein the feed ratio (mass ratio) of the first IgM antibody to the biotin is 1:2-10.

[0041] In some embodiments, the method for preparing the acridinium ester-labeled second IgM antibody comprises: mixing the second IgM antibody with acridinium ester, wherein the feed ratio (mass ratio) of the second IgM antibody to the acridinium ester is 1:10-30.

[0042] In a seventh aspect, the present invention further provides a method for improving the detection sensitivity of pathogenic microorganisms, characterized in that it comprises the following steps:

[0043] Obtaining samples to be tested;

[0044] Adding a detection reagent to the sample to be detected;

[0045] Using a chemiluminescence method to detect the concentration of pathogenic microorganisms in the sample to be detected;

[0046] The detection reagent comprises the above-mentioned IgM antibody or detection reagent.

[0047] In some embodiments, the pathogenic microorganism is a coxsackievirus.

[0048] In some embodiments, the detection reagent specifically includes:

[0049] Magnetic beads-streptavidin conjugate;

[0050] a biotin-modified primary IgM antibody; and

[0051] acridinium ester-labeled secondary IgM antibody;

[0052] The first IgM antibody and the second IgM antibody are selected from the IgM antibodies described in the second aspect or the fourth aspect, and the first IgM antibody and the second IgM antibody are different.

[0053] In some embodiments, the method specifically comprises:

[0054] Mixing the magnetic bead-streptavidin conjugate and the biotin-modified first IgM antibody to obtain a premix;

[0055] Mixing the premix with the sample to be detected to obtain a magnetic bead-first IgM antibody-antigen conjugate;

[0056] Mixing the magnetic bead-first IgM antibody-antigen conjugate with the acridinium ester-labeled second IgM antibody to obtain a first IgM antibody-antigen-second IgM antibody sandwich complex;

[0057] The concentration of Coxsackie virus in the sample to be detected is detected using a chemiluminescence method.

[0058] In an eighth aspect, the present invention further provides a method for improving the detection sensitivity of biomarkers, characterized in that it comprises the following steps:

[0059] S101 immunizing an animal with an antigen, CpG and manganese chloride to induce the animal to produce IgG antibodies;

[0060] S102 fusing the B lymphocytes of the animal with myeloma cells to obtain hybridoma cells;

[0061] S103 screening the hybridoma cells to obtain positive hybridoma cells;

[0062] S104 collecting IgG monoclonal antibodies from the culture of the positive hybridoma cells;

[0063] S105 sequencing the IgG monoclonal antibody to obtain a sequence of a variable region of the IgG monoclonal antibody;

[0064] S106 Recombining the heavy chain variable region sequence of the IgG monoclonal antibody, the constant region sequence fragment CH1 of the mouse or rabbit IgG antibody, and the constant region fragment CH2-CH4 of the mouse or rabbit IgM antibody to obtain a heavy chain expression vector;

[0065] S107 expressing the heavy chain expression vector and the light chain expression vector in cells and purifying the culture supernatant to obtain purified IgM recombinant antibodies;

[0066] S108 Detecting a biomarker using the IgM recombinant antibody.

[0067] In some embodiments, the antibody titer of the animal in S102 is ≥10 4 .

[0068] In some embodiments, the variable region comprises a heavy chain variable region and a light chain variable region.

[0069] In some embodiments, the biomarkers include pathogenic microorganisms, such as one or more of bacteria, viruses, fungi, and parasites.

[0070] In some embodiments, the pathogenic microorganism is a coxsackievirus.

[0071] In some embodiments, the purification is a one-step protein G purification.

[0072] In some embodiments, the IgM antibody comprises a heavy chain constant region, a light chain constant region, a heavy chain variable region, and a light chain variable region.

[0073] In some embodiments, the heavy chain constant region comprises a first constant region, a second constant region, a third constant region, and a fourth constant region, wherein the first constant region is derived from a murine IgG antibody.

[0074] In some embodiments, the amino acid sequence of the first constant region is shown in SEQ ID NO:13.

[0075] In some embodiments, the second constant region, the third constant region, and the fourth constant region are derived from a murine IgM antibody.

[0076] In some embodiments, the amino acid sequences of the second constant region, the third constant region, and the fourth constant region are shown as SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.

[0077] In some embodiments, the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0078] In some embodiments, the amino acid sequences of the complementarity determining regions CDRH1-CDRH3 of the heavy chain variable region are shown as SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequences of the complementarity determining regions CDRL1-CDRL3 of the light chain variable region are shown as SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0079] In some embodiments, the IgM antibody is coupled to magnetic beads.

[0080] In some embodiments, the IgM antibody is labeled with an acridinium ester.

[0081] In some embodiments, the IgM antibody is conjugated to biotin.

[0082] In some embodiments, the recombinant IgM antibody comprises a first IgM antibody and a second IgM antibody.

[0083] In some embodiments, the first IgM antibody is coupled to magnetic beads.

[0084] In some embodiments, the second IgM antibody is labeled with an acridinium ester.

[0085] In some embodiments, the first IgM antibody is conjugated to avidin.

[0086] Compared with the prior art, the beneficial effects of the present invention include at least the following aspects:

[0087] Although the immunological detection method based on the specific binding of antigen and antibody has the advantages of simple operation, convenience and rapidity, it still has the disadvantages of insufficient sensitivity and easy to cause false positive and false negative results. In order to improve the detection sensitivity of coxsackie virus, the present invention provides an IgG antibody that specifically binds to coxsackie virus, which not only has good specificity and can specifically identify coxsackie virus, but also has a high level of sensitivity and stability.

[0088] Based on the above IgG antibody, the present invention also provides an IgM antibody transformed from the above IgG antibody. In many prior arts (e.g., CN112538111A, CN117821510A), when transforming IgG antibodies into IgM antibodies, the heavy chain constant region of the IgM antibody usually does not involve the CH1 part of the heavy chain constant region, or completely uses the CH1-CH4 of the heavy chain constant region of the IgM antibody. The present invention finds that the CH1 of the heavy chain constant region of the IgM antibody is actually crucial for the expression and purification of the IgM antibody, the spatial structure, and the affinity for the antigen. The IgM antibody provided by the present invention not only retains the CH1 part of the heavy chain constant region, but also creatively replaces it with the CH1 of the heavy chain constant region of the IgG antibody (e.g., the amino acid sequence shown in SEQ ID NO: 13). The results show that the IgM antibody provided by the present invention can be purified by protein G in one step to reach a purity of more than 95% (in actual operation, this purity often requires more than two steps of purification methods to achieve), and can be directly used for subsequent chemiluminescence detection.

[0089] In addition, it has been experimentally verified that compared with the IgM antibody prepared by the existing method (for example, CN118772272A), the IgM antibody provided by the present invention can improve the sensitivity of detecting Coxsackie virus by 4-5 times compared with IgG antibody while ensuring the original stability, specificity and affinity of IgG antibody. The sensitivity of IgM antibody coupled with biotin for detecting Coxsackie virus can be improved by 16-20 times compared with IgG antibody, that is, the sensitivity and accuracy are high and the non-specific adsorption and detection limit are low, which avoids the occurrence of false positive and false negative results, and is suitable for low-abundance detection of Coxsackie virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0091] Figure 1 is a schematic diagram of the structure of the heavy chain expression vector;

[0092] Figure 2 Schematic diagram of the structure of the light chain expression vector;

[0093] Figure 3 This is a diagram of the thermal acceleration stability verification results. DETAILED DESCRIPTION

[0094] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. 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 making creative work are within the scope of protection of the present invention.

[0095] In this document, the directions or positional relationships indicated by the terms "upper", "lower", "inner", "outer", "front", "rear", "one end", "the other end", etc. are based on the directions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0096] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0097] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0098] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0099] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0100] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0101] Embodiment 1

[0102] 1.1 Animal immunization

[0103] Immunized animals: 4 SJL female mice;

[0104] Immunization scheme: Before immunization, 0.5 mL of blood was collected from mice, and serum was separated as a pre-immunization control. On day 0, the mice were given a primary immunization by subcutaneous injection in the armpits and back. After 14 days from the primary immunization, the mice were given a secondary immunization by subcutaneous injection in the armpits and back, for a total of three immunizations. The injection dose for each immunization was 100 μL / mouse.

[0105] Specific immunization methods include:

[0106] Before immunization, 0.5 mL of blood was collected and centrifuged at 4000 rpm for 10 min to separate serum;

[0107] The antigen and adjuvant are mixed and emulsified in a volume ratio of 2:1 for later use;

[0108] On day 0, mice were initially immunized with 10 μg CpG, 50 μg manganese chloride, and 50 μg coxsackie antigen, injected subcutaneously at 100 μL per mouse;

[0109] On day 14, mice were immunized twice with 10 μg CpG, 50 μg manganese chloride, and 25 μg coxsackie antigen, 100 μL / mouse by subcutaneous injection;

[0110] On day 28, mice were immunized three times with 10 μg CpG, 50 μg manganese chloride, and 12.5 μg coxsackie antigen, injected subcutaneously at 100 μL per mouse.

[0111] After immunization of mice, blood was collected 10 days later, and the mouse serum was diluted in multiples. The dilution endpoint was determined by indirect ELISA method. The highest dilution determined as positive was the serum binding antibody titer. The results of three immune titer tests are shown in Table 1.

[0112] Table 1: Binding antibody titers of mouse sera from three immunizations (×10 4 )

[0113]

[0114] In the method of activating the mouse immune system to produce an immune response, the current CpG immunization method mostly uses aluminum hydroxide as an adjuvant. Although aluminum adjuvant is a recognized safe adjuvant that can effectively promote antigen-induced humoral immune response, it is difficult to induce cellular immune response. At the same time, aluminum adjuvant also has certain side effects, which can cause inflammation and allergic reactions at the injection site. The present invention uses manganese adjuvant (manganese chloride) instead of aluminum adjuvant to perform CpG immunization on mice, which can obtain a more excellent immune effect and highly efficiently activate immune cells with less antigen loading and lower injection usage.

[0115] 1.2 Cell screening

[0116] (1) Sorting of mouse B lymphocytes:

[0117] The results of ELISA determination of mouse serum antibody titers are shown in Table 1, indicating that the serum titers of immunized mice were greater than 1:10. 4 , which can meet the requirements of cell fusion. Take mice with immune titers that meet the requirements to sort B lymphocytes;

[0118] After collecting blood from the orbit, serum was separated and used as a positive control;

[0119] To avoid splenic infarction, mice were killed by rapid cervical dislocation and then immersed in 75% alcohol;

[0120] The spleen was removed under sterile conditions, and the surrounding fat and connective tissue were stripped off to separate the spleen;

[0121] Grind the spleen with a grinder, mix it with DMEM and pass it through a sieve to make a spleen cell suspension (or use tweezers to gently comb and scrape, suck the cell suspension into a centrifuge tube and let it settle naturally for 5 minutes, transfer the suspension to another centrifuge tube, discard the larger tissue pieces, and centrifuge to precipitate the cells; or place the spleen on a 200-mesh stainless steel net, gently grind the spleen with a syringe needle to obtain a cell suspension; or crush the spleen with tweezers, add 400 u / ml collagenase (type III) 5ml / spleen, digest at 37 degrees for 20 minutes, filter with a nylon mesh to obtain a single cell suspension), and centrifuge at 2000 rpm for 10 minutes;

[0122] The supernatant was discarded, the cells were washed with DMEM, and centrifuged at 2000 rpm for 10 min.

[0123] Discard the supernatant, resuspend in DMEM and store at 37°C for later use.

[0124] (2) Fusion of myeloma cells (SP2 / 0) and B lymphocytes:

[0125] SP2 / 0 cells were resuscitated one week before fusion;

[0126] When the SP2 / 0 cells are cultured to a full morphology, a large number, and a good condition, they are expanded and cultured at a ratio of 1:5;

[0127] When the SP2 / 0 cells were expanded and cultured to the logarithmic growth phase, cell fusion was performed;

[0128] SP2 / 0 cells and mouse B lymphocytes were mixed at a ratio of 1:2 and centrifuged at 1000 rpm for 10 min;

[0129] Discard the supernatant and gently knock off the cell clumps at the bottom to allow the SP2 / 0 cells and mouse B lymphocytes to mix and spread evenly at the bottom of the tube;

[0130] Add 1 mL of preheated 50% PEG1450 (PH=8.0) dropwise evenly within 1 min and react at 37°C for 1 min.

[0131] Add 10 mL of DMEM to terminate the reaction;

[0132] Centrifuge at 1000 rpm for 10 min, discard the supernatant, and resuspend the cells with HAT;

[0133] 200 μL / well of cell suspension was taken and added to a 96-well culture plate containing feeder cells for culture, and then transferred to a 37°C, 5% CO2 constant temperature incubator for culture and observation;

[0134] After 3–7 days of HAT culture, the culture medium was replaced with HT, 200 μL per well;

[0135] Depending on the culture conditions, fusion-positive screening was performed using ELISA 4 to 7 days later.

[0136] (3) Screening of positive hybridoma cells:

[0137] The hybridoma cells were diluted multiple times and seeded on a 96-well cell culture plate;

[0138] The antibodies secreted by cells in the supernatant of each well were detected by ELISA method, and the culture wells whose supernatants could specifically bind to specific antigens were positive wells;

[0139] Subclone the cells in the positive wells using the limiting dilution method, and test whether each culture well is a positive well. Repeat 3-4 times until the positive rate of monoclonal clones reaches 100%;

[0140] Expand subculture;

[0141] After centrifugation, the supernatant was collected and affinity chromatography was performed on a Protein G column to obtain IgG monoclonal antibodies with a high purity of 95% from the hybridoma culture supernatant sample.

[0142] The variable regions of mouse IgG antibodies were sequenced.

[0143] The amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region of the obtained monoclonal antibody 4B9 are shown in SEQ ID NO: 1-SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4-SEQ ID NO: 6, respectively.

[0144] SEQ ID NO: 1: DYYGEK

[0145] SEQ ID NO: 2: DINPKNMDTFYNQGFKG

[0146] SEQ ID NO: 3: WTQTHFAMDY

[0147] SEQ ID NO: 4: KKSQSLLNSRTRKMYLA

[0148] SEQ ID NO: 5: WKSTRES

[0149] SEQ ID NO: 6: KQSYDLWT

[0150] The amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region of the obtained monoclonal antibody 1C5 are shown in SEQ ID NO: 7-SEQ ID NO: 9, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10-SEQ ID NO: 12, respectively.

[0151] SEQ ID NO: 7: GYSTTSGY

[0152] SEQ ID NO: 8: SYDGR

[0153] SEQ ID NO: 9: AMAY

[0154] SEQ ID NO: 10: RSSQSLIHSNGNYY

[0155] SEQ ID NO: 11: KVSNRFS

[0156] SEQ ID NO: 12: SQSTHVPWT

[0157] Embodiment 2

[0158] 2.1 IgM transformation of IgG antibodies

[0159] The heavy chain variable region sequence of mouse IgG antibody (monoclonal antibody 4B9 and monoclonal antibody 1C5) and the heavy chain constant region sequence fragment CH1 of mouse IgG antibody were genetically recombined with the heavy chain constant regions CH2, CH3, and CH4 of mouse IgM antibody, and after codon optimization, they were constructed into the expression vector pcDNA3.4. The structure of the heavy chain expression vector is shown in the figure. Figure 1 shown. Figure 1 The various regions from the 5' end to the 3' end are shown, including L (light chain), IGHV (immunoglobulin heavy chain variable region gene), D (diversity region), J (joining region), mlgG1-CH1 (constant region 1 of membrane-bound IgG1), CH2, CH3, CH4 (constant regions 2, 3, 4 of membrane-bound IgM). mlgG1-CH1 together with mIgM-CH2, CH3, and CH4 constitute the heavy chain constant region of the modified IgM antibody.

[0160] The structure of the light chain expression vector is as follows Figure 2 shown. Figure 2 The various regions from the 5' end to the 3' end are shown, including V (immunoglobulin light chain variable region gene), J (joining region) and IGKC (constant region gene)

[0161] Extract plasmids for cell transfection.

[0162] Among them, the amino acid sequence of mlgG1-CH1 is: AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG (SEQ ID NO: 13)

[0163] The amino acid sequence of mIgM-CH2 is: AVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTIENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCA (SEQ ID NO: 14)

[0164] The amino acid sequence of mIgM-CH3 is: ASPSTDILTFTIPPSFADIFLSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEFVCTVTHRDLPSPQKKFISKPN (SEQ ID NO: 15)

[0165] The amino acid sequence of mIgM-CH4 is: EVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQRGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKSTGGTCY (SEQ ID NO: 16)

[0166] The amino acid sequence of the light chain constant region is: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 17)

[0167] 2.2 Expression of recombinant antibodies in CHO cells

[0168] (1) Resuscitation of CHO cells:

[0169] Thaw the frozen CHO cells in a 37°C water bath;

[0170] Centrifuge at 1200 rpm for 2 min;

[0171] Remove the supernatant and resuspend the CHO cells in fresh medium;

[0172] 1 mL of cells was taken daily for counting and viability testing;

[0173] Before transfection, the density was controlled at 3.0×10 6 cells / mL.

[0174] (2) Transfection-grade plasmid extraction:

[0175] 200 μL competent cell suspension, add 80 ng expression plasmid;

[0176] The competent cells after adding the plasmid were heat-shocked at 42°C, transformed for 90 seconds, and cooled on ice for 5 minutes;

[0177] Add 1 mL of LB liquid medium and culture at 37°C and 200 rpm for 30 min;

[0178] 100 μL of cell suspension was applied to the screening plate containing ampicillin resistance and cultured statically at 37°C for 24 h;

[0179] Pick a single clone of cells and inoculate it into 200 mL LB medium and culture it at 37°C and 200 rpm for 16 h;

[0180] Collect the bacterial solution, centrifuge, remove the supernatant, add 250 μL RB solution, and shake on an oscillator to fully suspend the bacteria;

[0181] Add 250 μL LB solution and incubate at 37°C for 2 min;

[0182] Add 250 μL NB solution, incubate at 37°C for 2 min, and centrifuge at 15,000 rpm for 15 min;

[0183] The supernatant was collected and transferred to an adsorption column, and centrifuged at 15,000 rpm for 30 s;

[0184] The supernatant was discarded, 700 μL of WB solution was added to the adsorption column, and centrifuged at 15,000 rpm for 30 s, and repeated twice;

[0185] Place the adsorption column in a 1.5 mL centrifuge tube, add 50 μL Elution Buffer, incubate at 37°C for 2 min;

[0186] The plasmid was collected by centrifugation at 15000 rpm for 1 min.

[0187] (3) Transfection:

[0188] Add light chain and heavy chain expression plasmids to the transfection buffer, dilute them in a 1:1 mass ratio, mix with the transfection reagent, and incubate at room temperature for 5-20 min.

[0189] Add the transfection reagent mixture to the cells to be transfected and culture at 37°C and 120 rpm for 5-6 days;

[0190] Centrifuge and collect the supernatant.

[0191] 2.3 Purification of IgM antibodies after IgG antibody transformation

[0192] (1) Cell supernatant is passed through a Protein G column for antibody purification:

[0193] All buffers used in chromatography were filtered through a 0.45 μm filter membrane;

[0194] The samples were centrifuged at 12,000 rpm for 15 min to remove larger cells and cell debris;

[0195] After diluting the sample with loading buffer at a ratio of 1:1, the diluted sample was filtered through a 0.45 μm filter membrane;

[0196] Use 0.1M NaOH, elute for 2CV and then pause for 15 minutes to clean impurities;

[0197] At a flow rate of 1 mL / min, equilibrate the column with 5-10 column volumes of loading buffer until the pH of the effluent is 7.4;

[0198] The filtered diluted sample was loaded onto the chromatography column at a flow rate of 1 mL / min, and 5 to 10 times the volume of the chromatography column was washed with loading buffer until the baseline was stable;

[0199] The column was eluted with the eluent. When an elution peak appeared, 3 mL of the effluent was collected in each tube until the elution peak returned to the baseline, and the pH value of the collected effluent in each tube was immediately adjusted to 7.0 with 1.0 mol / L Tris-HCL buffer (pH 9.0);

[0200] Elute the column with 3 to 5 times the volume of the column regeneration solution at a flow rate of 1 mL / min;

[0201] Re-equilibrate the column with 5-10 column volumes of loading buffer until the pH of the effluent is 7.4, and then re-equilibrate with 10 column volumes of pure water;

[0202] Collect the eluate in each tube.

[0203] (2) SDS-PAGE to identify the purity and stability of IgM antibodies:

[0204] The modified IgM antibody solution was tested for accelerated stability at 37°C for 0, 3, 7, and 14 days.

[0205] The samples were diluted or concentrated with PBS to the specified concentration, mixed with SDS (1:1) to adjust the loading volume, denatured after mixing, and subjected to SDS-PAGE to identify the purity;

[0206] Heat in a water bath for 5 min, cool to room temperature, centrifuge at 12000 rpm for 1 min, and take the supernatant for later use;

[0207] Load 10 μL of the prepared protein sample into the sample well of the electrophoresis tank;

[0208] After loading, add buffer and cap;

[0209] The voltage of the concentrated gel part is 90V for 15-20 min, and the voltage of the separation gel part is 180V for 30-50 min;

[0210] The gel was stained with Coomassie Brilliant Blue R250 for 15 min;

[0211] Add decolorizing solution and boil in a microwave oven for 1 min to accelerate decolorization. Repeat 3 times until the base color of the gel is almost colorless.

[0212] The results are as follows Figure 3 (Lane 1-9 are the thermal accelerated stability verification results of IgM antibody 1C5 on day 0, day 3, day 7 and day 14, and marker, IgM antibody 4B9 on day 0, day 3, day 7 and day 14, respectively) as shown in the present invention, the IgM antibody obtained by combining the CH1 fragment of the mouse IgG1 constant region with the mouse IgM constant region CH2 / 3 / 4 not only has good accelerated thermal stability, but also can achieve a purity of more than 95% based on the protein G one-step purification method.

[0213] 2.4 Sensitivity test results of IgG antibody and modified IgM antibody

[0214] The experiments in Sections 2.4-2.7 of this Example verified the conventional IgG system (specifically magnetic beads-IgG 4B9 / acridinium ester-IgG IC5, referred to as IgG antibody) and the IgM system (specifically magnetic beads-IgM 4B9 / acridinium ester-IgM IC5, referred to as IgM antibody).

[0215] The IgG antibody was diluted 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times, 6400 times, 12800 times, 25600 times, 51200 times, 102400 times, 204800 times, and 409600 times with Tris buffer; the modified IgM antibody was diluted 2000 times, 4000 times, 8000 times, 16000 times, 32000 times, 64000 times, 128000 times, 256000 times, 512000 times, 1024000 times, 2048000 times, 4096000 times, and 8192000 times with Tris buffer, and the sensitivity was detected on the chemiluminescence platform. The results are shown in Table 2 below.

[0216] Table 2

[0217]

[0218] The results show that the measured value of IgG antibody raw material can reach up to 1100000, and the measured value of IgM antibody raw material can reach up to 5800000; the linear maximum point of IgG antibody raw material is about 600000; the linear maximum point of IgM antibody raw material is about 2500000. In other words, the above results show that the sensitivity of IgG antibody prepared by the present invention can reach a higher level and can meet the needs of detection. The sensitivity of IgM antibody obtained by the modification of the present invention is further improved and can be controlled to 4-5 times that of IgG antibody.

[0219] 2.5 Precision testing of modified IgM antibodies

[0220] The modified IgM antibody was diluted 4500 times, 20000 times and 50000 times with Tris buffer, and the measurement was repeated 10 times for 1 negative reference, 1 positive reference and 1 minimum detection limit sample, and the coefficient of variation (CV(%) = SD (standard deviation) / mean (average value) × 100%) should not be greater than 10.0%.

[0221] Table 3

[0222]

[0223] The results are shown in Table 3. The IgM antibodies transformed by the present invention have positive test results for positive repeatability reference products and minimum detection limit reference products, and negative test results for negative repeatability reference products. The coefficient of variation CV can be controlled to be less than 5%.

[0224] 2.6 Batch-to-batch variation detection of modified IgM antibodies

[0225] Three batches of small-scale test samples were produced respectively, and 25 negative samples were tested simultaneously using the chemiluminescence method, and the relative deviation was calculated.

[0226] Table 4

[0227]

[0228] The results are shown in Table 4. The IgM antibodies transformed by the present invention have small batch differences, and the relative deviation can be controlled within ±5%.

[0229] 2.7 Stability testing of modified IgM antibodies

[0230] The stability of the modified IgM antibody was investigated on day 0, day 7 at 4°C, and day 7 at 37°C.

[0231] Table 5

[0232]

[0233] The results are shown in Table 5, and the IgM antibody obtained by the modification of the present invention meets the requirements of stability.

[0234] Embodiment 3

[0235] 3.1 Component 1 - Preparation of magnetic beads-streptavidin conjugate:

[0236] Use JSR Carboxyl MS160 magnetic beads, mix the beads, take 50 μL of carboxyl magnetic beads into a 1 mL centrifuge tube, add 300 μL of 0.01 M PBS buffer, vortex and shake for 5 min; magnetically absorb, remove the supernatant, and repeat twice;

[0237] Add 150 μL 100 mM pH 6.5 MES to dissolve and vortex for 30 seconds to mix;

[0238] Add 100 μL of freshly prepared 10 mg / mL EDC and 100 μL of 10 mg / mL sulfo-NHS, vortex again to mix, and activate at 25-37°C for 20 min-1 h, preferably at 25°C for 20 min;

[0239] Add 300 μL of 0.01 M PBS buffer and vortex for 30 s;

[0240] Magnetic suction, remove the supernatant, repeat 3 times;

[0241] Add 50 μg-300 μg streptavidin, add 500 μL 0.01 M PBS buffer, add 0.05% Tween20, mix gently, use a vertical mixer to mix by inversion, and couple at 25°C for 2 h;

[0242] Magnetic aspiration, removal of supernatant, re-suspending in 200 μL PBST solution (pH 7.2, containing 1% BSA), and blocking at 25°C for 1 h;

[0243] The cells were magnetically aspirated, the supernatant was removed, and the cells were washed three times with 200 µL PBS solution (pH 7.2) each time. The cells were then resuspended in 0.01 M PBS, 0.05% Tween 20, and 5% sucrose solution and stored at 4 °C.

[0244] 3.2 Component 2 - Preparation of IgM antibody 4B9-biotin conjugate:

[0245] Antibody preparation: Zeba desalting column, cut the bottom of the column, place it in a 2mL EP tube, loosen the column cap, centrifuge at 1500g for 1min, remove the liquid in the tube; add 300μL PBS buffer and centrifuge, repeat 3 times, remove the liquid in the tube each time; add the sample to the column; 1500g for 2min, collect the solution in the 1.5mL EP tube, which is the target product.

[0246] The replacement buffer was 0.1 M PBS pH 7.2 using a Zeba desalting column, and the concentration of IgM antibody 4B9 was adjusted to 2.0 mg / mL;

[0247] Take 500 μL of IgM antibody 4B9 and 0.33-6.66 μL of 10mM / L NHS-biotin (feed ratio is 1:2-1:10), vortex mix, and then rotate and mix at 25℃-37℃ for 30min-2h; preferably at 25℃ for 30min;

[0248] 300 μL of 0.1 M PBS buffer was added for dialysis and washed twice to obtain the biotin-modified antibody IgM antibody 4B9.

[0249] 3.3 Preparation of Component 3 - IgM Antibody 1C5-Acridinium Ester Conjugate:

[0250] Dilute IgM antibody 1C5 to 2 mg / mL using 0.01M pH 7.2 PBS;

[0251] Take 1 mL of IgM antibody 1C5 and add 400 μL of 0.25 mg / mL acridinium ester solution (feed ratio is 1:10-1:30) to react for 30 minutes, and then add lysine to terminate for 20 minutes;

[0252] Add to the dialysis bag and dialyze in 0.01M pH7.2 PBS, changing the dialysate every 2-3 hours;

[0253] The IgM antibody 1C5-acridinium ester conjugate was stored in a storage solution of 0.05 mol / L Tris pH 7.2, 2% BSA, 0.05% Tween-20, and 0.2% PC300.

[0254] 3.4 Capture and detection of pathogenic microorganisms in samples:

[0255] According to the calculation of the antibody mass, component 1 (i.e., magnetic beads-streptavidin conjugate) and component 2 (i.e., IgM antibody 4B9-biotin conjugate) are premixed at a ratio of 1:2 to form a magnetic bead-IgM antibody 4B9 complex;

[0256] 20 μL of magnetic beads-IgM antibody 4B9 complex was added to 100 μL of sample and mixed and incubated at 37°C for 10 min;

[0257] Magnetic suction, remove supernatant;

[0258] Add 200 μL of 0.1 M PBS buffer and wash twice;

[0259] Magnetic suction, remove supernatant;

[0260] Add 20 μL of pure water to resuspend and obtain magnetic beads-IgM antibody 4B9-antigen conjugate.

[0261] The magnetic beads-IgM antibody 4B9-antigen conjugate and component 3 (i.e., IgM antibody 1C5-acridinium ester conjugate) were incubated with rotation mixing at 37°C for 15 min to form a solid phase antibody-antigen-antibody sandwich complex.

[0262] Magnetic suction was performed, the supernatant was removed, 200 μL PBST solution (pH 7.2, containing 1% BSA) was added for resuspending, and the cells were washed three times repeatedly;

[0263] After adding pre-excitation solution and excitation solution, the instrument detects the number of photons and makes a numerical interpretation.

[0264] The collected clinical samples were diluted 25600, 51200 and 102400 times using sample diluent, and the measurement was repeated three times to calculate the average value. Then, the ratio of biotin signal amplification was calculated based on these data.

[0265] In this example, the IgG antibody coupled to biotin was verified by the magnetic beads-streptavidin / IgG 4B9-biotin / acridinium ester-IgG IC5 system.

[0266] Table 6

[0267]

[0268] The above results show that the sensitivity of the IgG antibody prepared by the present invention can be further amplified by biotin amplification, and the minimum detection limit is 1:25600; and the sensitivity of the IgM antibody modified by the present invention amplified by biotin can be increased to 16-20 times that of the IgG antibody, and the minimum detection limit is 1:102400.

[0269] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. An IgG antibody that specifically binds to a coxsackie virus, characterized in that: The amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region of the IgG antibody are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

2. An IgM antibody, characterized in that: The IgM antibody is transformed from the IgG antibody according to claim 1, and the IgM antibody comprises a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region, and the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the heavy chain constant region comprises a first constant region, a second constant region, a third constant region and a fourth constant region, wherein the first constant region is derived from a mouse IgG antibody, the amino acid sequence of the first constant region is shown in SEQ ID NO: 13, and the second constant region, the third constant region and the fourth constant region are derived from a mouse IgM antibody.

3. The IgM antibody according to claim 2, wherein The amino acid sequences of the second constant region, the third constant region and the fourth constant region are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

4. The IgM antibody according to claim 2, characterized in that The IgM antibody is coupled to magnetic beads.

5. The IgM antibody according to claim 2, characterized in that The IgM antibody was labeled with an acridinium ester.

6. The IgM antibody according to claim 2, characterized in that The IgM antibody is conjugated to biotin.

7. A method for improving the detection sensitivity of pathogenic microorganisms for non-disease diagnosis purposes, characterized in that: The following steps are involved: Obtaining samples to be tested; Adding a detection reagent to the sample to be detected; Using a chemiluminescence method to detect the concentration of Coxsackie virus in the sample to be detected; Wherein the detection reagent comprises the IgM antibody as described in any one of claims 2-6.

8. The method according to claim 7, characterized in that The detection reagent specifically includes: Magnetic beads-streptavidin conjugate; a biotin-modified primary IgM antibody; and acridinium ester-labeled secondary IgM antibody; wherein the first IgM antibody is the IgM antibody according to claim 2; the second IgM antibody comprises a heavy chain constant region, a light chain constant region, a heavy chain variable region and a light chain variable region, and the amino acid sequences of the complementary determining regions CDRH1-CDRH3 of the heavy chain variable region are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the amino acid sequences of the complementary determining regions CDRL1-CDRL3 of the light chain variable region are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the heavy chain constant region comprises a first constant region, a second constant region, a third constant region and a fourth constant region, wherein the first constant region is derived from a mouse IgG antibody, the amino acid sequence of the first constant region is shown in SEQ ID NO: 13, and the second constant region, the third constant region and the fourth constant region are derived from a mouse IgM antibody.

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