WO2022199107A1

WO2022199107A1 - Antibody complex, and preparation method therefor and detection kit therefof

Info

Publication number: WO2022199107A1
Application number: PCT/CN2021/134784
Authority: WO
Inventors: 刘陶旭; 钱纯亘; 林标杨; 祝亮; 程方明
Original assignee: 深圳市亚辉龙生物科技股份有限公司
Priority date: 2021-03-22
Filing date: 2021-12-01
Publication date: 2022-09-29
Also published as: CN113049805A; CN113049805B

Abstract

Disclosed is an antibody complex, and a preparation method therefor and a detection kit thereof. The antibody complex comprises a plurality of labeled antibodies, and the plurality of labeled antibodies are connected by means of a cross-linking agent.

Description

Antibody complex and its preparation method, detection kit

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of the Chinese patent application filed on March 22, 2021, with the application number 202110303562.3 and the invention titled "Antibody complexes, methods for their preparation, and detection kits", the entire contents of which are incorporated by reference This article.

technical field

The present application relates to the technical field of immunodetection, in particular to an antibody complex, a preparation method thereof, and a detection kit.

Background technique

Chemiluminescence immunoassay (CLIA) is a combination of highly sensitive chemiluminescence assay technology and highly specific immune response for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs It is the latest immunoassay technology developed after radioimmunoassay, enzyme-linked immunoassay, fluorescence immunoassay and time-resolved fluorescence immunoassay.

Chemiluminescence immunoassay consists of two parts: chemiluminescence analysis system and immune response system. The chemiluminescence analysis system uses the chemiluminescence substance to be catalyzed by a catalyst and oxidized by an oxidant to form an excited state intermediate. When this excited state intermediate returns to a stable ground state, it emits photons (hM) at the same time, using luminescence. The signal measuring instrument measures the photon yield. The immune response system utilizes luminescent substances directly labeled on antigens or antibodies (which can generate excited intermediates under the excitation of reactants), or enzymes that act on luminescent substrates.

Chemiluminescence immunoassays are classified into two types according to different labeling methods: (1) chemiluminescence labeling immunoassays; (2) chemiluminescence enzyme immunoassays using enzyme labeling and chemiluminescence substrates as signal reagents.

Chemiluminescence labeling immunoassay is an immunoassay method in which an antigen or antibody is directly labeled with a chemiluminescent agent. Commonly used chemiluminescent substances for labeling include acridinium ester (AE), and acridine esters emit light by initiating the action of luminescent reagents (NaOH, H ₂ O ₂ , etc.), which are fast scintillation luminescence.

Chemiluminescence enzyme immunoassay belongs to enzyme immunoassay. The substrate of the enzymatic reaction is a luminescent agent, and the enzyme-labeled biologically active substance (such as an antigen or antibody) is used for immunoreaction, and the enzyme on the immune reaction complex acts on the luminescent substrate. It emits light under the action of a signaling reagent. The commonly used labeling enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (ALP), which have their own luminescent substrates.

However, the sensitivity of bioactive substances labeled with chemiluminescent agents or enzyme-labeled labels still needs to be improved when performing chemiluminescent immunoassays.

SUMMARY OF THE INVENTION

According to some embodiments, an antibody complex is provided, the antibody complex comprising a plurality of label-labeled antibodies linked by a cross-linking agent.

The above-mentioned antibody complex is a complex formed by polymerizing a plurality of antibodies, and each antibody is labeled with a label. When used, one antigen can correspond to multiple labels and the luminescence signal is amplified. Therefore, when the antibody complex is used in chemiluminescence immunoassay, the signal-to-noise ratio can be increased and the sensitivity can be improved.

According to some embodiments, there is provided a method of preparing an antibody complex, comprising:

Using an activator to activate the antibody and react with a cross-linking agent to prepare a mixture containing multiple polymers, the multimer is formed by cross-linking at least two antibodies through the cross-linking agent;

screening the mixture for the desired multimer; and

The multimer of interest is labeled with a label to prepare an antibody complex.

According to some embodiments, there is provided a detection kit comprising the above-described antibody complex.

The above description is only an overview of the technical solutions of the present application. In order to understand the technical means of the present application more clearly and implement them in accordance with the contents of the description, the preferred embodiments of the present application and the accompanying drawings are described in detail below.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application or in the traditional technology, the following briefly introduces the accompanying drawings used in the description of the embodiments or the traditional technology. Obviously, the drawings in the following description are only the For some embodiments of the application, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is the preparation flow chart of the antibody complex of embodiment 1;

FIG. 2 is a purification separation map of the mixture containing multiple polymers of Example 1. FIG.

Detailed ways

In order to facilitate understanding of the present application, the present application will be described more fully below, which may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terms used herein in the specification of the present application are for the purpose of describing particular embodiments only, and are not intended to limit the present application.

Definition of Terms

An "antibody" is a class of immunoglobulins that bind specifically to an antigen. Generally, antibodies exist as one or more Y-shaped monomers, each Y-shaped monomer is composed of 4 polypeptide chains, including two identical heavy chains and two identical light chains, the light and heavy chains are based on named after their molecular weight. The top of the Y-shaped structure is the variable region, which is the antigen-binding site. Each heavy chain has two regions, the constant region and the variable region. The constant region of all antibodies of the same type is the same, but there are differences between antibodies of different types. Each light chain also has two consecutive domains, a constant region and a variable region.

One embodiment provides an antibody complex, the antibody complex includes a plurality of labeled antibodies, and the plurality of labeled antibodies are linked by a cross-linking agent. The antibody complex is a complex formed by cross-linking and polymerizing multiple antibodies through a cross-linking agent, and each antibody is labeled with a marker. When in use, one antigen corresponds to multiple markers and the luminescent signal is amplified. Therefore, when the antibody complex is applied to chemiluminescence immunoassay, the signal-to-noise ratio can be increased and the sensitivity can be improved.

Optionally, the label in the label-labeled antibody is selected from acridine ester, ruthenium terpyridine, adamantane, luminol, derivatives of luminol, isoluminol, derivatives of isoluminol , one of horseradish peroxidase and alkaline phosphatase. Of course, in other embodiments, the label in the label-labeled antibody is not limited to the above, and can also be other substances that can be used in a chemiluminescence immunoassay platform.

Optionally, the antibody in the labeled antibody is CHI3L1 antibody, procalcitonin antibody (PCT antibody) or cardiac troponin I antibody (cTn-I antibody). It can be understood that, in other embodiments, the antibody in the labeled antibody is not limited to the above, and can also be other substances that can be used in the chemiluminescence immunoassay platform. In this embodiment, the antibody is a monoclonal antibody. Of course, in other embodiments, the antibody may also be a polyclonal antibody. In addition, the antibody may also be an antibody fragment in which the variable region of the light chain and the variable region of the heavy chain are linked via their sulfhydryl groups to form a disulfide bond. At this time, the light chain variable region and the heavy chain variable region form an FV region with all antigen-binding sites through non-covalent bonding, and have antigen-binding ability, such as antigen-binding fragments (antigen-binding fragments, Fab fragments) or Fab' fragment.

Optionally, the cross-linking agent is a dihydrazide compound. The two antibodies are linked by the reaction between the two amino groups of the dihydrazide compound and the carboxyl group of the antibody. Specifically, the crosslinking agent is selected from at least one of maleic acid dihydrazide, oxalic acid dihydrazide and adipic acid dihydrazide.

Optionally, the antibody complex described above includes four labeled antibodies. It can be understood that, in other embodiments, the number of labeled antibodies in the antibody complex is not limited to 4, and can also be any other integer greater than 1. In some embodiments, the number of labeled antibodies in the above antibody complex is 2-6.

In an optional specific example, the above-mentioned complex comprises four acridinium ester-labeled CHI3L1 antibodies, and the four acridinium ester-labeled CHI3L1 antibodies are linked by adipic acid dihydrazide.

In addition, one embodiment also provides a method for preparing the above-mentioned antibody complex, the method comprising the following steps:

Step 1: The antibody is activated with an activator and reacted with a cross-linking agent to prepare a mixture containing multiple polymers.

Specifically, the activating agent is used to activate the carboxyl group of the antibody, and the cross-linking agent has at least two amino groups. The carboxyl group of the antibody is activated and reacted with the amino group of the cross-linking agent, so that a plurality of antibodies are linked through the cross-linking agent to form a polymer. Multimers are formed by cross-linking at least two antibodies with a cross-linking agent.

Optionally, the activating agent used to activate the antibody includes carbodiimide. Specifically, the activator used to activate the antibody is selected from dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N,N'-dicyclohexylcarbodiimide At least one of isopropylcarbodiimide.

In some embodiments, the activator for activating the antibody further comprises at least one of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (Sulfo-NHS). Through N-hydroxysuccinimide and/or N-hydroxysulfosuccinimide, the structure of the antibody after carbodiimide activation is more stable, which is more favorable for its reaction with the cross-linking agent. Further, the activator for activating the antibody includes carbodiimide and N-hydroxysuccinimide, wherein the molar ratio of carbodiimide and N-hydroxysuccinimide is 10:(1-200). Further, the molar ratio of carbodiimide and N-hydroxysuccinimide is 3: (1-30). In a specific example, the activators used to activate the antibody are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

Specifically, the crosslinking agent is a dihydrazide compound. Further, the cross-linking agent is selected from at least one of oxalic acid hydrazide, maleic acid dihydrazide and adipic acid dihydrazide.

In this embodiment, the antibody is a CHI3L1 monoclonal antibody. The molar amount of the activator used to activate the antibody is 5 to 1000 times the molar amount of the antibody. Further, the molar amount of the activator for activating the antibody is 5 times to 1000 times the molar amount of the antibody. It should be noted that the molar amount of the antibody is based on the number of sites where the antibody can bind to the antigen. It can be understood that, in other embodiments, the antibody is not limited to the monoclonal antibody of CHI3L1, but can also be other antibodies.

Specifically, antibodies are activated in acidic buffers. Optionally, the acidic buffer is selected from at least one of citric acid-sodium citrate buffer, acetic acid-sodium acetate buffer and 2-morpholinoethanesulfonic acid (MES) buffer. Further, the pH of the acidic buffer is 3 to 6.5. Still further, the acidic buffer is pH 5 2-morpholinoethanesulfonic acid buffer.

In one embodiment, the step of activating the antibody with an activating agent and reacting it with a cross-linking agent to prepare a mixture containing a plurality of polymers comprises: using carbodiimide and N-hydroxysuccinyl in an acidic buffer The imine-activated antibody is used to prepare the activated antibody; and the activated antibody is desalted and reacted with a cross-linking agent to prepare a mixture containing multiple polymers.

Step 2: Screen the mixture for the desired multimer.

According to the size of the molecular weight of the target multimer to be obtained, a protein purification apparatus is used to screen and purify the target multimer.

In one embodiment, a protein purifier is used, a pre-packed column that is easy to purify and separate large proteins and protein complexes is selected, and after equilibrating the pre-packed column, a high-purity target is obtained by purifying and separating a mixture containing multiple polymers. Multimers; the collected multimers of interest were then determined by SDS-PAGE electrophoresis experiments.

Step 3: Use a marker to label the target multimer to prepare an antibody complex.

In this embodiment, the acridine ester whose carboxyl group has been activated is mixed and reacted with the target multimer obtained in step 2 to prepare an antibody complex. The carboxyl group of the acridine ester reacts with the lysine residue on the antibody of the target multimer, so that the acridine ester is labeled on the antibody of the target multimer, and the antibody complex is prepared.

Specifically, the activator used to activate the acridine ester includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Optionally, the activator for activating the acridine ester further includes at least one of N-hydroxysuccinimide (NHS) and N-hydroxysulfosuccinimide (Sulfo-NHS). When using EDC to activate acridinium ester, EDC reacts with acridinium ester to obtain O-acyl isourea intermediate product, and the acridine ester is labeled to the target multimer by reacting the O-acyl isourea intermediate product with the antibody of the target polymer. on the aggregated antibody. However, the O-acylisourea intermediates have poor stability and are easily hydrolyzed. Therefore, by adding at least one of N-hydroxysuccinimide and N-hydroxysulfosuccinimide, the O-acylisourea is made The stability of the urea intermediate is improved, thereby increasing the labeling efficiency of the acridinium ester.

It can be understood that, in other embodiments, the activator for activating the acridine ester is not limited to the above, and can also be other substances that can activate the carboxyl group of the acridine ester. Of course, in other embodiments, the label is not limited to acridinium ester, but can also be other substances, as long as the substance can be labeled on the antibody of the target multimer.

Further, in this embodiment, a two-step method is used to label the acridinium ester to the antibody of the target multimer. Specifically, after the activator for activating the acridine ester is mixed and reacted with the acridine ester, the excess activator is removed by liquid chromatography (HPLC) to obtain the purified activated acridine ester; then, the purified The activated acridinium ester is mixed and reacted with the target multimer, so that the acridine ester is labeled on the antibody of the target multimer, and the antibody complex is prepared. It will be appreciated that, in some embodiments, a one-step method (i.e. the direct mixing reaction of the acridinium ester, the activator for activating the acridine ester, and the target polymer) can also be used to label the acridine ester to the target polymer. on the antibody.

In the present embodiment, the label is labeled on the antibody of the multimer of interest by reacting the carboxyl group of the label with the lysine residue of the antibody of the multimer of interest. It can be understood that, in other embodiments, the manner of linking the label to the antibody is not limited to the above, and the label can also be labeled to the multimeric antibody by other means.

In this embodiment, the antibody is formed into a multimer, and then the label is labeled to form an antibody complex. Of course, in other embodiments, an antibody complex can also be formed by first labeling the antibody and then cross-linking the labeled antibody.

The preparation method of the above-mentioned antibody complex is simple and easy to operate, and the antibody complex prepared according to the above-mentioned preparation method of the antibody complex comprises a plurality of labeled antibodies linked by a cross-linking agent. At the same time, one antigen can correspond to multiple markers, so that the luminescence signal during detection is amplified, thereby increasing the signal-to-noise ratio of detection and improving the sensitivity of detection.

The above-mentioned antibody complex can realize that one antigen corresponds to multiple labels during detection, which can improve the detection sensitivity. Therefore, an embodiment also provides an application of the above-mentioned antibody complex in the preparation of a detection kit.

In addition, an embodiment also provides a detection kit, which includes the above-mentioned antibody complex for detecting an antigen that can specifically bind to the antibody in the antibody complex.

Optionally, the above-mentioned detection kit further includes at least one of a buffer and a solid phase carrier.

In an optional specific example, the buffer is selected from at least one of phosphate buffer, carbonate buffer and borate buffer. The solid phase carrier is selected from one of magnetic beads and resin.

The above-mentioned detection kit includes the above-mentioned antibody complex, and has good sensitivity.

The following describes in detail with reference to specific embodiments. The following examples do not include other components except inevitable impurities unless otherwise specified. Unless otherwise specified, the reagents and instruments used in the examples are routinely selected in the art. The experimental methods for which specific conditions are not indicated in the examples are realized according to conventional conditions, such as conditions described in literatures, books or methods recommended by manufacturers. Hereinafter, unless otherwise specified, the reagents used were purchased from Sigma-aldrich Company, and the grades were of analytical grade. The anti-CHI3L1 monoclonal antibody used in the following was purchased from Hangzhou Puwang Biotechnology Co., Ltd.

Example 1

Referring to Figure 1, the preparation method of the antibody complex of the present embodiment includes but is not limited to the following steps:

(1) Dissolve 1 mg of anti-CHI3L1 monoclonal antibody for labeling in 1 mL of 50 mM MES buffer (pH 5.0), with a final concentration of 6.67 nmol/L, to prepare an antibody solution.

(2) To the antibody solution in step (1), add EDC (the final concentration of EDC in the mixed solution consisting of the antibody solution, EDC and NHS is 0.34 mmol/L) and NHS (NHS in the antibody solution, EDC and NHS) The final concentration in the mixed solution was 3.4 mmol/L), and after reacting for 30 minutes at 25°C, the AKTA purification equipment of GE company was used, and 5mL G25 prepacked purification column (GE company) was used, and 50mM MES (pH) was used. 5.0) Buffer was used as an equilibration buffer to remove unreacted EDC and NHS to obtain purified activated antibody.

(3) Add adipic acid dihydrazide to the activated antibody purified in step (2) (the final concentration of adipic acid dihydrazide in the mixture of antibody, buffer and adipic acid dihydrazide is 0.78 mmol/L), and reacted at 25 °C for 1 h to obtain a mixture containing multiple polymers.

(4) Pass the mixture containing multiple polymers prepared in step (3) through the AKTA equipment of GE Company, select a purification column SuperoseTM 6 Increase 10/300GL which is easy to separate protein complexes, and buffer it with 50mM PBS (pH 8.0). The solution was used as an elution buffer for purification and separation to obtain a high-purity homotetrameric anti-CHI3L1 monoclonal antibody. Among them, the purification and separation pattern of the mixture containing multiple multimers is shown in Figure 2, and the No. 3 peak in Figure 2 is the homotetrameric anti-CHI3L1 monoclonal antibody.

(5) To the homotetrameric anti-CHI3L1 monoclonal antibody in step (4), add 10 μL of 10 mmol/L acridine ester dissolved in DMSO solvent, react at 25° C. for 1 h, and then use GE’s AKTA purification equipment, Select 5mL G25 pre-packed purification column (GE company), use 50mM PBS (pH 8.0) buffer as elution buffer for elution, remove acridinium ester, DMSO, NHS and acridinium ester hydrolyzate acrinic acid to obtain this product. Implemented antibody complexes.

Example 2

The preparation method of the antibody complex in this example is roughly the same as that in Example 1. The difference is that the mixture containing multiple polymers obtained by crosslinking with a crosslinking agent in this example is not purified and separated, but only desalted to remove Unreacted crosslinker. The specific preparation steps include:

(2) To the antibody solution in step (1), add EDC (the final concentration of EDC in the mixed solution consisting of the antibody solution, EDC and NHS is 0.34 mmol/L) and NHS (NHS in the antibody solution, EDC and NHS) The final concentration in the mixed solution was 3.4 mmol/L), and after reacting for 30 minutes at 25°C, the AKTA purification equipment of GE company was used, and 5mL G25 prepacked purification column (GE company) was used, and 50mM MES (pH) was used. 5.0) The buffer is used as the equilibration buffer to remove free NHS and reaction by-products to obtain the purified activated antibody.

(4) Pass the mixture containing multiple polymers prepared in step (3) through the AKTA equipment of GE Company, select a purification column SuperoseTM 6 Increase 10/300GL which is easy to separate protein complexes, and buffer it with 50mM PBS (pH 8.0). The solution is used as an elution buffer for purification and separation to remove unreacted small molecule cross-linking agents and by-products to obtain a mixture solution containing multiple polymers.

(5) Add 10 μL of 10 mmol/L acridine ester dissolved in DMSO solvent to the mixture solution containing multiple polymers in step (4), react at 25° C. for 1 h, and then use the AKTA purification equipment of GE company to select 5mL G25 pre-packed purification column (GE company), eluted with 50mM PBS (pH 8.0) buffer as elution buffer to remove free acridinium ester, DMSO, NHS and acridinium ester hydrolyzate acridine acid, etc., The antibody complex of this example was obtained.

Comparative Example 1

In this comparative example, an acridinium ester-labeled anti-CHI3L1 monoclonal antibody was prepared. Specific steps include but are not limited to:

(1) Dissolve 1 mg of anti-CHI3L1 monoclonal antibody for labeling in 1 mL of 50 mM PBS buffer (pH 5.0), with a final concentration of 6.67 nmol/L, to prepare an antibody solution.

(2) Add 10 μL of 10 mmol/L commercial acridine ester (with NHS activating group, NHS-DMAE-NSP) dissolved in DMSO solvent to the antibody solution prepared in step (1), at 25°C The reaction was carried out for 1 h, and then the AKTA purification equipment of GE company was used, 5mL G25 pre-packed purification column (GE company) was selected, and 50mM PBS (pH 8.0) buffer was used as the elution buffer to remove free acridine ester, NHS, DMSO and acridinium ester hydrolyzed product acridic acid, etc., to obtain acridinium ester-labeled anti-CHI3L1 monoclonal antibody.

Experimental example 1

The antibody complexes prepared in each example and the acridinium ester-labeled anti-CHI3L1 monoclonal antibody prepared in Comparative Example 1 were used in the chemiluminescence immunoassay of CHI3L1. The concrete steps of each embodiment and comparative example 1 all include the following steps:

Take a serum sample containing 100 ng/mL of CHI3L1, add 50 μg of anti-CHI3L1 monoclonal antibody-coated magnetic beads, and then add 10 pmol of the antibody complex corresponding to each example or the acridine ester-labeled anti-CHI3L1 monoclonal corresponding to Comparative Example 1. Antibodies were incubated at 37°C for 10 min, then magnetically separated and washed 3 times, and then added 100 μL HNO ₃ -H ₂ O ₂ solution and 100 μL sodium hydroxide solution successively, and measured with iFlash3000 chemiluminescence immunoassay analyzer (Yahuilong Biotechnology). For the luminescence value, three measurements were performed in parallel, and the average value was taken. The results are shown in Table 1.

Table 1

组别group	平均发光值(RLU)Average Luminous Value (RLU)
实施例1Example 1	55245725524572
实施例2Example 2	32421543242154
对比例1Comparative Example 1	841269841269

The greater the luminescence value of the immunoassay results, the better the activity of the acridinium ester label. Therefore, it can be seen from Table 1 that the luminescence value of the antibody complex of Example 1 is the largest when CHI3L1 is detected, and the luminescence value of Example 1 is greater than that of Example 2, indicating that when the antibody complex of Example 1 is used to detect CHI3L1, the sensitivity will be higher. The sensitivity of the acridinium ester-labeled homotetrameric monoclonal antibody is higher than that of the other examples and comparative examples, and the sensitivity of the acridinium ester-labeled homopolymeric monoclonal antibody is higher than that of a mixture composed of acridinium ester-labeled homopolymeric monoclonal antibodies.

Experimental example 2

The antibody complexes prepared in each Example and the acridinium ester-labeled anti-CHI3L1 monoclonal antibody prepared in Comparative Example 1 were used in the chemiluminescence immunoassay of CHI3L1 concentration in blood samples. The concrete steps of each embodiment and comparative example 1 include the following steps:

To the CHI3L1 sample solutions of a series of concentrations, 50 μg of anti-CHI3L1 monoclonal antibody-coated magnetic bead reagent and 5 pmol of the antibody complex corresponding to each example or the acridine ester-labeled anti-CHI3L1 monoclonal antibody corresponding to Comparative Example 1 were added, 37 After incubation at ℃ for 10 min, magnetic separation and cleaning were performed three times. Then, 100 μL of HNO ₃ -H ₂ O ₂ solution and 100 μL of sodium hydroxide solution were added successively. Take the average value, and calculate the detection limit, repeatability and detection range, the results are shown in Table 2, where:

Repeatability: 1000μg/mL (±20%) and 6500μg/mL (±20%) repeatable reference materials were used for testing, each test was performed 10 times, and the average (M) and standard of the 10 measurement results were calculated. Difference (SD), coefficient of variation (CV) was obtained according to the formula CV=SD/M×100%.

The detection limit is carried out by the experimental method recommended in the CLSI EP17-A document;

Linear range detection: Dilute the high-value samples close to the upper limit of the linear range by 2 times, 4 times, 6 times, 8 times and 10 times, and the low value concentration samples are based on the detection limit, 2 times, 4 times, 6 times and 6 times the detection limit. times and 8 times. The samples of each concentration were tested 3 times, the average value was calculated, the average value of the results and the dilution ratio were linearly fitted by the least squares method, and the linear correlation coefficient r was calculated.

Table 2

	实施例1Example 1	实施例2Example 2	对比例1Comparative Example 1
检测限(ng/mL)Detection limit (ng/mL)	1.51.5	22	55
重复性(CV％)Repeatability (CV%)	3.2％3.2%	7.2％7.2%	8.7％8.7%

检测范围(ng/mL)Detection range (ng/mL)	1.5～20001.5～2000	5.1～20005.1～2000	10～200010～2000
rr	0.99970.9997	0.99900.9990	0.99780.9978

As can be seen from the results in Table 2, when using the antibody complexes of Examples 1 to 2 to detect CHI3L1, the detection limit is low, the sensitivity is high, the repeatability is good, the detection range is wide, and the linearity is good (r≥0.9900), which can be applied to chemiluminescence immunoassays. Analysis and results are good.

The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be regarded as the scope described in this specification.

The above-mentioned embodiments only represent several embodiments of the present application, and the descriptions thereof are relatively specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the scope of protection of the patent of the present application shall be subject to the appended claims.

Claims

An antibody complex comprising a plurality of label-labeled antibodies linked by a cross-linking agent.
The antibody complex according to claim 1, wherein the cross-linking agent is a dihydrazide compound.
The antibody complex according to claim 2, wherein the cross-linking agent is selected from at least one of maleic acid dihydrazide, oxalic acid dihydrazide and adipic acid dihydrazide.
The antibody complex according to claim 1, wherein the label in the label-labeled antibody is selected from acridine esters, ruthenium terpyridine, adamantane, luminol, derivatives of luminol, isoforms One of luminol, derivatives of isoluminol, horseradish peroxidase and alkaline phosphatase.
The antibody complex according to claim 1, wherein the antibody in the labeled antibody is CHI3L1 antibody, procalcitonin antibody or cardiac troponin I antibody.
The antibody complex according to any one of claims 1 to 5, wherein the antibody complex comprises four of the labeled antibodies.
The antibody complex of claim 1, wherein the antibody complex comprises four acridinium ester-labeled CHI3L1 antibodies linked by adipic acid dihydrazide.
A method of preparing an antibody complex, comprising:

Using an activator to activate the antibody and react with a cross-linking agent to prepare a mixture containing multiple polymers, wherein the multimer is formed by cross-linking at least two antibodies through the cross-linking agent;

screening the mixture for the desired multimer; and

The multimer of interest is labeled with a label to prepare an antibody complex.
The method of claim 8, wherein the activating agent is used to activate the carboxyl group of the antibody, and the cross-linking agent has at least two amino groups.
The method of claim 9, wherein the activator comprises carbodiimide, and the cross-linking agent is a dihydrazide compound.
The method of claim 10, wherein the activator further comprises at least one of N-hydroxysuccinimide and N-hydroxysulfosuccinimide.
The method of claim 11, wherein the activator comprises carbodiimide and N-hydroxysuccinimide, wherein the molar ratio of carbodiimide and N-hydroxysuccinimide is 10:(1 ~200).
The method of claim 8, wherein the molar amount of the activator is 5 times to 1000 times the molar amount of the antibody.
The method of claim 8, wherein the antibody is activated in an acidic buffer.
The method according to claim 8, wherein the marker is selected from acridine ester, ruthenium terpyridine, adamantane, luminol, derivatives of luminol, isoluminol, isoluminol One of the derivatives of horseradish peroxidase and alkaline phosphatase.
A detection kit, comprising the antibody complex according to any one of claims 1 to 7.
The detection kit according to claim 16, wherein the detection kit further comprises at least one of a buffer solution and a solid phase carrier.