Polymeric antibody for immunoturbidimetry as well as preparation method and application thereof

Abstract

The invention discloses a polymeric antibody for immunoturbidimetry as well as a preparation method and application thereof, relates to the technical field of in-vitro detection, and comprises a polymeric technology based on a polymer skeleton coupling antibody, and the polymeric antibody prepared by the technology has a multivalent effect, can replace the existing monoclonal antibody or secondary antibody immunoturbidimetry reagent, and can be applied to immunoturbidimetry. The HOOK-resistant monoclonal antibody can be well applied to various immunoturbidimetric biochemical reagents, not only has extremely high sensitivity to low-value samples, but also has an excellent HOOK-resistant effect on high-value samples, and meanwhile, avoids different stability problems caused by dynamic combination of a secondary antibody detection system.

Description

Technical Field

[0001] The present invention relates to the field of in vitro diagnosis, and particularly to polymeric antibodies for immunoturbidimetric detection, their preparation methods and applications. Background Art

[0002] Immunoturbidimetric detection technology is an optical analysis method based on the specific binding reaction between antigen and antibody. By detecting the turbidity change caused by the formation of antigen-antibody complexes in solution, quantitative analysis of target molecules can be achieved. Since its development in the 1970s, due to its advantages such as simple operation, no need for complex labeling, and fast detection speed, it has been widely used in clinical diagnosis (such as the quantification of C-reactive protein and immunoglobulins) and biomedical research fields. Its core principle is that when an antibody binds to a soluble antigen to form an immune complex, the particle size in the solution increases, resulting in a change in light scattering or transmitted light intensity. By measuring the absorbance or scattered light signal with a spectrophotometer or a dedicated turbidimeter, a concentration-signal standard curve is established to complete quantitative analysis.

[0003] To improve the detection sensitivity, traditional immunoturbidimetry often introduces a secondary antibody amplification system. The core role of the secondary antibody is to amplify the signal intensity of the antigen-antibody complex through a cascade reaction. In immunoturbidimetric detection, after the primary antibody binds to the target antigen, the secondary antibody recognizes the Fc segment of the primary antibody (such as goat anti-mouse IgG antibody) to form a larger complex network, such as the "primary antibody-antigen-primary antibody-secondary antibody" complex. The particle size of this complex increases significantly, resulting in a stronger change in light scattering or transmitted light signal, thereby improving the detection sensitivity from the μg / mL level of traditional methods to ng / mL or even lower. For example, in the detection of C-reactive protein (CRP), the introduction of the secondary antibody system can lower the detection limit to below 0.5 mg / L, meeting the clinical demand for accurate monitoring of low-concentration inflammatory markers.

[0004] However, the storage of secondary antibody reagents requires strict cold chain conditions, with relatively high transportation and storage costs. Moreover, repeated freezing and thawing easily lead to a decrease in antibody activity, affecting the detection consistency. In addition, the animal-derived characteristics of secondary antibodies may cause compatibility problems with samples of different species, requiring customized development and further increasing the reagent cost. In multiplex turbidimetric analysis for simultaneously detecting multiple antigens, the risk of cross-reaction of the secondary antibody system increases significantly. For example, if two primary antibodies are from the same species, the secondary antibody will recognize all primary antibodies simultaneously, resulting in signal crosstalk between different detection channels and severely limiting the throughput of multiplex detection.

[0005] In addition, the secondary antibody detection system relies on non-covalent interactions to maintain the binding relationship. Under conditions such as thermal acceleration, this non-covalent bond is extremely vulnerable to damage, resulting in a significant reduction in the stability of immune binding, thereby affecting the reliability of the detection results. At the same time, the secondary antibody detection system also has problems with poor detection linearity. When facing HbA1c samples in different concentration ranges, especially high-concentration or low-concentration extreme samples, it is difficult to maintain an accurate linear correspondence between the detection signal and the actual concentration, which limits the detection accuracy within a certain range and cannot meet all the requirements for high-precision detection. Finally, the operation of existing detection kits is complicated and time-consuming. From sample pretreatment, to the step-by-step addition and mixing of various reagents, to subsequent detection and data analysis, the entire operation process involves multiple steps and links. This not only requires high professional skills from the operators, but also takes a long time, increasing the labor cost and time cost, and is also prone to introducing human errors during the operation process.

[0006] CN110514848A and CN113125759A use protein substances such as bovine serum albumin, gelatin, protein G, and protein A as antibody carriers in immunoturbidimetric assays. However, it has been found in practice that such reagents have insufficient sensitivity in the detection of low-value samples, are prone to false-negative detection results, and are not conducive to the accurate diagnosis of early diseases. Summary of the Invention

[0007] Based on the disadvantages of immunoturbidimetry in detection, the present invention discloses a polymerization technology based on polymer backbone-coupled antibodies. The multimeric antibodies prepared by this technology have a multivalent effect and can replace existing monoclonal or secondary antibody immunoturbidimetric reagents. They can be well applied to immunoturbidimetric biochemical reagents and can be widely used in the detection of various biochemical indicators such as adiponectin, complement components, troponin I, troponin T, and HbA1c.

[0008] Therefore, in one aspect of the present invention, a polymeric antibody for immunoturbidimetric detection is provided. The polymeric antibody contains a polymer backbone and antibodies coupled to the polymer backbone. The molecular weight of the polymer backbone is 50 KDa - 800 KDa; further, the molecular weight of the polymer backbone is 60 KDa - 600 KDa, and more preferably 70 KDa - 450 KDa.

[0009] Furthermore, the polymer backbone material of the present invention is selected from hydrophilic polymer substances having active functional groups. However, the protein substances themselves have irregular structures, and the binding sites of antibodies on their surfaces also show irregularities, which may pose challenges to the consistency of detection results and the detection of low-value samples. Therefore, the polymer backbone material of the present invention is a non-protein carrier; further, the polymer backbone is selected from linear or dendritic polymers, including one or more of dextran and its modified derivatives, polyethyleneimine (PEI), and polyamidoamine (PAMAM) dendrimer. The dextran modified derivative is modified on the dextran backbone material to generate dextran with carboxyl, amino, or aldehyde groups, namely carboxyl dextran, amino dextran, and aldehyde dextran. Preferably, the polymer backbone is selected from one or more of dextran, carboxyl dextran, amino dextran, and aldehyde dextran.

[0010] The backbone material of the present invention has active functional groups on it. Utilizing the supporting effect of the backbone material, it can provide multiple binding sites for antibodies, and then couple multiple antibodies on the backbone material (a reaction process is as Figure 1 shown), which is beneficial to the rapid enrichment of latex microspheres, and then achieves the purpose of high-speed and sensitive detection.

[0011] For some backbone materials, a significant advantage is that the coupling of the backbone material and the antibody can be achieved without the bridging action of a cross-linking agent. Taking the HbA1c polymer antibody as an example, the aldehyde group carried by aldehyde dextran can spontaneously react with the amino group of the HbA1c antibody. After the reaction is sufficient at room temperature, sodium borohydride is used to terminate the reaction and block the remaining aldehyde groups to complete the reaction. In addition, for some backbone materials, such as amino dextran, it can also be activated with 2-iminothiolane (2-IT) or N-succinimidyl 4-cyclohexane-1-carboxylate (SMCC). At the same time, the antibody part is activated with SMCC or 2-IT, and then the backbone material and the antibody are reacted to prepare the polymer antibody. It can be understood that when the backbone material is dextran, in one implementation, a coupling agent is used to make the dextran backbone carry aldehyde or amino groups, and then react with the amino, carboxyl, or mercapto groups on the antibody. Specifically, the hydroxyl group on dextran reacts with the aldehyde group of the coupling agent CHO-Ph-PEG-NH-Fmo to form a conjugate. Fmoc is easily removed under acidic conditions, thereby releasing the free amino group as an active functional group to further react with the antibody. Another example is that amino siloxane generates silanol groups through hydrolysis, and then condenses with the hydroxyl groups on the surface of dextran, while retaining the amino functional group to achieve the introduction of amino groups. Those skilled in the art are fully aware based on the above examples that different functional groups can be introduced through other coupling agent methods, which does not pose a substantial technical obstacle to those skilled in the art.

[0012] On the other hand, the present invention provides an immunoturbidimetric reagent, which contains the polymeric antibody described in the present invention; further, the reagent also contains one or more of a buffer, inorganic salts, surfactants, preservatives and stabilizers.

[0013] Further, the buffer is selected from at least one of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-morpholinoethanesulfonic acid, borate, carbonate, citrate, Tris, and Good's buffer; the inorganic salts are selected from at least one of sodium sulfate, sodium chloride, and ammonium chloride; the surfactants are selected from at least one of Tween 20, Tween 80, cocamidopropyl betaine, imidazole, phosphate betaine, and carboxylate betaine; the preservatives are selected from at least one of sodium azide, ProClin 300, ProClin 950, gentamicin, and benzoates; the stabilizers are selected from at least one of sucrose, bovine serum albumin, D-trehalose, mannitol, and glycerol.

[0014] On the other hand, the present invention provides a kit, which contains the above-mentioned polymeric antibody reagent, denoted as R2.

[0015] Further, the kit also includes a reagent containing latex microspheres, denoted as R1. Further, the mass ratio of the latex microspheres to the polymeric antibody is 10:1 - 400:1; preferably, the mass ratio of the latex microspheres to the polymeric antibody is 15:1 - 300:1, more preferably 20:1 - 200:1, or 30:1, 50:1, 70:1, 100:1 or 150:1.

[0016] According to the different analytes, optionally, the latex microspheres are coated with an antibody or not coated with an antibody; preferably, the latex microspheres are coated with an antibody. In one embodiment, when the analyte is HbA1c, the latex microspheres are not coated with an antibody; in another embodiment, when the analyte is adiponectin or cTnT, the latex microspheres are coated with an anti-adiponectin or anti-cTnT antibody.

[0017] Further, the reagent R1 also includes one or more of a buffer, a surfactant and a preservative; further, the buffer is selected from at least one of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, Tris buffer, Good's buffer, borate buffer, and citrate buffer; the surfactant is selected from at least one of Tween 80, Tween 20, carboxylate betaine, cocamidopropyl betaine, imidazoles, and phosphate betaine; the preservative is selected from at least one of sodium azide, ProClin 300, ProClin 950, benzoates, and gentamicin.

[0018] The pH of the reagent R1 is 6.0 - 9.0, and the pH of the reagent R2 is 5.0 - 9.0; further, the pH of the reagent R1 is 6.5 - 8.5, and the pH of the reagent R2 is 5.5 - 8.5.

[0019] Another aspect of the present invention lies in providing an application of a polymeric antibody in the preparation of an immunoturbidimetric detection reagent. The polymeric antibody contains a polymer backbone and antibodies conjugated to the polymer backbone, and the molecular weight of the polymer backbone is 50 KDa - 800 KDa; further, the molecular weight of the polymer backbone is 60 KDa - 600 KDa, and more preferably 70 KDa - 450 KDa.

[0020] Further, the polymer backbone material is selected from hydrophilic polymer substances with active functional groups, and the polymer backbone material is a non - protein substance; preferably a linear or dendritic polymer, including one or more of dextran and its modified derivatives, polyethyleneimine (PEI), and dendrimer PAMAM. The dextran modified derivatives are obtained by modifying the dextran backbone material to generate dextran with carboxyl, amino, or aldehyde groups, namely carboxyl dextran, amino dextran, and aldehyde dextran. Preferably, the polymer backbone is selected from one or more of dextran, carboxyl dextran, amino dextran, and aldehyde dextran.

[0021] Further, the antibody is selected from one or more of an anti - adiponectin antibody, an anti - complement antibody, an anti - HbA1c antibody, an anti - apolipoprotein A1 antibody, an anti - apolipoprotein B antibody, and an anti - immunoglobulin antibody; the complement includes serum complement C1q, complement C3, or complement C4.

[0022] The antibody described in the present invention is selected from one of polyclonal antibody, monoclonal antibody, or antibody fragment; the antibody fragment is selected from any one of F(ab')2, Fab', Fab, Fv, and scFv; preferably a monoclonal antibody.

[0023] The inventors believe that in the two - antibody turbidimetric detection system, an antibody that can only bind two molecules has limited bridging effect. However, the polymeric antibody of the present invention has multiple antibodies, and the significant multivalent effect promotes larger and faster aggregation of microspheres, enabling sensitive capture even in the case of early - stage and trace analytes to be measured, and not easily missing early signals of some potential disease risks. Moreover, since the secondary antibody reagent part is omitted, problems such as the cumbersome operation and operation errors faced when reagent 2 and reagent 3 in the secondary antibody detection system are mixed by the inspector on - site are avoided. Therefore, the detection system of the present invention makes the detection results more accurate.

[0024] In addition, in the aggregated antibody, the antibody and the backbone material are covalently linked, greatly improving the stability of the detection reagent, thereby providing reliable results. Moreover, selecting a backbone material with a specific molecular weight contributes to the stability and reliability of the reagent during long-term storage.

[0025] The present invention selects a linear or dendritic regular polymer backbone material. Under the condition of the same molecular weight, the spatial size of the polymer backbone material described in the present invention is larger, and the multivalent effect is more prominent. Therefore, it is more conducive to the consistency of detection results and the high-sensitivity detection of low-value samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram for preparing an aggregated antibody of the present invention; Figure 2 Molecular sieve analysis diagram of the aggregated antibody based on aldehyde dextran backbone of the present invention; Figure 3 Molecular sieve analysis diagram of the aggregated antibody based on aminodextran backbone of the present invention; Figure 4 Reagent function test results of the aggregated antibody of HbA1c established by the present invention; wherein, the abscissa is the concentration (%), and the ordinate is the absorbance (OD value); Figure 5 Reagent thermal stability acceleration function test results of the aggregated antibody of HbA1c established by the present invention; wherein, the abscissa is the concentration (%), and the ordinate is the absorbance (OD value); Figure 6 Function test comparison results between the formulated reagent of the aggregated antibody of HbA1c established by the present invention and a commercially available kit; wherein, the abscissa is the concentration (%), and the ordinate is the absorbance (OD value); Figure 7 Function test comparison results between the formulated reagent of the aggregated antibody of serum complement (C1q) established by the present invention and a commercially available kit; wherein, the abscissa is the concentration (mg / L), and the ordinate is the absorbance (OD value). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0028] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0029] In this article, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0030] Example 1

[0031] In this example, aldehyde group dextran was used as the backbone, and HbA1c monoclonal antibody (Okai Biotech - k17t8) was used as the basic raw material to prepare a polyclonal antibody. The preparation method is as follows: 1) Take an appropriate amount of aldehyde group dextran (molecular weight about 100KDa) and dissolve it in PBS (20mM), and mix well; 2) Add HbA1c monoclonal antibody to the aldehyde group dextran solution, where the molar ratio of HbA1c monoclonal antibody to aldehyde group dextran is 20:1, and react at room temperature for 3 hours. After the reaction is complete, add an appropriate amount of sodium borohydride to terminate the reaction; 3) Dialyze the sample into a buffer system of pH 7.4, 20mM PBS, and perform molecular sieve detection on the obtained polymeric antibody. The results are shown in Figure 2.

[0032] This technical solution does not require the introduction of an activation operation step. The aldehyde group of dextran can directly undergo a covalent reaction with the amino group on the antibody. Figure 2 It can be seen that even under simple, direct and mild reaction conditions, the content of polyclonal antibody in the reaction product of aldehyde group dextran and antibody exceeds 85%, proving the effectiveness and high efficiency of the reaction system. And the peak shape is sharp, indicating a high content and high degree of homogeneity of antibodies with a high degree of polymerization.

[0033] Referring to the above method, aldehyde group dextrans with different molecular weights were selected as the backbone materials to prepare different polyclonal antibodies.

[0034] Example 2

[0035] In this example, amino group dextran was used as the backbone, and C1q monoclonal antibody (thermofisher - MA1 - 40312) was used as the basic raw material to prepare a polyclonal antibody.

[0036] 1) Take an appropriate amount of amino group dextran (molecular weight about 80KDa) and dissolve it in PBS; take an appropriate amount of 2 - IT and dissolve it in PBS (20mM PBS) solution; 2) Mix amino group dextran and 2 - IT in a molar ratio of 1:20 and mix well, and react at room temperature for 2 hours to obtain activated amino group dextran after the reaction; 3) Take an appropriate amount of Sulfo - SMCC and dissolve it in an aqueous solution, and feed it according to the molar ratio of antibody to Sulfo - SMCC of 1:10, and react at room temperature for 2 hours to obtain activated antibody; 4) Mix the activated antibody and the activated glucosamine in a molar ratio of 20:1. After reacting for 2 hours at room temperature, weigh an appropriate amount of maleimide and dissolve it in DMSO. The feeding amount is 10-fold molar equivalent of 2-IT for terminating the reaction. Finally, dialyze into a pH 7.4, 20 mM PBS solution and perform molecular sieve detection on the polymeric antibody.

[0037] The results are as Figure 3 shown. The content of the polyclonal antibody is 82.11%. From the peak shape, the content of the antibody with high polymerization degree is high, the valence state is high, and the peak shape is relatively sharp, indicating that the antibody is more homogeneous.

[0038] Example 3

[0039] Prepare a polyclonal antibody substance with BSA as the backbone material according to the method described in Example 1 of CN110514848A, where the molar ratio of BSA to the antibody is 1:20, and the antibody selected is the HbA1c monoclonal antibody.

[0040] Example 4

[0041] The HbA1c detection reagent includes reagent R1 and reagent R2.

[0042] a. Preparation of reagent R1 Prepare reagent R1 according to the following formula: the percentage is by mass fraction Latex microspheres (pH 8.0) 0.1% HEPES buffer 50 mM Tween 80 0.1% ProClin-300 0.2%; b. Preparation of reagent R2 Prepare reagent R2 according to the following formula: the percentage is by mass fraction The HbA1c polymeric antibody prepared in Example 1 50 mg / L HEPES buffer (pH 6.5) 80 mM Sodium chloride 0.95% Tween 20 0.1% BSA 0.5 g / L ProClin-300 0.2%.

[0043] At the same time, select the HbA1c polymeric antibody with BSA as the backbone prepared in Example 3 and prepare the R2 reagent according to the above method.

[0044] Example 5

[0045] The C1q detection reagent includes reagent R1 and reagent R2.

[0046] a. Preparation of Reagent R1 Prepare Reagent R1 according to the following formula: The percentages are mass fractions HEPES buffer (pH 7.0) 100 mM Emulsifier 3 g / L Tween 80 0.2% ProClin-300 0.1%, b. Preparation of Reagent R2 Prepare Reagent R2 according to the following formula: The percentages are mass fractions C1q polymerized antibody prepared in Example 2 1 mg / L MES buffer (pH 6.2) 100 mM Sodium chloride 1.5% Tween 20 0.2% BSA 0.5 g / L ProClin-300 0.2%.

[0047] Effect Example 1 Verify with the turbidimetric reagent prepared in Example 4 to prove the effectiveness of the polyclonal antibody. The verification scheme is as follows: a. Setting of reagent detection parameters Method: Endpoint method Reaction direction: Rising Samples: Reagent R1:Reagent R2 = 8:300:100 (volume ratio) Main wavelength: 660 nm Sub-wavelength: 800 nm Reaction temperature: 37 °C Reaction time: 10 min.

[0048] b. The sample addition method and measurement scheme are shown in Table 1.

[0049] Table 1 Measurement scheme

[0050] c. Calculation method Use the multi-point non-linear / semi-logarithmic calibration mode, with the spline function as the calculation mode. The abscissa is the concentration (%), and the ordinate is the OD absorbance. According to the values of the calibrator and the change value of the absorbance, a dose / response curve is made. The content of the target analyte in the sample can be calculated on the dose / response curve based on its absorbance change value. The calibration curve reflects the change of the absorbance with the change of the concentration. The greater the slope, the steeper the calibration curve, and the higher its analysis sensitivity.

[0051] The results are asFigure 4 As shown, the polymeric antibody with an aldehyde dextran backbone has a reading value of over 600 at the low-concentration sample S2 point, indicating that the polymeric antibody has high sensitivity, and the S4-S5 points at high concentration show exponential growth, predicting that the polymeric antibody has stronger anti-HOOK ability.

[0052] Effect Example 2 A thermal stability acceleration experiment was carried out on the raw materials and reagents prepared in Example 4 (the aldehyde dextran has a molecular weight of 100KDa), demonstrating the stability of the polyclonal antibody of the present invention at high temperatures.

[0053] According to the reagent R2 preparation scheme in Effect Example 1, 5 mL of the reagent was respectively prepared and placed in an environment at 4°C and 37°C for 7 days, and then a biochemical analyzer was used to explore the performance. The specific detection method is as in Effect Example 1, and the results are as Figure 5 shown. The horizontal axis therein is the target value of HbA1c of the standard product, and S1-S5 are 0, 4.8, 7.6, 10.6, and 13.8. The polymeric antibody has good stability under the thermal acceleration condition in the R2 reagent buffer, and the deviation of the readings of each point from the reagent under the 4°C condition is within 5%.

[0054] According to the above method, the stability of polyclonal antibody reagents with different molecular weights was tested, and the detection errors corresponding to 5 target values after 7 days in environments at 4°C and 37°C were evaluated. The more target value points with a deviation exceeding 5%, the worse the stability. The information and results of specific reagents are shown in Table 2.

[0055] Table 2 Comparison of the stability of polyclonal antibodies prepared from backbone materials with different molecular weights

[0056] It can be seen from Table 2 that the molecular weight of the backbone material has a certain influence on the test stability. When the molecular weight of the backbone material exceeds 600KDa, the error deviation points are still acceptable; when the molecular weight exceeds 800KDa, the number of deviation points further increases. The possible reason is that the larger backbone material provides more binding sites, and more conjugated antibodies bind. Long-term storage will affect the antibody conjugation effect, thereby leading to the deterioration of the stability of the polymeric antibody. The inventors also found similar results in other polymer backbone materials such as dextran and PEI. In addition, when the molecular weight of the backbone material is too small, the amount of conjugated antibody on its surface is effective, and the anti-Hook ability of high-concentration analytes is not significantly improved (the results are not shown). Therefore, considering indicators such as detection effect and storage stability, the backbone molecular weight should be 50KDa - 800KDa, preferably 60KDa - 600KDa.

[0057] Effect Example 3 To prove the effectiveness of the polymeric antibody of the present invention, the polymeric antibody reagent prepared by the present invention, the polymeric antibody disclosed in the prior art and the commercially available products were further compared and demonstrated. Taking HbA1c detection as an example, the reagent containing the polymeric antibody with a dextran derivative backbone material and the polymeric antibody with a BSA backbone material in Example 4 were selected, and 5 mL of each reagent was prepared for standby. The commercially available zybio glycated hemoglobin detection kit (secondary antibody detection system) was selected, and the performance of the three groups of experiments was explored using a biochemical analyzer (EXC2000, zybio). The specific detection method was as in Effect Example 1, and the results were as Figure 6 shown. Among them, the closer the sensitivity of the sample near the HbA1c diagnostic standard of 6.5% is, the smaller the CV value is, and the more accurate the detection result is. The horizontal axis is the target value of HbA1c of the standard product, S1-S5 are 0, 4.8, 7.6, 10.6, 13.8, and the vertical axis is the absorbance.

[0058] It can be seen from the experimental results that, compared with the commercially available kit, the functional detection result of the polymeric antibody based on the polymeric backbone is significantly better than that of the secondary antibody system. The reading values of each point tested by the polymeric antibody reagent of the present invention are about twice that of the commercially available kit, and the S2 point is increased by 3.6 times, indicating that the polymeric antibody prepared based on the polymer backbone material forms turbidity faster and has higher turbidity. At the same time, compared with the polymeric antibody with a protein-based backbone material represented by BSA, the regular backbone material of the present invention has higher sensitivity, especially the absorbance is greater than 500 in the low-value region (5%), with significant detectability, and significantly improves the missed detection situation caused by insufficient sensitivity of low-value samples.

[0059] Effect Example 4 To verify the broad effectiveness of the polyclonal antibody of the present invention in immunoturbidimetry, this effect example used a biochemical analyzer (EXC2000, zybio) to compare the effects of the reagent containing the polymeric antibody with a dextran derivative backbone material prepared in Example 5 and the commercially available C1q detection kit (zybio). The test scheme is as follows: a. Reagent detection parameter settings Method: Endpoint method Reaction direction: Rising Sample: The ratio of reagent R1: reagent R2 is 4:240:60 (volume ratio) Main wavelength: 340 nm Sub-wavelength: 700 nm Reaction temperature: 37 °C Reaction time: 10 min b. Sample addition method and measurement scheme Table 3 C1q measurement scheme

[0060] c. Calculation method Use the multi-point non-linear / semi-logarithmic calibration mode, with the spline function as the calculation mode. The abscissa is the concentration (mg / L), and the ordinate is the absorbance (OD value). According to the values of the calibrator and the change value of the absorbance, a dose / response curve is made. The content of the target analyte in the sample can be calculated on the dose / response curve based on its absorbance change value. The calibration curve reflects the change of absorbance with the change of concentration. The greater the slope, the steeper the calibration curve, and the higher its analysis sensitivity.

[0061] The results are as Figure 7 shown, where the horizontal axis is the target value of C1q of the standard product, S1 - S5 are 0, 74, 137, 282, 507, and the vertical axis is the absorbance. The results show the comparison results of the functions of the reagent prepared from the polyclonal antibody based on the polymer backbone of the present invention and a commercially available kit. Compared with the commercially available reagent, each point of the functional test of the polyclonal antibody has a higher reading value and stronger turbidity-forming ability. Compared with the commercially available kit, the results of the functional test of the C1q polyclonal antibody show that the reading value of each point is about twice that of the commercially available product, indicating that the C1q polyclonal antibody forms turbidity faster and has a higher turbidity.

[0062] The above preparation examples and effect examples only exemplarily explain and prove the effects of the technical solutions described in the present invention. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit of the present invention and the scope protected by the claims. All of these belong to the protection scope of the present invention. The inventors have also observed the significant effectiveness of the polyclonal antibody raw material with the structure described in the present invention in immunoturbidimetric assays for other analytes such as adiponectin, C3 and other complements, apolipoproteins, and immunoglobulins.

Claims

1. An immunoturbidimetric reagent, characterized in that: The reagent contains a polymerized antibody, which contains a polymer skeleton and an antibody coupled to the polymer skeleton, and the molecular weight of the polymer skeleton is 50 KDa-800 KDa; wherein the polymer skeleton material is a non-protein substance.

2. The reagent according to claim 1, characterized in that The polymer backbone is selected from one or more of dextran, carboxydextran, aminodextran, aldehydedextran, polyethyleneimine PEI and dendrimer PAMAM.

3. The reagent according to claim 1 or 2, characterized in that The polymer skeleton is selected from one or more of dextran, carboxydextran, aminodextran and aldehydedextran.

4. An immunoturbidimetric kit, characterized in that: The kit comprises a reagent R2, and the reagent R2 contains the reagent according to any one of claims 1-3.

5. The kit according to claim 4, characterized in that The kit further comprises a reagent R1, which contains latex microspheres.

6. The kit according to claim 5, characterized in that The mass ratio of the latex microspheres to the polymerized antibody is 10:1-400:

1.

7. The kit according to claim 5 or 6, characterized in that Reagent R1 further contains one or more of a buffer, a surfactant or a preservative, and / or reagent R2 further contains one or more of a buffer, an inorganic salt, a surfactant, a preservative and a stabilizer.

8. The kit according to claim 7, characterized in that The pH of reagent R1 is 6.0-9.0, and the pH of reagent R2 is 5.0-9.

0.

9. Use of a polymerized antibody in the preparation of an immunoturbidimetric detection reagent, wherein the polymerized antibody comprises a polymer backbone and an antibody coupled to the polymer backbone, wherein the molecular weight of the polymer backbone is 50 KDa-800 KDa; wherein: The polymer skeleton material is a non-protein substance.

10. The use according to claim 9, characterized in that The antibody is selected from one or more of anti-adiponectin antibody, anti-complement component antibody, anti-HbA1c antibody, anti-cTnT antibody, anti-cTnI antibody, anti-apolipoprotein A1 antibody, anti-apolipoprotein B antibody and anti-immunoglobulin antibody.

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