CN111793122B - Method for opening V-domain of natural beta 2 glycoprotein I, antigen prepared by using method, application and kit - Google Patents

Method for opening V-domain of natural beta 2 glycoprotein I, antigen prepared by using method, application and kit Download PDF

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CN111793122B
CN111793122B CN202010690439.7A CN202010690439A CN111793122B CN 111793122 B CN111793122 B CN 111793122B CN 202010690439 A CN202010690439 A CN 202010690439A CN 111793122 B CN111793122 B CN 111793122B
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glycoprotein
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CN111793122A (en
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徐顺澍
杨晶仪
沈佳燕
邓京
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Zhuhai Livzon Diagnostics Inc
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Abstract

The invention provides a method for opening the V-th structural domain of a natural beta 2 glycoprotein I, an antigen prepared by the method, an application and a kit, and relates to the technical field of antigen preparation. The method for opening the V domain of the natural beta 2 glycoprotein I comprises the following steps: carrying out first dialysis on the natural beta 2 glycoprotein I, then mixing the natural beta 2 glycoprotein I with the bacterial lipoid A, and reacting to obtain the beta 2 glycoprotein I with the V structural domain opened; the dialysate used for the first dialysis comprises buffer A; the buffer solution A contains sodium ions with the concentration of 0.75-2M, and the pH value is 9-12. Compared with the natural beta 2 glycoprotein I antigen and the recombinant beta 2 glycoprotein I antigen, the sensitivity of the beta 2 glycoprotein I opened by the V structural domain prepared by the method is greatly improved.

Description

Method for opening V-domain of natural beta 2 glycoprotein I, antigen prepared by using method, application and kit
Technical Field
The invention relates to the technical field of antigen preparation, in particular to a method for opening the V-th domain of a natural beta 2 glycoprotein I, an antigen prepared by the method, application and a kit.
Background
The anti-beta 2 glycoprotein I antibody is an important laboratory index in the classification standard of the anti-phospholipid syndrome, is widely applied clinically at present, and becomes one of the most common autoantibody detection projects in clinical laboratories. The beta 2 glycoprotein I is the most central key raw material of the antibody kit for detecting the beta 2 glycoprotein I.
The molecular weight of the natural beta 2 glycoprotein I is about 50kD, the natural beta 2 glycoprotein I consists of 326 amino acid residues and comprises 5 structural domains, and D I, D II, D III, D IV and D V regions are arranged from an amino terminal to a carboxyl terminal in sequence. The first 4 domains of β 2 glycoprotein I are highly homologous, each containing about 60 amino acid residues, and each region has four cysteines (Cys), which make up two disulfide bonds. The V-domain (carboxy-terminus) of β 2 glycoprotein I contains 82 amino acids, of which 6 cysteines make up three pairs of disulfide bonds. Studies have shown that the V domain of β 2 glycoprotein I binds to negatively charged substances, such as cardiolipin, and is recognized by anti- β 2 glycoprotein I antibodies or anti-cardiolipin antibodies, which are key causes of thrombosis in antiphospholipid syndrome.
The natural beta 2 glycoprotein I is in the blood, and the I domain and the V domain form a closed loop structure, as shown in fig. 1; when autoimmunity is problematic, the beta 2 glycoprotein I is combined with phospholipids, and the ring structure is opened, as shown in FIG. 2, to expose hidden antigenic determinants, thereby causing antiphospholipid syndrome.
The traditional detection methods for detecting anti-beta 2 glycoprotein I antibodies are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLIA). The key raw material used by the method is natural beta 2 glycoprotein I antigen or recombinant beta 2 glycoprotein I antigen. And the natural extracted untreated beta 2 glycoprotein I antigen is a closed loop structure, the key V structural domain is not opened, the sensitivity is low, and the omission factor is easily caused. The beta 2 glycoprotein I has a plurality of disulfide bonds to form a complex space conformation, and the recombinant beta 2 glycoprotein I antigen cannot reproduce the complex conformation, so the reactivity is poor, the stability is inferior to that of the natural beta 2 glycoprotein I antigen, and the cost is far higher than that of the natural beta 2 glycoprotein I antigen.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for opening the V domain of the natural beta 2 glycoprotein I, which is simple and efficient and can specifically open the V domain of the natural beta 2 glycoprotein I.
The second objective of the invention is to provide a method for preparing a V domain-opened beta 2 glycoprotein I antigen by using the method, wherein the sensitivity of the V domain-opened beta 2 glycoprotein I antigen prepared by using the method to an anti-beta 2 glycoprotein I antibody is greatly improved compared with that of a natural beta 2 glycoprotein I antigen and a recombinant beta 2 glycoprotein I antigen.
The third purpose of the invention is to provide the application of the V domain opened beta 2 glycoprotein I antigen in detection.
It is a fourth object of the present invention to provide a kit comprising a V domain opened β 2 glycoprotein I antigen.
In order to solve the technical problems, the following technical scheme is adopted:
in a first aspect, the present invention provides a method for opening the V domain of a native β 2 glycoprotein I, comprising the steps of:
carrying out first dialysis on the natural beta 2 glycoprotein I, then mixing the natural beta 2 glycoprotein I with the bacterial lipoid A, and reacting to obtain the beta 2 glycoprotein I with the V structural domain opened;
wherein the dialysate used for the first dialysis comprises buffer A;
the buffer solution A contains sodium ions with the concentration of 0.75-2M, and the pH value is 9-12.
Further, the buffer solution A is a HEPES buffer solution, the sodium ion concentration of the HEPES buffer solution is 1.15M, and the pH value is 11.5.
Furthermore, the concentration of the HEPES buffer solution is 10-40mM, and preferably 20 mM.
Further, the dialysis process time is 36-60h, preferably 48 h.
Further, the natural beta 2 glycoprotein I is firstly dialyzed for the first time, then dialyzed for the second time, and then mixed and reacted with the bacterial lipoid A;
the dialysate used for the second dialysate is buffer B, wherein: the concentration of sodium ions in the buffer solution B was 0.15M, and the pH was 7.4. The second dialysis changes the environment of the beta 2 glycoprotein I from pH 9-12 and sodium ion concentration of 0.75-2M to a milder environment with pH 7.4 and sodium ion concentration of 0.15M. The second dialysis can be replaced by desalting process, but desalting will cause protein loss, resulting in less protein produced than dialysis, so the present scheme changes the environment of the beta 2 glycoprotein I antigen from pH 9-12, sodium ion concentration of 0.75-2M, to a milder environment of pH 7.4, sodium ion concentration of 0.15M, preferably using dialysis.
Further, the buffer B is HEPES buffer with the concentration of 20 mM;
the dialysis time of the second dialysis was 8 h.
Furthermore, the mass ratio of the beta 2 glycoprotein I prepared by the second dialysis to the bacterial lipid A is 100-750: 1;
preferably, the mass ratio of β 2 glycoprotein I to bacterial lipid a prepared by the second dialysis is 500: 1.
In a second aspect, the invention provides a V domain opened β 2 glycoprotein I antigen.
In a third aspect, the invention provides the use of a V domain opened β 2 glycoprotein I antigen in the detection of β 2 glycoprotein I antibodies.
In a fourth aspect, the present invention provides a kit comprising a V domain-opened β 2 glycoprotein I antigen.
Compared with the prior art, the method for opening the V-th structural domain of the natural beta 2 glycoprotein I and the antigen, the application and the kit prepared by the method have the following beneficial effects:
the invention provides a method for opening the V structural domain of a natural beta 2 glycoprotein I, which comprises the steps of firstly dialyzing the natural beta 2 glycoprotein I under the extreme conditions of pH 9-12 and sodium ion concentration of 0.75-2M, changing the spatial structure conformation of the natural beta 2 glycoprotein I, and opening the binding site part of the I structural domain and the V structural domain in the natural beta 2 glycoprotein I; after the second dialysis, the environment of the beta 2 glycoprotein I with the partially opened binding site of the I domain and the V domain is changed to pH 7.4 and the sodium ion concentration is 0.15M, and the milder reaction environment is more suitable for bacterial lipid A to completely open the binding site of the I domain and the V domain of the beta 2 glycoprotein I and is beneficial to the restoration of the spatial structure of other beta 2 glycoprotein I except the binding site of the I domain and the V domain. And mixing the beta 2 glycoprotein I with the binding site part opened by the I domain and the V domain after the second dialysis with the bacterial lipoid A, reacting, and completely opening the connection between the V domain and the I domain of the beta 2 glycoprotein I to prepare the beta 2 glycoprotein I with the opened V domain. The method is simple and efficient, and can specifically open the V structural domain of the natural beta 2 glycoprotein I without damaging the protein structures of other regions.
The invention provides a beta 2 glycoprotein I antigen with an opened V-th structural domain, because the V-th structural domain of the beta 2 glycoprotein I antigen is opened, compared with a natural beta 2 glycoprotein I antigen which is a closed ring structure, the binding site of the beta 2 glycoprotein I antigen and an anti-beta 2 glycoprotein I antibody is more fully exposed, and the steric hindrance when the beta 2 glycoprotein I antigen is combined with the anti-beta 2 glycoprotein I antibody is reduced, so that the sensitivity of the antigen is greatly improved compared with the sensitivity of the natural beta 2 glycoprotein I antigen, and experiments prove that the sensitivity of the beta 2 glycoprotein I antigen with an opened V-th structural domain prepared by the method for resisting the beta 2 glycoprotein I antibody is greatly improved compared with the sensitivity of the natural beta 2 glycoprotein I antigen and the sensitivity of a recombinant beta 2 glycoprotein I antigen.
The beta 2 glycoprotein I antigen with the opened V structural domain provided by the invention is applied to the detection of a beta 2 glycoprotein I antibody, and the sensitivity is high.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic structural diagram of a native beta 2 glycoprotein I;
FIG. 2 is a schematic diagram of the structure of domain V-opened beta 2 glycoprotein I.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The beta 2 glycoprotein I belongs to antigen proteins, and because the antigen and the antibody are relative concepts, and the invention adopts the detection principle of antigen and antibody specific recognition, the beta 2 glycoprotein I in the invention is understood as beta 2 glycoprotein I protein, and can also be understood as beta 2 glycoprotein I antigen capable of being specifically recognized and combined with an anti-beta 2 glycoprotein I antibody.
In a first aspect, the present invention provides a method for opening the V domain of a native β 2 glycoprotein I, comprising the steps of:
carrying out first dialysis on the natural beta 2 glycoprotein I, then mixing the natural beta 2 glycoprotein I with the bacterial lipoid A, and reacting to obtain the beta 2 glycoprotein I with the V structural domain opened; wherein the dialysate for the first dialysis comprises a buffer solution A, wherein the buffer solution A contains sodium ions with the concentration of 0.75-2M, and the pH value is 9-12.
The first dialysis referred to in the present invention includes only one dialysis or a plurality of dialysis, and in the case of a plurality of dialysis, each dialysis is independent of each other, and each dialysis may use a dialysate of different components or may use the same dialysate.
Firstly, selecting natural beta 2 glycoprotein I with the molecular weight of 45-60 kDa, wherein the molecular weight is 50kDa in the invention. The structure of the natural beta 2 glycoprotein I is shown in figure 1, the natural beta 2 glycoprotein I is dialyzed for the first time, the dialyzate contains a buffer solution A, the buffer solution A contains sodium ions with the concentration of 0.75-2M, and the pH value is 9-12. Under the conditions that the sodium ion concentration is 0.75-2M and the pH is 9-12, the structural conformation of the natural beta 2 glycoprotein I is changed, and the connection between the V structural domain and the I structural domain of the natural beta 2 glycoprotein I is opened; the dialyzed beta 2 glycoprotein I is then mixed with bacterial lipid A and reacted to completely open the junction of the V domain and the I domain of beta 2 glycoprotein I to give a V domain-opened beta 2 glycoprotein I having the structure shown in FIG. 2.
Buffer A is HEPES buffer, namely, Hydroxyethyl piperazine ethanethiosulfonic acid (2- [4- (2-hydroxyethaneyl) -1-piperazinyl ] ethanesulfonic acid) buffer, which is prepared by dissolving Hydroxyethyl piperazine ethanethiosulfonic acid (2- [4- (2-hydroxyethaneyl) -1-piperazinyl ] ethanesulfonic acid) in ultrapure water. The concentration of sodium ions in buffer A may typically, but not exclusively, be 0.75M, 1M, 1.25M, 1.5M, 1.75M or 2M. The pH of buffer A may typically, but not exclusively, be 9.0, 9.5, 10.0, 10.5, 11.0, 11.5 or 12.0. In this embodiment, buffer a is preferably a HEPES buffer with a sodium ion concentration of 1.15M and a pH of 11.5.
The concentration of the HEPES buffer is 10-40mM, and in the present invention, the concentration of the HEPES buffer may typically, but not limited to, be 10mM, 20mM, 30mM or 40mM, and preferably 20 mM.
The first dialysis process time is 36-60h, in the present invention, the first dialysis time can be typically but not limited to 36h, 42h, 48h, 54h or 60h, preferably 48 h.
Firstly, performing first dialysis on natural beta 2 glycoprotein I, then performing second dialysis, and then mixing the natural beta 2 glycoprotein I with bacterial lipoid A for reaction; the dialysate used for the second dialysate was buffer B, wherein the sodium ion concentration in buffer B was 0.15M and the pH was 7.4.
The environment of the beta 2 glycoprotein I subjected to the first dialysis is changed from the environment with the pH of 9-12 and the sodium ion concentration of 0.75-2M into the environment with the milder pH of 7.4 and the sodium ion concentration of 0.15M through the second dialysis, so that the beta 2 glycoprotein I antigen with the V structural domain and the I structural domain partially connected and opened after the first dialysis is more easily reacted with bacterial lipoid A, the connection point of the V structural domain and the I structural domain of the beta 2 glycoprotein I is completely opened, and the restoration of other spatial structures of the beta 2 glycoprotein I except the connection point of the V structural domain and the I structural domain is facilitated. Under the conditions of pH 7.4 and sodium ion concentration of 0.15M, the reaction efficiency of the bacterial lipoid A is higher, the other beta 2 glycoprotein I protein structures except the binding site of the V domain and the I domain are not damaged, and compared with a recombinant beta 2 glycoprotein I antigen and a natural beta 2 glycoprotein I antigen, the sensitivity of the beta 2 glycoprotein I with the opened V domain to an anti-beta 2 glycoprotein I antibody is greatly improved.
The mass ratio of the beta 2 glycoprotein I prepared by the second dialysis to the bacterial lipid A is 100-750:1, the mass ratio of the beta 2 glycoprotein I to the bacterial lipid A can be typically, but not limited to, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1 or 750:1, and preferably, the mass ratio of the beta 2 glycoprotein I prepared by the second dialysis to the bacterial lipid A is 500: 1.
In the invention, the beta 2 glycoprotein I and the bacterial lipoid A can react more efficiently by further optimizing and adjusting the mass ratio of the beta 2 glycoprotein I to the bacterial lipoid A, and unnecessary waste of raw materials is avoided.
It should be noted that the mixing manner of the dialyzed β 2 glycoprotein I and the bacterial lipid a includes, but is not limited to, shaking a mixer for mixing, or mixing by other mixing methods known to those skilled in the art. For example, the mixed β 2 glycoprotein I and bacterial lipid a are placed in a shaking mixer, and the operating parameters are set as follows: the temperature is 18-40 ℃, the rotating speed is 100-1500 rpm, and the mixture can be fully mixed and reacted after shaking for 0.5-6 h.
After the beta 2 glycoprotein I and the bacterial lipoid A are uniformly mixed and react, a high-purity beta 2 glycoprotein I with the V structural domain opened is obtained through a separation and purification step. The means for separation and purification includes, but is not limited to, chromatographic purification, or other means for separation and purification suitable for proteins known to those skilled in the art. The column for chromatographic purification includes, but is not limited to, a HiTrap chelating column, or a chromatographic column known to those skilled in the art that can be used to separate the β 2 glycoprotein I from bacterial lipid a.
The method is simple and efficient, and can specifically open the V structural domain of the natural beta 2 glycoprotein I antigen. Firstly, performing primary dialysis on natural beta 2 glycoprotein I under the extreme conditions of pH 9-12 and sodium ion concentration of 0.75-2M, changing the spatial structure conformation of the natural beta 2 glycoprotein I, and partially opening the binding site of an I structural domain and a V structural domain in the natural beta 2 glycoprotein I; then after the second dialysis, the environment of the beta 2 glycoprotein I with the binding site of the I domain and the V domain partially opened is changed into the environment with the pH value of 7.4 and the sodium ion concentration of 0.15M, and the milder reaction environment is more suitable for bacterial lipid A to completely open the binding site of the I domain and the V domain of the beta 2 glycoprotein I antigen and is beneficial to restoring the spatial structure of other proteins of the beta 2 glycoprotein I except the binding site of the I domain and the V domain. And mixing the beta 2 glycoprotein I with the binding site part opened by the I domain and the V domain after the second dialysis with the bacterial lipoid A, reacting, and completely opening the connection between the V domain and the I domain of the beta 2 glycoprotein I to prepare the beta 2 glycoprotein I with the opened V domain. The method is simple and efficient, and can specifically open the V structural domain of the natural beta 2 glycoprotein I without damaging the protein structures of other regions.
Because the V-th domain of the beta 2 glycoprotein I antigen is opened, compared with a closed loop structure of the natural beta 2 glycoprotein I antigen, the binding site of the beta 2 glycoprotein I antigen and the anti-beta 2 glycoprotein I antibody is more fully exposed, and the steric hindrance when the beta 2 glycoprotein I antigen is bound with the anti-beta 2 glycoprotein I antibody is reduced, so that the sensitivity of the antigen is greatly improved compared with the natural beta 2 glycoprotein I antigen, and experiments confirm that the sensitivity of the beta 2 glycoprotein I antigen with the V-th domain opened, which is prepared by using the method, on the anti-beta 2 glycoprotein I antibody is greatly improved compared with the natural beta 2 glycoprotein I antigen and the recombinant beta 2 glycoprotein I antigen.
Preferably, the concentration of the HEPES buffer solution is 10-40mM, and preferably 20 mM.
In the present invention, the temperature of the first dialysis and the second dialysis is 2 to 25 ℃, and the temperature of the first dialysis and the second dialysis can be typically, but not limited to, 2 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃ or 25 ℃, preferably 2 to 8 ℃.
It should be noted that there are various ways of mixing β 2 glycoprotein I with bacterial lipid a, for example, β 2 glycoprotein I is dissolved in buffer B before bacterial lipid a is added; alternatively, bacterial lipid A can be dissolved in buffer B before the addition of β 2 glycoprotein I. For the former, one possible mixing approach is: and (3) carrying out second dialysis on the beta 2 glycoprotein I subjected to the first dialysis in a buffer solution B containing sodium ions with the concentration of 0.15M and the pH value of 7.4, and adding the bacterial lipoid A into the beta 2 glycoprotein I solution subjected to the second dialysis after the dialysis is completed.
In some preferred embodiments, the mixing and reacting further comprise a preservation step; the preservation step is to dilute the beta 2 glycoprotein I in a preservation solution to 0.01-5 mg/mL, preferably 0.5-1.5 mg/mL.
In the present invention, the storage concentration of β 2 glycoprotein I may be typically, but not limited to, 0.01mg/mL, 0.02mg/mL, 0.04mg/mL, 0.06mg/mL, 0.08mg/mL, 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1mg/mL, 2mg/mL, 4mg/mL or 5mg/mL, preferably 0.5 to 1.5 mg/mL.
In the present invention, the preservation temperature is-85 to-60 deg.C, and the preservation temperature may be typically, but not limited to, -85 deg.C, -80 deg.C, -75 deg.C, -70 deg.C, -65 deg.C or-60 deg.C, and preferably-80 deg.C.
In the invention, the preservation solution is a solution which can maintain the stability of the prepared protein and has a protection effect on the protein, the beta 2 glycoprotein I is preserved by adopting the method, which is beneficial to keeping the activity of the protein and avoiding the closure of the I structural domain and the V structural domain of the prepared beta 2 glycoprotein I.
In a second aspect, the present invention provides a β 2 glycoprotein I antigen with an open V domain prepared by the above method, wherein, as the V domain of the β 2 glycoprotein I antigen is open, the binding site of the β 2 glycoprotein I antigen and the anti- β 2 glycoprotein I antibody is more fully exposed than the closed loop structure of the natural β 2 glycoprotein I antigen, and the steric hindrance when the β 2 glycoprotein I antigen is bound to the anti- β 2 glycoprotein I antibody is reduced, so that the sensitivity of the antigen is greatly improved compared with the sensitivity of the natural β 2 glycoprotein I antigen, and experiments confirm that the sensitivity of the β 2 glycoprotein I antigen with an open V domain prepared by the above method against the anti- β 2 glycoprotein I antibody is greatly improved compared with the sensitivity of the natural β 2 glycoprotein I antigen and the recombinant β 2 glycoprotein I antigen.
In a third aspect, the invention provides the use of a V domain opened β 2 glycoprotein I antigen in the detection of β 2 glycoprotein I antibodies.
The application of the V-domain opened beta 2 glycoprotein I antigen comprises the application of the antigen in technical platforms such as colloidal gold, enzyme-linked immunosorbent assay (ELISA), immunochromatography, immunoturbidimetry, chemiluminescence, time resolution and the like, and comprises the application of the antigen in solid phase carriers such as nitrocellulose membranes (NC membranes), filter papers, ELISA96 pore plates, latex microspheres, magnetic particles/magnetic beads and the like, wherein the latex microspheres and the magnetic particles/magnetic beads comprise groups such as amino groups, carboxyl groups, streptavidin, Tosyl (Tosyl), EPOXY groups (EPOXY) and the like which are not modified. Including but not limited to reaction systems in which biotin, FITC, etc., labeled therewith, are used to enhance the signal or assist in labeling.
In a fourth aspect, the present invention provides a kit comprising a V domain-opened β 2 glycoprotein I antigen.
The beta 2 glycoprotein I antigen with the V structural domain opened, which is prepared by the method of the invention, can be used for preparing an anti-beta 2 glycoprotein I antibody detection kit by enzyme-linked immunosorbent assay (ELISA) or chemiluminescence assay (CLIA). Compared with a kit prepared by using an untreated natural beta 2 glycoprotein I antigen and a kit prepared by using a recombinant beta 2 glycoprotein I antigen, the beta 2 glycoprotein I antigen treated by the method has strong reactivity and high sensitivity, and the cost is greatly reduced.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
A method of opening the V domain of a native β 2 glycoprotein I comprising the steps of:
step 1, selecting natural human-derived beta 2 glycoprotein I with the molecular weight of 50 kDa.
Step 2. native β 2 glycoprotein I was dialyzed against 20mM HEPES (hydroxyethylpiperazine ethanethiosulfonic acid) buffer containing 1.15M NaCl, pH 11.5, at 4 ℃ for 48 hours, and exchanged once at 24 hours.
Step 3. the β 2 glycoprotein I obtained in step 2 was dialyzed against 20mM HEPES buffer containing 0.15M NaCl at pH 7.4 at 4 ℃ for 8 hours.
And 4, adding the bacterial lipoid A into the beta 2 glycoprotein I obtained in the step 3, wherein the mass ratio of the obtained beta 2 glycoprotein I to the bacterial lipoid A is 500:1, placing the mixture into a shaking and mixing device, and reacting for 1 hour at 37 ℃ and 1000 rpm.
And 5, separating and purifying the beta 2 glycoprotein I obtained in the step 4 by using a HiTrap chelate column.
And 6, quantifying the beta 2 glycoprotein I purified in the step 5 by using an ultraviolet spectrophotometer, diluting the solution to 1mg/mL by using 20mM HEPES buffer solution containing 0.15M NaCl and having the pH value of 7.0, and storing the solution at the temperature of-80 ℃.
The method for quantifying the ultraviolet spectral width comprises the following steps:
step 1: after purification, the absorbance of the beta 2 glycoprotein I is measured by an ultraviolet spectrophotometer.
Step 2: and preparing standard substance points by BSA, and respectively measuring the absorbance of each standard substance point to establish a standard curve.
And step 3: and (3) bringing the absorbance of the purified beta 2 glycoprotein I obtained in the step (1) into a standard curve to obtain the concentration of the beta 2 glycoprotein I.
And (3) coating the prepared beta 2 glycoprotein I antigen with carboxyl magnetic beads by a chemiluminescence method, and then detecting the reactivity of the carboxyl magnetic beads.
The coating method comprises the following steps:
step 1: 0.1ml of 10mg/ml carboxyl magnetic beads was removed by magnetic separation and the supernatant was aspirated.
Step 2: 0.1ml of 0.1M MES buffer solution with pH5.0 was added, and then 10ug of the above-prepared beta 2 glycoprotein I antigen was added thereto, followed by mixing well and shaking at room temperature for 30 minutes.
And step 3: EDC was dissolved in MES buffer described in step 2 at a concentration of 10mg/ml and was ready for use.
And 4, step 4: and (3) adding 10ul of the EDC solution obtained in the step (3) into the magnetic beads obtained in the step (2), fully and uniformly mixing, and shaking at room temperature for 3 hours.
And 5: the supernatant was aspirated by magnetic separation.
Step 6: 0.1ml of 25mM TBS-T solution was added to mix the beads well and washed.
And 7: and repeating the step 5 and the step 6 three times.
And 8: after the supernatant was aspirated, the magnetic beads were diluted to 0.2mg/ml with a magnetic bead diluent at 2-8 ℃ for further use.
The detection method comprises the following steps:
step 1: 50ul of sample is added into the magnetic beads coated by the method, and the mixture is fully and uniformly mixed. Incubate at 37 ℃ for 10 min.
Step 2: the supernatant was removed by magnetic separation, 200ul of washing solution was added and mixed well, and then the supernatant was removed by magnetic separation.
And step 3: repeat step 2, three times.
And 4, step 4: 100ul of mouse anti-human monoclonal antibody labeled with alkaline phosphatase was added and mixed well. Incubate at 37 ℃ for 10 min.
And 5: and (5) repeating the steps 2 and 3.
Step 6: 200ul of AMPPD (3- (2-spiroadamantane) -4-methoxy-4- (3-phosphonooxy) -phenyl-1, 2-dioxetane disodium salt) was added as substrate and read. Alkaline phosphatase excites the substrate to produce photons, the intensity of which is proportional to the concentration of the antibody to be detected. And substituting the luminous intensity into the standard curve to obtain the concentration of the antibody to be detected in the sample.
Comparative example 1
The concentration of anti- β 2 glycoprotein I antibody in the sample was measured under the same conditions as in example 1, using carboxyl magnetic beads coated with untreated native β 2 glycoprotein I antigen.
Comparative example 2
The concentration of anti-beta 2 glycoprotein I antibody in the sample was determined under the same conditions as in example 1 after coating carboxyl magnetic beads with untreated, untreated recombinant beta 2 glycoprotein I antigen (DIARECT, Inc., cat. No. 11300).
Comparative example 3
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of HEPES buffer in step 2 is 1 mM.
Comparative example 4
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of HEPES buffer in step 2 is 5 mM.
Example 2
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of HEPES buffer in step 2 is 10 mM.
Example 3
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of HEPES buffer in step 2 is 40 mM.
Comparative example 5
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of HEPES buffer in step 2 is 80 mM.
Comparative example 6
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of HEPES buffer in step 2 is 100 mM.
Comparative example 7
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of NaCl in the HEPES buffer in step 2 is 0.1M.
Comparative example 8
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of NaCl in the HEPES buffer in step 2 is 0.15M.
Comparative example 9
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of NaCl in the HEPES buffer in step 2 is 0.5M.
Example 4
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of NaCl in the HEPES buffer in step 2 is 0.75M.
Example 5
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of NaCl in the HEPES buffer in step 2 is 2M.
Comparative example 10
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the concentration of NaCl in the HEPES buffer in step 2 is 4M.
Comparative example 11
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 7.
Comparative example 12
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 7.4.
Comparative example 13
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 8.
Example 6
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 9.
Example 7
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 10.
Example 8
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 12.
Comparative example 14
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the pH of the HEPES buffer in step 2 is 12.5.
Comparative example 15
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the dialysis time in step 2 is 12 hours.
Comparative example 16
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the dialysis time in step 2 is 24 hours.
Example 9
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the dialysis time in step 2 is 36 hours.
Example 10
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the dialysis time in step 2 is 60 hours.
Comparative example 17
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the dialysis time in step 2 is 72 hours.
Comparative example 18
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that no bacterial lipid a is added in step 4.
Comparative example 19
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the mass ratio of β 2 glycoprotein I to bacterial lipid a in step 4 is 1000: 1.
Example 11
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the mass ratio of β 2 glycoprotein I to bacterial lipid a in step 4 is 750: 1.
Example 12
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the mass ratio of β 2 glycoprotein I to bacterial lipid a in step 4 is 250: 1.
Example 13
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the mass ratio of β 2 glycoprotein I to bacterial lipid a in step 4 is 250: 1.
Example 14
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the mass ratio of β 2 glycoprotein I to bacterial lipid a in step 4 is 100: 1.
Comparative example 20
This example provides a method for opening the V domain of native β 2 glycoprotein I, which differs from example 1 in that the mass ratio of β 2 glycoprotein I to bacterial lipid a in step 4 is 10: 1.
Data analysis
1. Selection of first dialysis buffer concentration
The light emitting value of a positive sample of a low-concentration HEPES buffer solution (1 mM-10 mM) is low, and the reactivity is slightly poor; the background luminescence value of a negative sample of a high-concentration HEPES buffer solution (40 mM-100 mM) is higher, and the negative and positive distinguishing degree is influenced. Therefore, 10-40mM is selected as the concentration of HEPES buffer solution used in the first dialysis, and 20mM is the optimum concentration.
TABLE 1 Effect of different HEPES concentrations on beta 2 glycoprotein I reactivity
Figure BDA0002588299770000171
2. Selection of NaCl concentration in first dialysis buffer
With the increase of NaCl use concentration (0.1-0.75M), the connection site of the V-th domain and the I-th domain of the beta 2 glycoprotein I is gradually opened, the light-emitting value of the positive sample is gradually increased, and the reactivity is gradually enhanced. From the results, it was found that the linker site opening effect of the V-domain and the I-domain of the β 2 glycoprotein I was the best and the reactivity was the best when the NaCl concentration was 1.15M. While the concentration is continuously increased, the luminous value of the NaCl (2M-4M) positive sample is gradually reduced, and the beta 2 glycoprotein I is gradually inactivated. Therefore, 0.75-2M is selected as the NaCl concentration in the HEPES buffer solution in the first dialysis, and 1.15 is the optimal concentration.
TABLE 2 Effect of different NaCl concentrations on the reactivity of beta 2 glycoprotein I
Figure BDA0002588299770000172
3. Selection of pH concentration of first dialysis buffer
The pH value is gradually increased from neutral to alkaline (pH7.0-11.5), the luminous value of the positive sample is gradually increased, the connecting site of the V-th domain and the I-th domain of the beta 2 glycoprotein I is gradually opened, and the reactivity is gradually enhanced. When the pH value is 12.5, the part of the beta 2 glycoprotein I is inactivated, and the luminous value of the positive sample is reduced. Therefore, 9-12 was selected as the pH of HEPES buffer in the first dialysis, and 11.5 was the optimum pH.
TABLE 3 Effect of different pH on the reactivity of beta 2 glycoprotein I
Figure BDA0002588299770000173
4. Selection of first dialysis time
With the increase of the first dialysis time (12 h-60 h), the light emitting value of the positive sample gradually increases, the connecting site of the V domain and the I domain of the beta 2 glycoprotein I is gradually opened, the reactivity is gradually enhanced, and the reactivities are close at 48h and 60 h. When the first dialysis time is further increased to 72 hours, the beta 2 glycoprotein I is partially inactivated, the luminous value of the positive sample is reduced, and the reactivity is reduced. Therefore, 36-60 hours are selected as the dialysis time for the first dialysis, and 48 hours is the optimal dialysis time.
TABLE 4 Effect of different dialysis times on the reactivity of beta 2 glycoprotein I
Figure BDA0002588299770000181
5. Selection of the ratio of beta 2 glycoprotein I to bacterial lipid A
When the bacterial lipoid A is not added, the connection site of the V-domain and the I-domain of the beta 2 glycoprotein I is not completely opened, the luminous value of a positive sample is very low, and the reactivity is very poor. With the increase of the dosage of the bacterial lipoid A (the mass ratio of the beta 2 glycoprotein I to the bacterial lipoid A is 750: 1-500: 1), the connecting site of the V-domain and the I-domain of the beta 2 glycoprotein I is gradually opened, the luminous value of the positive sample is gradually increased, and the reactivity is gradually enhanced. And with the further increase of the dosage of the bacterial lipoid A (the mass ratio of the beta 2 glycoprotein I to the bacterial lipoid A is 500: 1-100: 1), the light-emitting value of the positive sample has no obvious change. In view of cost, the mass ratio of the beta 2 glycoprotein I to the bacterial lipid A is 100-750:1, and the ratio is 500:1 is the optimum ratio.
TABLE 5 Effect of varying amounts of bacterial lipid A used on the reactivity of beta 2 glycoprotein I
Figure BDA0002588299770000182
From the above experimental groups, it is known that the synergistic effect of the five conditions of HEPES buffer concentration, NaCl concentration, pH, dialysis time and β 2 glycoprotein I and bacterial lipid a ratio obtained by the second dialysis in the first dialysis results in high sensitivity of the prepared V domain opened β 2 glycoprotein I against β 2 glycoprotein I antibodies, and any condition without using an optimal reaction condition results in poor sensitivity of the final β 2 glycoprotein I V domain against β 2 glycoprotein I antibodies due to insufficient opening, or poor sensitivity of the β 2 glycoprotein I antibodies due to damage to the remaining spatial structures of β 2 glycoprotein I except the binding site of the V domain and the I domain, thereby reducing β 2 glycoprotein I activity.
Performing a first dialysis of native β 2 glycoprotein I for 48 hours in an extreme environment of 20mM HEPES buffer at pH 11.5 and a sodium ion concentration of 1.15M, changing the spatial structural conformation of native β 2 glycoprotein I, and partially opening the binding site between domain I and domain V in native β 2 glycoprotein I; then after the second dialysis, the environment of the beta 2 glycoprotein I with the binding site of the I domain and the V domain partially opened is changed to a mild environment with pH 7.4, sodium ion concentration 0.15M and 20mM HEPES buffer solution, which is more suitable for bacterial lipid A to completely open the binding site of the I domain and the V domain of the beta 2 glycoprotein I antigen and restore the spatial structure of the residual protein of the beta 2 glycoprotein I except the binding site of the V domain and the I domain. And (3) contacting the beta 2 glycoprotein I with bacterial lipid a partially opened at the binding site of domain I and domain V after the second dialysis at a ratio of 500:1, reacting, and completely opening the connection of the V-th domain and the I-th domain of the beta 2 glycoprotein I to prepare the beta 2 glycoprotein I with the V-th domain opened. The method is simple and efficient, and can specifically open the V structural domain of the natural beta 2 glycoprotein I without damaging the protein structures of other regions.
6. Reactivity test
Reactivity was measured by chemiluminescence using the beta 2 glycoprotein I antigen coated carboxyl beads of comparative example 1 and example 1, respectively, using the same coating technique and coating concentration (10ug antigen/mg beads).
It can be seen that the β 2 glycoprotein I antigen of example 1, for samples at a concentration of 5-400RU/mL, had an emission value of about 4 times that of the untreated native β 2 glycoprotein I antigen after treatment with the method of the present invention.
Because the V domain of the beta 2 glycoprotein I antigen is opened, compared with the closed loop structure of the natural beta 2 glycoprotein I antigen, the binding site of the beta 2 glycoprotein I antigen and the anti-beta 2 glycoprotein I antibody is more fully exposed, and the steric hindrance caused when the beta 2 glycoprotein I antigen is combined with the anti-beta 2 glycoprotein I antibody is reduced, so that the antigen sensitivity is greatly improved compared with the natural beta 2 glycoprotein I antigen.
TABLE 6 beta 2 glycoprotein I antigen reactivity of example 1 and comparative example 1
Figure BDA0002588299770000201
7. Clinical compliance rate test
The beta 2 glycoprotein I antigens of example 1 and comparative example 1 (untreated native beta 2 glycoprotein I antigen), comparative example 2 (recombinant beta 2 glycoprotein I antigen) were coated with carboxymagnetic beads using chemiluminescence, respectively. When the serum of 122 patients with positive phospholipid syndrome and the serum of 131 patients with negative phospholipid syndrome are diagnosed, a large number of false negative samples can appear in the beta 2 glycoprotein I antigen of the comparative examples 1 and 2. 54 and 66 samples of the beta 2 glycoprotein I antigen positive samples of comparative example 1 and comparative example 2 were missed, respectively, and the anti-beta 2 glycoprotein I antibody sensitivity of the untreated native beta 2 glycoprotein I antigen was only 53.27%, and the anti-beta 2 glycoprotein I antibody sensitivity of the recombinant beta 2 glycoprotein I antigen was only 43.44%, which were 44.27% and 54.1% lower than those of example 1, respectively. The sensitivity of the V domain opened beta 2 glycoprotein I antigen prepared by the method is 97.54 percent, and is improved by nearly one time compared with the sensitivity of untreated natural beta 2 glycoprotein I antigen and recombinant beta 2 glycoprotein I antigen.
Because the method used in the present invention does not change the spatial structure of the β 2 glycoprotein I antigen for the β 2 glycoprotein I antigen, except for opening the binding site of the I domain and the V domain, the method has less change to the native β 2 glycoprotein I antigen compared to the recombinant β 2 glycoprotein I antigen, and thus the sensitivity of the anti- β 2 glycoprotein I antibody is greatly improved compared to the recombinant β 2 glycoprotein I antigen.
The sensitivity of the kit prepared from the beta 2 glycoprotein I antigen treated by the method is far higher than that of untreated natural beta 2 glycoprotein I antigen and recombinant beta 2 glycoprotein I antigen, which has great significance for improving the clinical detection rate of phospholipid-resistant syndrome.
TABLE 7 clinical compliance rates of beta 2 glycoprotein I antigen preparation kits for comparative examples 1,2 and example 1
Figure BDA0002588299770000211
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (8)

1. A method of opening the V domain of a native β 2 glycoprotein I comprising the steps of:
carrying out first dialysis on the natural beta 2 glycoprotein I, then mixing the natural beta 2 glycoprotein I with the bacterial lipoid A, and reacting to obtain the beta 2 glycoprotein I with the V structural domain opened;
wherein the dialysate used for dialysis is buffer solution A;
the buffer solution A contains sodium ions with the concentration of 0.75-2M, and the pH value of the buffer solution A is 9-12;
the buffer solution A is HEPES buffer solution;
the concentration of the HEPES buffer solution is 10-40 mM;
the first dialysis time is 36-60 h.
2. The method of opening the V domain of native β 2 glycoprotein I of claim 1, wherein the HEPES buffer has a sodium ion concentration of 1.15M and a pH of 11.5.
3. The method of opening the V domain of native β 2 glycoprotein I according to claim 1, wherein the HEPES buffer is at a concentration of 20 mM.
4. The method of opening the V domain of native β 2 glycoprotein I of claim 1, wherein the first dialysis time is 48 h.
5. The method for opening the Vth domain of a native β 2 glycoprotein I of any of claims 1-4, wherein the native β 2 glycoprotein I is subjected to a first dialysis, followed by a second dialysis, and then mixed with bacterial lipid A and reacted;
the dialysate used for the second dialysis is buffer B, wherein:
the concentration of sodium ions in the buffer solution B was 0.15M, and the pH was 7.4.
6. The method of opening the V domain of native β 2 glycoprotein I according to claim 5,
the buffer solution B is HEPES buffer solution with the concentration of 20 mM;
the dialysis time of the second dialysis was 8 h.
7. The method for opening the V domain of a natural beta 2 glycoprotein I according to claim 6, wherein the mass ratio of the beta 2 glycoprotein I prepared by the second dialysis to the bacterial lipid A is 100-750: 1.
8. The method of claim 7, wherein the mass ratio of β 2 glycoprotein I produced by the second dialysis to bacterial lipid a is 500: 1.
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