阅读说明：本技术 完全抗原及其制备方法、应用和产品 (Complete antigen and preparation method, application and product thereof ) 是由 黄记有 阳馨滢 李瑞净 周峻 任凯瑜 于 2019-11-05 设计创作，主要内容包括：本发明涉及免疫化学领域,具体而言,提供了一种完全抗原及其制备方法、应用和产品。本发明提供的完全抗原的制备方法,是在表面活性剂存在的条件下,将疏水性半抗原与载体进行偶联反应,得到完全抗原。本发明提供的该方法可以显著改善沉淀现象,偶联效率也得到大幅度提升,完全抗原活性高倍增加,极大地简化了制备工艺并且降低了成本。(The invention relates to the field of immunochemistry, and particularly provides a complete antigen, and a preparation method, application and a product thereof. The preparation method of the complete antigen provided by the invention is to carry out coupling reaction on hydrophobic hapten and carrier in the presence of surfactant to obtain the complete antigen. The method provided by the invention can obviously improve the precipitation phenomenon, greatly improve the coupling efficiency, highly increase the complete antigen activity, greatly simplify the preparation process and reduce the cost.)

1. A method for preparing a complete antigen is characterized in that a hydrophobic hapten and a carrier are subjected to coupling reaction in the presence of a surfactant to obtain the complete antigen.

2. The method of claim 1, wherein the hydrophobic hapten comprises vitamin D and its derivatives, aldosterone, or estradiol;

preferably, the hydrophobic hapten is vitamin D and derivatives thereof, and further preferably is 25-hydroxy vitamin D3 and derivatives thereof or 1, 25-dihydroxyvitamin D3 and derivatives thereof.

3. The method of claim 1, wherein the lipophilic end of the surfactant contains a straight chain alkyl group having 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms;

preferably, the surfactant comprises an ionic surfactant having a straight-chain alkyl group having 10 to 18 carbon atoms at the oleophilic end, preferably an ionic surfactant having a straight-chain alkyl group having 12 to 16 carbon atoms at the oleophilic end;

preferably, the surfactant comprises cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, sodium dodecyl sulphate or sodium dodecyl benzene sulphonate;

preferably, the final usage amount of the hexadecyl trimethyl ammonium chloride is 5mg/ml to 50 mg/ml;

preferably, the final usage amount of the hexadecyl trimethyl ammonium bromide is 5mg/ml to 50 mg/ml;

preferably, the final usage amount of the sodium dodecyl sulfate is 2mg/ml to 20 mg/ml;

preferably, the final usage amount of the sodium dodecyl benzene sulfonate is 2mg/ml to 20 mg/ml.

4. The method according to claim 1, wherein the carrier comprises BSA, KHL, OVA, HSA, GST, Poly-Lys, or Poly-PEG.

5. The method according to claim 1, wherein the coupling method comprises a mixed acid anhydride method, a carbodiimide-based condensing agent method, an onium salt-based condensing agent method, an organophosphorus-based condensing agent method, a glutaraldehyde method, a diazotization method, a succinic anhydride method, a carbonyl diimidazole method, a disulfide bond method, or a periodic acid oxidation method.

6. The preparation method according to claim 1, wherein the coupling reaction comprises activating hydrophobic hapten, adding surfactant after the hydrophobic hapten is activated, mixing uniformly, and coupling with a carrier;

preferably, the coupling reaction comprises activating the hydrophobic hapten, and adding a carrier containing a surfactant for coupling after the hydrophobic hapten is activated.

7. The production method according to claim 1, wherein the surfactant increases the solubility of the complete antigen and/or the competitive activity of the complete antigen;

preferably, the surfactant is such that no precipitation of the complete antigen occurs;

preferably, the surfactant increases the competitive activity of the complete antigen by at least 10%, preferably by at least 50%, more preferably by at least 100%, even more preferably by at least 120%.

8. A complete antigen produced by the production method according to any one of claims 1 to 7.

9. Use of the complete antigen of claim 8 for the preparation of antibodies or immunological assays.

10. A kit comprising the complete antigen of claim 8.

Technical Field

The invention relates to the field of immunochemistry, in particular to a complete antigen, and a preparation method, application and a product thereof.

Background

In the production of monoclonal antibodies, in vitro immunization is often required. One feature of most small molecule compounds (molecular weight less than 1 ten thousand daltons) is: it is also a hapten because it is reactogenic but not immunogenic and does not allow direct immunization of animals to produce specific antibodies. The solution to this problem is to conjugate these small molecule compounds to a macromolecular carrier, and the resulting conjugate is called an artificial antigen or a complete antigen. Another feature of small molecule compounds is: and (4) the hydrophobic property is stronger, such as vitamin D, aldosterone, estradiol and the like. In the process of synthesizing the complete antigen, the small molecular compound often causes protein precipitation or turbidity due to the enhancement of the hydrophobicity of the coupled protein after the coupling of the carrier protein, so that the activity of the complete antigen is reduced or the difficulty of controlling the batch-to-batch difference of the whole production is increased, and the use amount of the hapten small molecular compound has to be increased for counteracting the precipitation phenomenon and the inactivation condition, which undoubtedly greatly increases the research and development cost or the production cost for small molecules which are difficult to obtain or expensive.

Complete antigen agglutination and activity reduction in the immune monoclonal antibody field is a common problem, even in the market of many complete antigen type products are precipitated or turbid, such as DIASource products in the market also have certain turbidity. Therefore, it is very promising to solve the precipitation problem in the complete antigen production process and is necessary to optimize the product performance.

In response to such precipitation or turbidity problems, the current conventional solution is to adjust the PH up or down, i.e. to use more acidic or more basic buffer systems, which virtually increases the risk of complete antigen denaturation or inactivation. Another solution is to adjust the buffer pair or the carrier concentration and the amount of the small molecule compound, and consider the comprehensive optimum point of the three with multiple factors, and the exploration of the condition requires a relatively complicated workload. Furthermore, where a high activity or high coupling ratio of the complete antigen is required, the prior art has had to increase the amount of hydrophobic small molecule compound, which in turn further increases the hydrophobicity of the final conjugate or conjugate, leading to a large increase in the ease of sedimentation, which cannot even be optimized for and adjusted for PH by adjusting the buffer. Therefore, a simple, efficient and low-cost method is urgently needed to be found.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a preparation method of complete antigen, which can relieve the precipitation phenomenon of the complete antigen in the preparation process and the storage process in the prior art, and has the problems of low overall immunocompetence of the complete antigen, high difficulty in controlling the batch-to-batch difference, large usage amount of hydrophobic hapten and high cost.

The second purpose of the invention is to provide the complete antigen prepared by the preparation method, so as to solve the problems of easy agglutination, poor stability and low use activity of the complete antigen in the prior art.

The third purpose of the invention is to provide the application of the complete antigen in preparing antibodies or immunological detection.

The fourth object of the present invention is to provide a kit containing the complete antigen provided by the present invention.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a process for preparing complete antigen includes such steps as coupling reaction between hydrophobic hapten and carrier in the presence of surfactant to obtain complete antigen.

Further, the hydrophobic hapten comprises vitamin D and derivatives thereof, aldosterone or estradiol;

preferably, the hydrophobic hapten is vitamin D and derivatives thereof, and further preferably is 25-hydroxy vitamin D3 and derivatives thereof or 1, 25-dihydroxyvitamin D3 and derivatives thereof.

Further, the lipophilic end of the surfactant contains a straight-chain alkyl group of 10 to 18 carbon atoms, preferably a straight-chain alkyl group of 12 to 16 carbon atoms;

preferably, the surfactant comprises an ionic surfactant having a straight-chain alkyl group having 10 to 18 carbon atoms at the oleophilic end, preferably an ionic surfactant having a straight-chain alkyl group having 12 to 16 carbon atoms at the oleophilic end;

preferably, the surfactant comprises cetyltrimethylammonium chloride, cetyltrimethylammonium bromide, sodium dodecyl sulphate or sodium dodecyl benzene sulphonate;

preferably, the final usage amount of the hexadecyl trimethyl ammonium chloride is 5mg/ml to 50 mg/ml;

preferably, the final usage amount of the hexadecyl trimethyl ammonium bromide is 5mg/ml to 50 mg/ml;

preferably, the final usage amount of the sodium dodecyl sulfate is 2mg/ml to 20 mg/ml;

preferably, the final usage amount of the sodium dodecyl benzene sulfonate is 2mg/ml to 20 mg/ml.

Further, the carrier comprises BSA, KHL, OVA, HSA, GST, Poly-Lys or Poly-PEG.

Further, the coupling reaction method includes a mixed acid anhydride method, a carbodiimide-based condensing agent method, an onium salt-based condensing agent method, an organic phosphorus-based condensing agent method, a glutaraldehyde method, a diazotization method, a succinic anhydride method, a carbonyl diimidazole method, a disulfide bond method, or a periodic acid oxidation method.

Further, the coupling reaction comprises activating hydrophobic hapten, adding a surfactant to mix uniformly after the hydrophobic hapten is activated, and coupling with a carrier;

preferably, the coupling reaction comprises activating the hydrophobic hapten, and adding a carrier containing a surfactant for coupling after the hydrophobic hapten is activated.

Further, the coupling reaction comprises activating hydrophobic hapten, adding a surfactant to mix uniformly after the hydrophobic hapten is activated, and coupling with a carrier;

preferably, the coupling reaction comprises activating the hydrophobic hapten, and adding a carrier containing a surfactant for coupling after the hydrophobic hapten is activated.

The complete antigen prepared by the preparation method.

The complete antigen provided by the invention is applied to the preparation of antibodies or immunological detection.

A kit containing the complete antigen provided by the invention.

Compared with the prior art, the invention has the beneficial effects that:

the preparation method of the complete antigen provided by the invention is to carry out coupling reaction on hydrophobic hapten and carrier in the presence of surfactant to obtain the complete antigen. In the invention, in the conventional complete antigen coupling preparation process, the addition of a surfactant increases a dissolution promotion link, hydrophobic hapten and a hydrophobic end of the surfactant interact to form a specific soluble micelle, the micelle is a dynamically balanced micelle, and the hydrophobic hapten can be released when the micelle meets a carrier, so that the coupling reaction can be smoothly carried out, and meanwhile, because the interaction still exists, the surfactant still exists around the coupled complete antigen, and the complete antigen is prevented from precipitating due to overlarge conjugation degree. That is, the surfactant increases the water solubility of the hydrophobic hapten on one hand, thereby reducing the repulsive hydrolysis generated by the insoluble hapten in the activated state, and indirectly increasing the activation efficiency; on the other hand, the solubility of the generated complete antigen is increased, so that a plurality of hydrophobic haptens are continuously coupled to the original conjugate, the microscopic angles are relatively uniform, the titer of the product in macroscopic use can be improved, and the inter-batch difference of the product is reduced. The method provided by the invention can obviously improve the precipitation phenomenon, greatly improve the coupling efficiency, highly increase the complete antigen activity, greatly simplify the preparation process and reduce the cost.

The complete antigen prepared by the preparation method provided by the invention has good stability, no turbidity or precipitation, prolonged effective period, good antigen activity, higher activity under low coating concentration and greatly reduced cost. The complete antigen is suitable for being prepared into a kit for preparing an antibody or immunologically detecting, and has good effect.

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 diagram of the interaction mechanism of a surfactant CTAC and a hydrophobic semi-antigen vitamin D derivative provided by the invention;

FIG. 2 is a schematic diagram of an extended model of the interaction between linear surfactants and hydrophobic haptens provided by the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

A process for preparing complete antigen includes such steps as coupling reaction between hydrophobic hapten and carrier in the presence of surfactant to obtain complete antigen.

The invention adds a dissolution promotion link in the conventional complete antigen coupling preparation process, hydrophobic hapten can interact with the hydrophobic end of a surfactant to form a specific soluble micelle, the micelle is a dynamically balanced micelle, and can release the hydrophobic hapten when meeting a carrier, so that the coupling reaction can be smoothly carried out, and meanwhile, because the interaction still exists, the surfactant still exists around the coupled complete antigen, thereby avoiding the complete antigen from precipitating due to overlarge conjugation degree. That is, on one hand, the surfactant increases the water solubility of the hydrophobic hapten, so that the repulsive hydrolysis of the activated ester of the hapten, which is not easy to dissolve, is reduced, and the activation efficiency is indirectly increased; on the other hand, the solubility of the generated complete antigen is increased, so that a plurality of hydrophobic haptens are continuously coupled to the original conjugate, the microscopic angles are relatively uniform, the titer of the product in macroscopic use can be improved, and the batch difference of the product can be reduced. The method provided by the invention can obviously improve the precipitation phenomenon, greatly improve the coupling efficiency, highly increase the complete antigen activity, greatly simplify the preparation process and reduce the cost.

Note that the "hapten": small molecule substances with antigenicity and no immunogenicity, which is referred to herein as small molecules with a molecular weight of less than 1 ten thousand daltons, such as vitamin D and the like. "hydrophobic" refers to the physical property of a molecule or some portion of a molecule that repels water, and the hydrophobic molecule or hydrophobic portion of a molecule typically contains carbon chains or rings that do not contain hydrogen bonding elements such as N or O, such as alkyl groups, aliphatic groups, and the like. The carrier means a macromolecular substance imparting immunogenicity to the hapten, and may be a protein or the like.

In the technical scheme of the present invention, conventional methods for activating hydrophobic haptens, coupled carriers and coupling methods thereof, etc. in the prior art can be used, and the present invention is not limited thereto.

In a preferred embodiment, the hydrophobic hapten comprises vitamin D and its derivatives, aldosterone or estradiol.

In a preferred embodiment, the hydrophobic hapten is vitamin D and derivatives thereof, more preferably 25-hydroxyvitamin D3 and derivatives thereof or 1, 25-dihydroxyvitamin D3 and derivatives thereof.

In a preferred embodiment, the lipophilic end of the surfactant contains a straight chain alkyl group of 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms.

In a preferred embodiment, when the hydrophobic hapten is vitamin D, the lipophilic end of the surfactant contains a straight chain alkyl group of 10 to 18 carbon atoms, preferably 12 to 16 carbon atoms. The inventor finds through experimental research that the surfactant with lipophilic end containing straight-chain alkyl with 10-18 carbon atoms can play a good effect of promoting solubility, the lipophilic structure and the structure of vitamin D can possibly form a certain dynamic equilibrium micelle, for example, fig. 1 is an interaction mechanism diagram of the surfactant CTAC and the hydrophobic semi-antigen vitamin D derivative, and explains the principle that the surfactant CTAC is added to increase the solubility promotion and improve the immunocompetence when the hydrophobic semi-antigen vitamin D derivative is coupled with a carrier; FIG. 2 is a schematic diagram of an extended model of the interaction between a linear surfactant and a hydrophobic hapten, which can well reflect the interaction between hydrophilic ends of different molecules and the interaction between hydrophobic ends of different molecules, so that a relatively stable micelle form can be formed in a water system, and the micelle is wholly in a state that the hydrophilic ends are exposed and the hydrophobic ends are hidden, so that the water solubility can be increased, and the immunogenicity of the hydrophobic ends of the hapten can be maintained, namely the activity of the final conjugate is improved. The structure preferentially containing the linear chain hydrophobic end in the surfactant can form a medium hydrophobic combined acting force with the linear chain structure and the hydrophobic structure in the vitamin D micromolecule, so that a micelle state can be formed to solve the precipitation problem, and the influence of the overhigh hydrophobic combined acting force on immune site combination in the immune process can be avoided.

In a preferred embodiment, the surfactant comprises an ionic surfactant having a straight chain alkyl group of 10 to 18 carbon atoms at the oleophilic end, preferably an ionic surfactant having a straight chain alkyl group of 12 to 16 carbon atoms at the oleophilic end.

In preferred embodiments, the surfactant comprises cetyltrimethylammonium chloride (CTAC), cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulphate (SLS) or sodium dodecyl benzene sulphonate (LAS). Wherein, the final usage amount of CTAC is preferably 5mg/ml-50mg/ml, the final usage amount of CTAB is preferably 5mg/ml-50mg/ml, the final usage amount of SLS is preferably 2mg/ml-20mg/ml, and the final usage amount of LAS is preferably 2mg/ml-20 mg/ml. The final consumption of CTAC means that 5-50mg of CTAC is added into each 1ml of reaction system; similarly, the final consumption of CTAB means that 5-50mg of CTAB is added into each 1ml of reaction system; the final SLS usage amount is 2-20mg of SLS per 1ml of the reaction system; similarly, the final amount of LAS used means that 2-20mg LAS per 1ml reaction system is added.

In a preferred embodiment, the carrier comprises BSA (bovine serum albumin), KHL (keyhole limpet hemocyanin), OVA (ovalbumin), HSA (human serum albumin), GST (glutathione S transferase), Poly-Lys (polylysine), or Poly-PEG (polyethylene glycol). It is understood that the carrier of the present invention may be prepared by a method conventional in the art, and is not limited thereto, and is preferably BSA.

In a preferred embodiment, the method for coupling the hydrophobic hapten to the carrier includes a mixed acid anhydride method, a carbodiimide-based condensing agent method, an onium salt-based condensing agent method, an organophosphorus-based condensing agent method, a glutaraldehyde method, a diazotization method, a polybasic acid anhydride method, a carbonyl diimidazole method, a disulfide bond method, or a periodic acid oxidation method. The coupling method of the present invention may be a method generally used in the art, and is not limited thereto.

The mixed anhydride method mainly uses free carboxyl in hapten to react with isobutyl chloroformate or ethyl chloroformate to generate mixed anhydride, and then the mixed anhydride reacts with free amino in a carrier.

The carbodiimide-based condensing agent method, in which coupling of an amino group and a carboxyl group compound is performed using a carbodiimide-based condensing agent such as EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide), EDCI (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide), DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), may add an acylation catalyst or an activating agent to reduce the formation of by-products. The method can introduce N-hydroxysuccinimide (NHS) or N-hydroxythiosuccinimide to improve coupling efficiency.

The onium salt condensing agent method comprises subjecting carboxyl group and amino group to amide condensation with a carbonium salt condensing agent or phosphonium salt condensing agent, such as O- (7-azabenzotriazole-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HATU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HBTU), O- (5-chlorobenzotriazole-1-yl) -bis (dimethylamino) carbonium Hexafluorophosphate (HCTU), O- (benzotriazol-1-yl) -bis (dimethylamino) carbonium tetrafluoroborate (TBTU), O- (N-succinimidyl) -bis (dimethylamino) carbonium tetrafluoroborate (TSTU), O- (N-endo-5-norbornene-2, 3-dicarboximide) -bis (dimethylamino) carbonium tetrafluoroborate (TNTU), and the like.

The organophosphorus condensing agent method is a method of subjecting a carboxyl group and an amino group of a molecule to amide condensation using a phosphate ester or phosphoramide condensing agent, and DECP, BOP-Cl, and the like are commonly used.

The polyanhydride method is mainly used for reacting hydroxyl of hapten with succinic anhydride in anhydrous pyridine to obtain succinic acid half ester (intermediate with carboxyl), and then combining with protein amino by a carbodiimide method or a mixed anhydride method, and inserting a succinyl group between the hapten and a protein carrier, so the polyanhydride method is also called as the succinic anhydride method.

The diazotization method is mainly used for hapten of which the active group is aromatic amino, and amino is firstly diazotized and then coupled with tyrosine residue of carrier protein.

Glutaraldehyde as homotype double-function reagent is combined with hapten and amino group on carrier separately, and the intermediate primer is a connecting bridge of 5 carbon chains.

The periodate oxidation method is to oxidize sugar ring into aldehyde group and then couple with amino group on carrier protein.

Carbonyl diimidazole method: n, N' -carbonyldiimidazole is a highly reactive reagent for introducing carbonyl groups, and a hydroxyl-containing molecule reacts with carbonyldiimidazole to form an intermediate imidazolecarboxylate, which can react with an N-nucleophile to give an N-alkylated formate bond, and proteins generally form uncharged urethane-like derivatives via the N-terminus (. alpha. -amino) and the (. epsilon. -amino) of lysine side chains and molecules, with excellent chemical stability.

Disulfide bond method: the hapten can also be linked to the protein molecule by disulfide bond formation by hydrogen peroxide in an acetic acid buffer at pH4.0 with the carrier protein.

In a preferred embodiment, the coupling reaction comprises activating hydrophobic hapten, adding a surfactant after the hydrophobic hapten is activated, uniformly mixing, and coupling with a carrier; or the coupling reaction comprises activating the hydrophobic hapten, and adding a carrier containing a surfactant for coupling after the hydrophobic hapten is activated. According to the technical scheme, the surfactant can be mixed with the activated hydrophobic hapten or the carrier, and then the mixture is subjected to coupling reaction with the carrier or the activated hydrophobic hapten, so that the surfactant always exists in the coupling reaction process, the agglutination is avoided to the greatest extent, and the coupling efficiency is improved.

In preferred embodiments, the surfactant increases the solubility of the complete antigen and/or the competitive activity of the complete antigen. In the preparation of the complete antigen, the surfactant may act to improve the complete antigen in two ways, i.e., to increase the solubility of the complete antigen and/or the competitive activity of the complete antigen, separately or simultaneously. In the present invention, the improvement of the solubility of the complete antigen means that the complete antigen obtained by the preparation method with a surfactant has improved solubility compared to the complete antigen obtained by the preparation method without adding a surfactant under the same other operation conditions; the improvement of the competitive activity of the complete antigen means that the complete antigen obtained by the preparation method with the surfactant has improved competitive activity compared with the complete antigen obtained by the preparation method without adding the surfactant under the same other operation conditions.

In a preferred embodiment, the surfactant renders the complete antigen precipitate-free. Due to the addition of surface activity, the solubility of complete antigen is effectively improved, preferably completely dissolved, no precipitate is generated, and the stability is obviously improved.

In a preferred embodiment, the surfactant increases the competitive activity of the complete antigen by at least 10%, preferably by at least 50%, more preferably by at least 100%, even more preferably by at least 120%. The competitive activity of the complete antigen obtained by adding the surface activity is improved by more than 10%, or by not limited to 20%, 30%, 50%, 70%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, and the like, and it is understood that the more the competitive activity of the complete antigen is improved, the better the performance of the complete antigen is.

In a preferred embodiment, a method for preparing a vitamin D complete antigen may comprise:

dissolving 1mg VD derivative in 50-200 mu l N' N-dimethylformamide, adding 1.1-1.5 equivalent of EDC and 1.1-3.0 equivalent of NHS, reacting at room temperature for 1-3 hours, slowly adding the activating solution into 3-6ml buffer solution containing surface activity, shaking, adding the mixed solution into 3-6ml buffer solution containing BSA with concentration of 1-10 mg/ml, reacting overnight, dialyzing to remove unreacted small molecular impurities the next day, and centrifuging to obtain supernatant to evaluate complete antigen activity.

In a preferred embodiment, the method for preparing the vitamin D complete antigen may be:

dissolving 1mgVD derivative in 50-200 μ l N' N-dimethylformamide, adding 1.1-1.5 equivalent of EDC and 1.1-3.0 equivalent of NHS, reacting at room temperature for 1-3 hr to obtain activation solution; mixing 3-6ml buffer solution containing surfactant with 3-6ml buffer solution containing BSA with concentration of 1mg/ml-10mg/ml to obtain carrier solution containing surfactant. Slowly adding the reaction solution into a carrier solution containing a surfactant, reacting overnight, taking out the reaction solution the next day, dialyzing to remove unreacted micromolecular impurities, and finally centrifuging to take the supernatant to evaluate the complete antigen activity.

The "equivalent" refers to a multiple of the amount of a substance in a specific chemical reaction relative to a reference raw material, and herein refers to a multiple of the molar amount of a VD derivative. For example, the molar mass of the VD derivative is 344.24g/mol, and 1mg of the VD derivative is 2.9X 10^-3mmol, 1.1 equivalents means that EDC is added in an amount of 2.9X 1.1X 10^-3mmol。

The invention also provides the complete antigen prepared by the preparation method. The complete antigen has good stability, does not generate turbidity or precipitation, prolongs the effective period, has good antigen activity, has higher activity under low coating concentration, and greatly reduces the cost. The complete antigen is suitable for being prepared into a kit for preparing an antibody or immunologically detecting, and has good effect.

The invention also provides the application of the complete antigen in preparing antibodies or immunological detection.

The invention finally provides a kit containing the complete antigen.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

EXAMPLE 1CTAC hexadecyltrimethylammonium chloride

1mg VD derivative was dissolved in 50. mu. l N' N-dimethylformamide, 1.1 equivalents of EDC and 3.0 equivalents of NHS were added and reacted at room temperature for 3 hours, then the activating solution was slowly added to 6.4ml of buffer containing surface active CTAC, after shaking up, the mixture was added to 6.4ml of 10mM pH7.4 PB buffer containing BSA at a concentration of 6mg/ml, then the reaction was carried out overnight, the next day was taken out and dialyzed to remove unreacted small molecular impurities, and finally the supernatant was centrifuged to evaluate the complete antigenic activity.

Plate luminescence evaluation of the coated plates was performed on the complete antigen VD-BSA from different experimental groups, the coated plate used was VD-BSA corresponding to 30ng/ml antibody, conjugate: goat anti-mouse IgG-HRP (diluted 3000 times), quality control: the newly purchased 25OH-VD molecule was not coupled and the immunization program was: mu.l sample + 50. mu.l antibody/100. mu.l conjugate/100. mu.l luminol, 30min/30min/60 s. (sample-complete antigen, antibody-monoclonal antibody, conjugate-HRP labeled goat secondary antibody, Luminol substrate of Luminol-HRP, reaction time: complete antigen and monoclonal antibody react for 30min, reaction time of monoclonal antibody and goat secondary antibody is 30min, and reaction time of Luminol and HRP enzyme is 60 seconds).

The results for different coating concentrations are shown in tables 1 and 2. Note that the different CTAC concentrations in table 1 and table 2 are the final use concentrations of CTAC in the reaction system.

When the surfactant was CTAC, the precipitation of the prepared vitamin D complete antigen and the results of the detection of the antigenic activity were shown in table 1/2 below, wherein "+++" indicates the most significant precipitation, "+++" indicates the presence of precipitation, "+" indicates the presence of slight precipitation, "-" indicates the absence of precipitation, the upper data in the table are the competitive activity of the complete antigen in competition with the quality control substance for binding to the antibody, and the lower data are the percentage increase of the competitive activity of the experimental group to which CTAC was added relative to the competitive activity of the experimental group to which CTAC was not added.

Table 1 results for coating concentrations of 0.5. mu.g/ml.

TABLE 1

TABLE 2 results at a coating concentration of 0.1. mu.g/ml.

TABLE 2

From the data in the table, it can be seen that CTAC in the reaction system can improve the generation of the precipitate and increase the competitive activity of the complete antigen, in the examples, the average increase rate of the competitive activity of the antigen is up to more than 180%, and in the preferred concentration range, CTAC can completely eliminate the precipitate.

Example 2SLS sodium dodecyl sulfate

1mg VD derivative was dissolved in 200. mu. l N' N-dimethylformamide, 1.5 equivalents of EDC and 1.1 equivalents of NHS were added and reacted at room temperature for 1 hour, then the activated solution was slowly added to 6ml of a buffer containing surface active SLS, after shaking, the mixture was added to 6ml of 10mM PH7.4 PB buffer containing BSA at a concentration of 10mg/ml, followed by overnight reaction, dialysis was taken the next day to remove unreacted small molecular impurities, and finally the supernatant was centrifuged to evaluate complete antigenic activity.

Plate luminescence evaluation of the coated plates was performed on the complete antigen VD-BSA from different experimental groups, the coated plate used was VD-BSA corresponding to 30ng/ml antibody, conjugate: goat anti-mouse IgG-HRP (diluted 3000 times), quality control: the newly purchased 25OH-VD molecule was not coupled and the immunization program was: mu.l sample + 50. mu.l antibody/100. mu.l conjugate/100. mu.l luminol, 30min/30min/60 s. (sample-complete antigen, antibody-monoclonal antibody, conjugate-HRP labeled goat secondary antibody, Luminol substrate of Luminol-HRP, reaction time: complete antigen and monoclonal antibody react for 30min, reaction time of monoclonal antibody and goat secondary antibody is 30min, and reaction time of Luminol and HRP enzyme is 60 seconds).

The results for different coating concentrations are shown in tables 3 and 4. It should be noted that the different SLS concentrations in tables 1 and 2 are the final SLS concentrations used in the reaction system.

When the surfactant was SLS, the precipitation of the prepared vitamin D complete antigen and the results of the detection of the antigenic activity were shown in table 3/4 below, where "+++" indicates the most significant precipitation, "+++" indicates the presence of precipitation, "+" indicates the presence of slight precipitation, and "-" indicates the absence of precipitation, the upper data in the table are the competitive activity of the complete antigen in competition with the quality control substance for binding to the antibody, and the lower data are the percentage increase in the competitive activity of the SLS-added test group relative to the non-added test group.

TABLE 3 results at a coating concentration of 0.5. mu.g/ml.

TABLE 3

TABLE 4 results for coating concentrations of 0.1. mu.g/ml.

TABLE 4

From the data in the table, it can be seen that SLS in the reaction system can improve the generation of precipitate and increase the competitive activity of complete antigen, the average increase rate of the antigen competitive activity in the examples is as high as more than 200%, and SLS can completely eliminate the precipitate in the preferred concentration range.

Example 3 sodium LAS dodecylbenzene sulfonate

1mg VD derivative is dissolved in 100 u l N' N-dimethylformamide, add 1.3 equivalent EDC and 1.3 equivalent NHS, room temperature reaction for 2 hours, then slowly add the activation solution into 6ml buffer solution containing surface active LAS, shake the mixture, then add the mixture into 6ml buffer solution containing BSA with concentration of 8mg/ml, then overnight reaction, the next day take out dialysis to remove unreacted small molecular impurities, finally centrifuge the supernatant to evaluate complete antigen activity.

Plate luminescence evaluation of the coated plates was performed on the complete antigen VD-BSA from different experimental groups, the coated plate used was VD-BSA corresponding to 30ng/ml antibody, conjugate: goat anti-mouse IgG-HRP (diluted 3000 times), quality control: the newly purchased 25OH-VD molecule was not coupled and the immunization program was: mu.l sample + 50. mu.l antibody/100. mu.l conjugate/100. mu.l luminol, 30min/30min/60 s. (sample-complete antigen, antibody-monoclonal antibody, conjugate-HRP labeled goat secondary antibody, Luminol substrate of Luminol-HRP, reaction time: complete antigen and monoclonal antibody react for 30min, reaction time of monoclonal antibody and goat secondary antibody is 30min, and reaction time of Luminol and HRP enzyme is 60 seconds).

The results for different coating concentrations are shown in tables 5 and 6. The different LAS concentrations in tables 1 and 2 are the final LAS concentrations used in the reaction system.

When the surfactant was LAS, the precipitation of the prepared vitamin D complete antigen and the results of the detection of the antigenic activity were shown in table 5/6 below, in which "+++" indicates the most significant precipitation, "++" indicates the presence of precipitation, "+" indicates the presence of slight precipitation, "-" indicates the absence of precipitation, the upper data in the table are the competitive activity of the complete antigen in competition with the quality control substance for binding to the antibody, and the lower data are the percentage increase in the competitive activity of the LAS-added test group relative to the non-added test group.

TABLE 5 results at a coating concentration of 0.5. mu.g/ml.

TABLE 5

Table 6 results at a coating concentration of 0.1. mu.g/ml.

TABLE 6

From the data in the table, it can be seen that LAS in the reaction system can improve the generation of precipitate and increase the competitive activity of complete antigen, the average increase rate of the competitive activity of antigen in the examples is as high as 120% or more, and LAS can completely eliminate the precipitate in the preferred concentration range.

Example 4CHAPS

1mg of VD derivative was dissolved in 50. mu. l N' N-dimethylformamide, 1.1 equivalents of EDC and 3.0 equivalents of NHS were added and reacted at room temperature for 3 hours, then the activating solution was slowly added to 6.4ml of a buffer containing surface active CHAPS, after shaking up, the mixture was added to 6.4ml of 10mM pH7.4 PB buffer containing BSA at a concentration of 6mg/ml, and then reacted overnight, and then removed by dialysis the next day to remove unreacted small molecular impurities, and finally the supernatant was centrifuged to evaluate the complete antigenic activity.

Plate luminescence evaluation of the coated plates was performed on the complete antigen VD-BSA from different experimental groups, the coated plate used was VD-BSA corresponding to 30ng/ml antibody, conjugate: goat anti-mouse IgG-HRP (diluted 3000 times), quality control: the newly purchased 25OH-VD molecule was not coupled and the immunization program was: mu.l sample + 50. mu.l antibody/100. mu.l conjugate/100. mu.l luminol, 30min/30min/60 s. (sample-complete antigen, antibody-monoclonal antibody, conjugate-HRP labeled goat secondary antibody, Luminol substrate of Luminol-HRP, reaction time: complete antigen and monoclonal antibody react for 30min, reaction time of monoclonal antibody and goat secondary antibody is 30min, and reaction time of Luminol and HRP enzyme is 60 seconds).

The results of the different coating concentrations are shown in tables 7 and 8. Note that the different CHAPS concentrations in tables 1 and 2 are the final use concentrations of CHAPS in the reaction system.

When the surfactant was CHAPS, the precipitation of vitamin D complete antigen and the results of the detection of the antigenic activity were shown in Table 7/8 below, wherein "+++" indicates the most significant precipitation, "+ +" indicates the presence of precipitation, "+" indicates the presence of slight precipitation, "-" indicates the absence of precipitation, the upper data in the table are the competitive activity of complete antigen in competition with the quality control substance for binding to the antibody, and the lower data are the percentage increase in the competitive activity of the test group with CHAPS over the test group without CHAPS.

Table 7 results at a coating concentration of 0.5. mu.g/ml.

TABLE 7

TABLE 8 results at a coating concentration of 0.1. mu.g/ml.

TABLE 8

As can be seen from the results of tables 7 and 8, CHAPS in the reaction system improved the precipitation, and in the preferred concentration range, CHAPS completely eliminated the precipitation, but attenuated the full antigenic activity to various degrees.

Example 5 Sparban 80

1mg VD derivative is dissolved in 200 u l N' N-dimethylformamide, adding 1.5 equivalent EDC and 1.1 equivalent NHS, room temperature reaction for 1 hours, then slowly adding the activation solution into 6ml buffer solution containing surface active span 80, shaking, adding the mixture into 6ml 10mM PH7.4 PB buffer solution containing BSA with concentration of 10mg/ml, then overnight reaction, the next day taking dialysis to remove unreacted small molecular impurities, finally centrifuging and taking supernatant to evaluate complete antigen activity.

Plate luminescence evaluation of the coated plates was performed on the complete antigen VD-BSA from different experimental groups, the coated plate used was VD-BSA corresponding to 30ng/ml antibody, conjugate: goat anti-mouse IgG-HRP (diluted 3000 times), quality control: the newly purchased 25OH-VD molecule was not coupled and the immunization program was: mu.l sample + 50. mu.l antibody/100. mu.l conjugate/100. mu.l luminol, 30min/30min/60 s. (sample-complete antigen, antibody-monoclonal antibody, conjugate-HRP labeled goat secondary antibody, Luminol substrate of Luminol-HRP, reaction time: complete antigen and monoclonal antibody react for 30min, reaction time of monoclonal antibody and goat secondary antibody is 30min, and reaction time of Luminol and HRP enzyme is 60 seconds).

The results of the different coating concentrations are shown in tables 9 and 10. It should be noted that the different span 80 concentrations in table 1 and table 2 are the final use concentrations of span 80 in the reaction system.

When the surfactant is span 80, the precipitation of the prepared vitamin D complete antigen and the results of the detection of the antigenic activity are shown in the following table 9/10, wherein "+++" indicates the most significant precipitation, "+++" indicates the presence of precipitation, "+" indicates the presence of slight precipitation, "-" indicates the absence of precipitation, the upper data in the table are the competitive activity of the complete antigen in competition with the quality control substance for binding the antibody, and the lower data are the percentage increase of the competitive activity of the experimental group with span 80 in comparison with the experimental group without the addition.

TABLE 9 results at a coating concentration of 0.5. mu.g/ml.

TABLE 9

TABLE 10 results at a coating concentration of 0.1. mu.g/ml.

Watch 10

From the results in tables 9 and 10, it can be seen that span 80 in the reaction system improves the precipitation, and that span 80 completely eliminates the precipitation within the preferred concentration range, but attenuates the complete antigenic activity to varying degrees.

Example 6Triton X-100

1mg VD derivative is dissolved in 100 mu l N' N-dimethylformamide, 1.3 equivalents of EDC and 1.3 equivalents of NHS are added, the reaction is carried out at room temperature for 2 hours, then the activating solution is slowly added into 6ml of buffer solution containing surface active Triton X-100, the mixture solution is added into 6ml of buffer solution containing BSA with the concentration of 8mg/ml after shaking up, then the reaction is carried out overnight, the reaction solution is taken out the next day, dialysis is carried out to remove unreacted small molecular impurities, and finally the supernatant is centrifuged to evaluate the complete antigen activity.

Plate luminescence evaluation of the coated plates was performed on the complete antigen VD-BSA from different experimental groups, the coated plate used was VD-BSA corresponding to 30ng/ml antibody, conjugate: goat anti-mouse IgG-HRP (diluted 3000 times), quality control: the newly purchased 25OH-VD molecule was not coupled and the immunization program was: mu.l sample + 50. mu.l antibody/100. mu.l conjugate/100. mu.l luminol, 30min/30min/60 s. (sample-complete antigen, antibody-monoclonal antibody, conjugate-HRP labeled goat secondary antibody, Luminol substrate of Luminol-HRP, reaction time: complete antigen and monoclonal antibody react for 30min, reaction time of monoclonal antibody and goat secondary antibody is 30min, and reaction time of Luminol and HRP enzyme is 60 seconds).

The results of the different coating concentrations are shown in tables 11 and 12. Note that the different Triton X-100 concentrations in tables 1 and 2 are the final use concentrations of Triton X-100 in the reaction system.

When the surfactant was Triton X-100, the precipitation of the prepared vitamin D complete antigen and the results of the detection of the antigen activity were shown in table 11/12 below, in which "+++" indicates the most significant precipitation, "+++" indicates the presence of precipitation, "+" indicates the presence of slight precipitation, "-" indicates the absence of precipitation, the upper data in the table are the competitive activity of the complete antigen in competing with the quality control for the binding antibody, and the lower data in the table are the percentage increase in the competitive activity of the test group to which Triton X-100 was added, compared to the test group to which no addition was made.

TABLE 11 results at a coating concentration of 0.5. mu.g/ml.

TABLE 11

TABLE 12 results at a coating concentration of 0.1. mu.g/ml (conclusions above).

TABLE 12

As can be seen from the results in tables 9 and 10, Triton X-100 in the reaction system improved the precipitation, and in the preferred concentration range, Triton X-100 completely eliminated the precipitation, but attenuated the complete antigen activity to various degrees.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.