A kit for measuring IgE using immunoglobulin E antibody and latex-enhanced immunoturbidimetry

Abstract

The present invention relates to the field of biological detection technology and provides an immunoglobulin E antibody comprising a heavy chain and a light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain is shown in SEQ ID NO. 2. The present invention also provides a kit for determining IgE using a latex-enhanced immunoturbidimetric assay comprising the immunoglobulin E antibody. The present invention has the advantages of simple operation, high linearity, good stability, and high sensitivity, accuracy, and specificity when using the immunoglobulin E antibody of the present invention in combination with a corresponding latex-enhanced immunoturbidimetric assay kit for determining IgE.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, in particular to an immunoglobulin E antibody and a kit for determining IgE by latex-enhanced immunoturbidimetry. Background Art

[0002] Immunoglobulin E (IgE), a class of Ig discovered in 1966, has a relative molecular mass of 160,000 and is the least abundant immunoglobulin subtype in the body, comprising only 0.002% of total serum Ig. The biological functions of IgE are mediated by the interaction between allergens and IgE receptors on immune cells. IgE plays a crucial role in both the early and late stages of allergic immune responses. Basophils and mast cells are the primary players in the early stages of allergic reactions. These cells contain preformed intracellular granules that, after cross-linking of FcεRI by allergen IgE, are rapidly expelled from the cell membrane, leading to degranulation and the release of mediators such as histamine, mediating type I allergic reactions.

[0003] IgE, though present in low serum levels, is clinically significant and primarily used in the diagnosis of allergic diseases. Testing IgE levels can identify allergic reactions, such as parasitic infections and skin allergies. Elevated IgE levels are often associated with conditions such as atopic dermatitis, allergic asthma, allergic rhinitis, urticaria, and parasitic infections.

[0004] IgE plays a key role in the development of allergic diseases. Testing IgE levels helps clinicians make differential diagnoses and treatment assessments of the patient's etiology. IgE testing includes total serum IgE testing and allergen-specific IgE testing. Currently, commonly used detection methods include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence. Among them, the operation process of ELISA is cumbersome and has a high error rate. The shortcomings of chemiluminescence are, on the one hand, poor selectivity and the reaction to a series of compounds rather than a single compound; on the other hand, the emission intensity of chemiluminescence depends on various environmental factors, and various external factors must be strictly controlled.

[0005] Latex-enhanced immunoturbidimetry (PETIA) utilizes the specific binding of antigens and antibodies to crosslink or physically adsorb antibodies on the surface of nanoscale latex microspheres. After the crosslinked antibody microspheres in the reagent reaction solution bind to the antigens in the sample, they rapidly aggregate within a short period of time, forming turbidity, thereby changing the light scattering or light transmittance properties of the reaction solution. This method can reflect the concentration of the antigen being tested within the linear range. The advantage of latex-enhanced immunoturbidimetry is its ease of operation, with results available in minutes. Furthermore, the simplified operating procedures of the fully automated immunoturbidimetry method avoid interference from many human factors and external factors such as reagents and the environment. It offers excellent stability and repeatability, and can more accurately reflect the content of the substance being tested.

[0006] Therefore, it is urgent to develop a new IgE detection method based on latex enhanced immunoturbidimetry. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an immunoglobulin E antibody and a kit for determining IgE by latex-enhanced immunoturbidimetry based on the antibody. The immunoglobulin E antibody is used in combination with a corresponding latex-enhanced immunoturbidimetry kit for determining IgE, which has the advantages of simple operation, high linearity, good stability, and high sensitivity, accuracy, and specificity.

[0008] The present invention adopts the following technical solutions to solve the above technical problems:

[0009] An immunoglobulin E antibody comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO.1, and the amino acid sequence of the light chain is shown in SEQ ID NO.2.

[0010] As one of the preferred aspects of the present invention, the immunoglobulin E antibody is capable of binding to immunoglobulin E in serum.

[0011] A kit for determining IgE by latex-enhanced immunoturbidimetry comprises the above-mentioned immunoglobulin E antibody.

[0012] As one of the preferred embodiments of the present invention, the kit for determining IgE by latex-enhanced immunoturbidimetry specifically comprises R1 reagent and R2 reagent;

[0013] The R1 reagent includes: 20-50 mmol / L R1 buffer, 0.2-1.5 g / L surfactant, 10-30 g / L stabilizer, and 0.5-1 mL / L preservative;

[0014] The R2 reagent includes: 10-50 mmol / L HEPES buffer, 10-40 mg / L immunoglobulin E antibody, 1-15 mL / L 100-200 nm latex microsphere particles, 2-30 mL / L 200-300 nm latex microsphere particles, 5-30 g / L stabilizer, and 0.05-2 g / L preservative.

[0015] As one of the preferred embodiments of the present invention, the R1 buffer is one or more of boric acid buffer, sodium borate buffer, and Tris buffer.

[0016] As one of the preferred embodiments of the present invention, the surfactant is Tween-20.

[0017] As one of the preferred embodiments of the present invention, the stabilizer is one or more of trehalose, NaCl, and BSA.

[0018] As one of the preferred embodiments of the present invention, the preservative is PC-300.

[0019] As one of the preferred embodiments of the present invention, in the R2 reagent, the immunoglobulin E antibody is coupled to 100-200 nm latex microsphere particles to form first latex particles coated with IgE antibodies; and the immunoglobulin E antibody is coupled to 200-300 nm latex microsphere particles to form second latex particles coated with IgE antibodies.

[0020] As one of the preferred embodiments of the present invention, the specific preparation method of the first latex particles coated with IgE antibodies and the second latex particles coated with IgE antibodies is as follows:

[0021] ① Add latex microspheres with a particle size of "100-200 nm" and latex microspheres with a particle size of "200-300 nm" to the activation buffer at a ratio of 1:2 and mix evenly; then, place in a constant temperature stirrer at 30-37°C for 20-30 minutes, with a temperature range of 30-37°C;

[0022] ② Add activator EDC solution, mix well, and react in a constant temperature stirrer for 0.5 to 1 hour;

[0023] ③ Add 10-40 mg / L of IgE antibody to the coupling buffer, mix well, and place in a constant temperature water bath for 10-30 minutes;

[0024] ④ Place the solution in step ③ into the activated latex reagent and couple for 1 to 2 hours;

[0025] ⑤ Add blocking solution, mix well, and place in a constant temperature stirrer to react for 1 to 2 hours;

[0026] ⑥After the reaction is completed, ultrasonic treatment is performed to disperse the particles and gel storage buffer is added;

[0027] ⑦After adding the gel storage solution, ultrasonicate it and place it in a constant temperature stirrer for aging for 16 to 24 hours.

[0028] The advantages of the present invention over the prior art are:

[0029] (1) The immunoglobulin E antibody designed by the present invention can bind to immunoglobulin E in serum, and after binding, it can increase the activity of immunoglobulin E, thereby significantly increasing the sensitivity, specificity, accuracy, repeatability and stability of the reagent in the immunoglobulin E latex immunoturbidimetric assay;

[0030] (2) The present invention is based on latex-enhanced immunoturbidimetry. After the antigen and antibody react, the absorbance value of the reaction solution is directly measured, eliminating the cumbersome operation steps of the enzyme-linked immunosorbent assay and obtaining the results in a few minutes, saving time and labor. At the same time, compared with the chemiluminescence immunoassay, it has strong applicability and convenient testing, and can be widely used in clinical testing.

[0031] (3) The present invention is simple to operate, eliminates the tedious centrifugation step, and can maintain the performance of the reagents in compliance with the requirements. It is low-cost, highly applicable, and convenient to test. At the same time, the kit can achieve a wider linear range for the performance of the immunoglobulin E assay kit by combining latex microspheres of different particle sizes, thereby achieving accurate detection of immunoglobulin E.

[0032] (4) The present invention utilizes the low-cost latex immunoturbidimetric method to detect immunoglobulin E, and at the same time adds immunoglobulin E antibodies to the existing immunoglobulin E detection kit, which not only improves the sensitivity and specificity of the kit but also greatly saves costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the standard curve graph of the control group and experimental group in Experimental Example 1;

[0034] Figure 2 is the linear analysis result graph of the control group in Experimental Example 2;

[0035] Figure 3 This is the linear analysis result graph of the experimental group in Experimental Example 2. DETAILED DESCRIPTION

[0036] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the scope of protection of the present invention is not limited to the following embodiments. At the same time, the reagents and experimental methods used in the following embodiments are conventional reagents or methods in the art unless otherwise specified, and will not be repeated here.

[0037] Example 1

[0038] The immunoglobulin E antibody of this embodiment comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain is shown in SEQ ID NO. 2. The immunoglobulin E antibody of this embodiment can bind to immunoglobulin E in serum and, upon binding, can increase the activity of immunoglobulin E.

[0039] Example 2

[0040] This embodiment is a preparation of immunoglobulin E antibodies in the above embodiment:

[0041] (1) Expression of immunoglobulin E antibodies in CHO-K1 suspension expression system

[0042] Cell Thawing: Thaw a vial of CHO cells (approximately 1 x 10 cells) frozen in liquid nitrogen at 37°C in a water bath. Centrifuge at 300g for 5 minutes. Wipe the vial with alcohol wipes and place in a laminar flow hood. Aspirate the supernatant, resuspend in 20 mL of pre-warmed complete CHO medium, and culture in a 125 mL shake flask at 37°C, 5% CO2, and 120-130 rpm.

[0043] Plasmid extraction: Inoculate the DH5a strain containing the pCDNA4.2 vector for immunoglobulin E antibody (gene synthesized by a biological company, the amino acid sequence of immunoglobulin E antibody is shown in SEQ ID NO.1 and SEQ ID NO.2) in advance, culture at 37°C overnight, and extract the plasmid using a commercial plasmid extraction kit.

[0044] Cell transfection: When cell viability is greater than 95%, there are no obvious cell clumping, and the cell density is between 2 and 3 x 10E6, harvest 4 x 10E7 CHO cells and centrifuge at 300g for 5 minutes. Remove the supernatant and resuspend the CHO cells in 400µL of Celetrix commercial electroporation buffer. Add 25µg of plasmid to deplete 2 x 10E7 CHO cells. Electroporate using a Celetrix cell electroporator at 1250V. Allow the cells to recover for 24 hours.

[0045] Cell transfer and fed-batch culture: After cell recovery, transfer the cells to fermentation medium at a density of 0.5x10E6. Culture until Day 5, then start fed-batch culture. Cool down the culture until Day 7.

[0046] Collect supernatant: Culture until Day 15 or when cell viability drops below 60%. Centrifuge the culture supernatant, remove the cell pellet, and retain the supernatant.

[0047] (2) Affinity purification of "immunoglobulin E antibodies"

[0048] Affinity column filling: calculate the required commercial nickel filler, load the filler into the column, and wash the loaded filler with PBS equilibration buffer.

[0049] Loading and washing: The cell supernatant collected by centrifugation was loaded at a low flow rate. After loading, 10 column volumes of equilibration buffer were used to wash the column, and 10 column volumes of pre-elution buffer A containing 20 mM imidazole were used to wash the column.

[0050] Elution and dialysis: After washing the filler with the pre-elution solution, elute it with 250mM imidazole-containing Tris elution solution. The eluted protein is dialyzed overnight with 20mM PBS buffer. After dialysis and changing the buffer, the antibody is aliquoted and stored.

[0051] Example 3

[0052] The kit for determining IgE by latex-enhanced immunoturbidimetry of this embodiment includes reagent R1 and reagent R2.

[0053] R1 reagent includes: 20 mmol / L boric acid buffer (pH 7.5), 0.2 g / L Tween-20, 10 g / L trehalose, and 0.5 mL / L PC-300.

[0054] R2 reagent includes: 10 mmol / L HEPES buffer (pH 7.5), 10 mg / L immunoglobulin E antibody, 1 mL / L 100 nm latex microsphere particles, 2 mL / L 200 nm latex microsphere particles, 5 g / L trehalose, and 0.05 g / L PC-300.

[0055] Preparation method:

[0056] (1) Prepare R1 reagent:

[0057] According to the component content of R1 reagent, each component is mixed in the same container, and after mixing evenly, R1 reagent is prepared.

[0058] (2) Preparing first latex particles coated with IgE antibodies and second latex particles coated with IgE antibodies:

[0059] ① Add 100nm and 200nm latex microspheres to the activation buffer in a ratio of 1:2 and mix well. Then, place in a 30℃ constant temperature stirrer and reheat for 30 minutes at 30℃.

[0060] ② Add activator EDC solution, mix well, and react in a constant temperature stirrer for 0.5h;

[0061] ③ Add 10 mg / L IgE antibody to coupling buffer (Hepes 20 mmol / L), mix well, and place in a constant temperature water bath for 10 minutes;

[0062] ④ Place the solution in step ③ into the activated latex reagent and couple for 1 hour;

[0063] ⑤ Add blocking solution (BSA 10g / L), mix well, and place in a constant temperature stirrer to react for 1 hour;

[0064] ⑥After the reaction is completed, ultrasonic treatment is performed to disperse the particles and gel storage buffer is added;

[0065] ⑦After adding gel storage solution, ultrasonicate and place in a constant temperature stirrer for aging for 16 hours.

[0066] (3) Prepare R2 reagent:

[0067] According to the component contents of the R2 reagent, the first latex particles coated with IgE antibodies, the second latex particles coated with IgE antibodies and the remaining components are mixed in the same container and mixed evenly to prepare the R2 reagent.

[0068] Example 4

[0069] The kit for determining IgE by latex-enhanced immunoturbidimetry of this embodiment includes reagent R1 and reagent R2.

[0070] R1 reagent includes: 50 mmol / L sodium borate buffer (pH 7.5), 1.5 g / L Tween-20, 14.5 g / L trehalose, 15 g / L NaCl, 0.5 g / L BSA, and 1 mL / L PC-300.

[0071] R2 reagent includes: 50 mmol / L HEPES buffer (pH 7.5), 40 mg / L immunoglobulin E antibody, 15 mL / L 200 nm latex microsphere particles, 30 mL / L 300 nm latex microsphere particles, 14.5 g / L trehalose, 15 g / L NaCl, 0.5 g / L BSA, and 2 g / L PC-300.

[0072] Preparation method:

[0073] (1) Prepare R1 reagent:

[0074] According to the component content of R1 reagent, each component is mixed in the same container, and after mixing evenly, R1 reagent is prepared.

[0075] (2) Preparing first latex particles coated with IgE antibodies and second latex particles coated with IgE antibodies:

[0076] ① Add 200nm and 300nm latex microspheres to the activation buffer at a ratio of 1:2 and mix well. Then, place in a 37℃ thermostatic stirrer and reheat for 20 minutes at 37℃.

[0077] ② Add activator EDC solution, mix well, and react in a constant temperature stirrer for 1 hour;

[0078] ③ Add 40 mg / L IgE antibody to coupling buffer (Hepes 20 mmol / L), mix well, and place in a constant temperature water bath for 30 minutes;

[0079] ④ Place the solution in step ③ into the activated latex reagent and couple for 2 hours;

[0080] ⑤ Add blocking solution (BSA 10g / L), mix well, and place in a constant temperature stirrer to react for 2h;

[0081] ⑥After the reaction is completed, ultrasonic treatment is performed to disperse the particles and gel storage buffer is added;

[0082] ⑦After adding gel storage solution, ultrasonicate and place in a constant temperature stirrer for aging for 24 hours.

[0083] (3) Prepare R2 reagent:

[0084] According to the component contents of the R2 reagent, the first latex particles coated with IgE antibodies, the second latex particles coated with IgE antibodies and the remaining components are mixed in the same container and mixed evenly to prepare the R2 reagent.

[0085] Example 5

[0086] The kit for determining IgE by latex-enhanced immunoturbidimetry of this embodiment includes reagent R1 and reagent R2.

[0087] R1 reagent includes: 45 mmol / L Tris buffer (pH 7.5), 1.0 g / L Tween-20, 10 g / L trehalose, 14.5 g / L NaCl, 0.5 g / L BSA, and 0.7 mL / L PC-300.

[0088] R2 reagent includes: 20 mmol / L HEPES buffer (pH 7.5), 20 mg / L immunoglobulin E antibody, 10 mL / L 150 nm latex microsphere particles, 20 mL / L 250 nm latex microsphere particles, 10 g / L trehalose, 14.5 g / L NaCl, 0.5 g / L BSA, and 1.0 g / L PC-300.

[0089] Preparation method:

[0090] (1) Prepare R1 reagent:

[0091] According to the component content of R1 reagent, each component is mixed in the same container, and after mixing evenly, R1 reagent is prepared.

[0092] (2) Preparing first latex particles coated with IgE antibodies and second latex particles coated with IgE antibodies:

[0093] ① Add 150nm and 250nm latex microspheres to the activation buffer in a ratio of 1:2 and mix well. Then, place in a 35℃ thermostatic stirrer and reheat for 25 minutes at 35℃.

[0094] ② Add activator EDC solution, mix well, and react in a constant temperature stirrer for 0.6h;

[0095] ③ Add 20 mg / L IgE antibody to the coupling buffer, mix well, and place in a constant temperature water bath for 20 minutes;

[0096] ④ Place the solution in step ③ into the activated latex reagent and couple for 1.5 hours;

[0097] ⑤ Add blocking solution, mix well, and place in a constant temperature stirrer to react for 1.5 hours;

[0098] ⑥After the reaction is completed, ultrasonic treatment is performed to disperse the particles and gel storage buffer is added;

[0099] ⑦After adding gel storage solution, ultrasonicate and place in a constant temperature stirrer for aging for 20 hours.

[0100] (3) Prepare R2 reagent:

[0101] According to the component contents of the R2 reagent, the first latex particles coated with IgE antibodies, the second latex particles coated with IgE antibodies and the remaining components are mixed in the same container and mixed evenly to prepare the R2 reagent.

[0102] Example 6

[0103] A method for using the kit for determining IgE using the latex-enhanced immunoturbidimetric assay of this embodiment:

[0104] Pipette 10uL of sample, add 240uL of reagent R1, and incubate at 37℃ for 3-5min;

[0105] Then add 60uL of reagent R2 and incubate at 37°C;

[0106] After incubation for 20 seconds, the absorbance value A1 was measured at a wavelength of 600 nm using an automatic biochemical analyzer; after incubation for another 5 minutes, the absorbance value A2 was measured at the same wavelength;

[0107] The absorbance change value △A was calculated according to △A=A2-A1, and △A was substituted into the linear relationship formula between the absorbance change value and the IgE concentration to convert the IgE content.

[0108] The linear relationship formula between the absorbance change value and the IgE concentration was obtained by the following method:

[0109] Calibrator buffer contains:

[0110] Tris: 1.21 g / L, pH 7.4;

[0111] Betaine: 15g / L;

[0112] Sodium azide: 0.9g / L.

[0113] Prepare calibrants with various concentration gradients

[0114] Dilute the purified IgE antigen with calibrator buffer to prepare calibrator concentrations of: 0 IU / mL, 187.5 IU / mL, 350 IU / mL, 750 IU / mL, and 1500 IU / mL.

[0115] A calibration curve was drawn and the corresponding formula was obtained: 240 μL of reagent R1 was added to the obtained 10 μL IgE standard, mixed, and incubated at 37°C for 3-5 minutes; then, 60 μL of reagent R2 was added and incubated at 37°C; after incubation for 20 seconds, the absorbance value Al was measured at a wavelength of 600 nm using a fully automatic biochemical analyzer; after another 5 minutes of incubation, the absorbance value A2 was detected at the same wavelength; the absorbance change value △A = A2-A1 of the standard was calculated, and a calibration curve was drawn; the calibration curve was fitted with multi-point nonlinear fitting to obtain the linear relationship formula between the absorbance change value and the IgE concentration.

[0116] Test Example 1

[0117] This test example is used to test the effect of the addition of immunoglobulin E antibodies on the calibration curve range of the kit product of the present invention.

[0118] Experimental and control groups were set up and tested using the method of Example 6. The experimental group consisted of the "immunoglobulin E detection kit of the present invention (using the kit of Example 5 as an example)" and the control group consisted of the "kit without the addition of immunoglobulin E antibody (i.e., the kit of Example 5 without the addition of immunoglobulin E antibody, with the antibody portion replaced by PBS buffer)."

[0119] The calibration results of the experimental group and the control group are shown in Table 1. The calibration curves are shown in Figure 1 shown.

[0120] Table 1 Calibration results (calibration point unit: IU / mL)

[0121]

[0122] From Table 1 and Figure 1 It can be seen that the calibration curve of the experimental group (with self-developed antibody reagent) has a wider range and higher sensitivity.

[0123] Test Example 2

[0124] This test example is used to test the effect of the addition of immunoglobulin E antibodies on the performance indicators of the kit product of the present invention.

[0125] Experimental and control groups were set up, and the following performance index tests were conducted using the method of Example 6. The experimental group was the immunoglobulin E detection kit of the present invention (using the kit of Example 5 as an example), and the control group was the kit supplemented with Haiyuan immunoglobulin E antibody (i.e., the kit of Example 5 supplemented with Haiyuan immunoglobulin E antibody "Cat. No.: 4Tr26 / 4Tr26cc", with all other components remaining the same).

[0126] 1. Reagent blank absorbance (under the condition of light path of 1.0 cm, the reagent blank absorbance value should be ≤1.500A):

[0127] Purified water was used as a sample, and the absorbance was measured at a dominant wavelength of 600 nm and a light path of 1.0 cm. The measurement was repeated twice, and the average value was taken. The results are shown in Table 2.

[0128] Table 2 Blank absorbance results (unit: △A)

[0129]

[0130] As shown in Table 2, both the experimental group and the control group meet the requirements, but the blank absorbance value of the experimental group (with the antibody reagent of the present invention) is smaller and the reagent sensitivity is higher.

[0131] 2. Accuracy Analysis:

[0132] The reagent of this study (Example 5) was compared with three levels of quality control products of Landau quality control. The deviation range within ±15% was considered to be no interference, and the deviation range exceeding ±15% was considered to be interference.

[0133] Table 3 Landau quality control test results (unit: IU / ml)

[0134]

[0135] As shown in Table 3, the deviations between the experimental group and the Landau quality control target values ​​were all less than ±15%, which met the requirements, while the deviations of two levels in the control group were greater than 15%.

[0136] 3. Sensitivity analysis: When the concentration of the analyte is 375IU / mL, the absorbance change rate should be ≥0.005A:

[0137] Test instrument: Hitachi 7180 fully automatic biochemical analyzer. Test sample: 1 IgE antigen sample at a concentration of 375 IU / mL. Using the kit prepared according to the present invention (using the kit of Example 5 as an example) and a control kit, absorbance was measured twice at a dominant wavelength of 600 nm and an optical path of 1.0 cm. The results are shown in Table 4.

[0138] Table 4 Sensitivity results

[0139]

[0140] It can be seen from Table 4 that both the experimental group and the control group meet the requirements, but the analytical sensitivity value of the experimental group is higher, indicating that the reagent is more stable.

[0141] 4. Precision analysis:

[0142] Two clinical serum samples were tested using a Hitachi 7180 fully automatic biochemical analyzer, with a low-value sample (150 IU / mL) and a high-value sample (600 IU / mL) being tested respectively. Each test sample was tested 10 times using the reagent of this study (Example 5) and the control reagent, and the mean, standard deviation, and CV were calculated, as shown in Table 5 below.

[0143] Table 5 Precision analysis

[0144]

[0145] The results showed that the precision of the kit of the present invention was as follows: the low CV value was 2.07% and the high CV value was 0.81%; while the low CV value of the control reagent was 3.56% and the high CV value was 1.00%. The sensitivity of the reagent of the present invention was better than that of the control reagent.

[0146] 5. Linear analysis:

[0147] The results were tested using a Hitachi 7180 fully automatic biochemical analyzer. The linear correlation coefficient was ≥0.990 within the range of 0.1 IU / mL to 1500 IU / mL. The linear deviation was within 22.5 IU / mL when the concentration was ≤187.5 IU / mL and within ±15.0% when the concentration was >187.5 IU / mL. Figure 2 、 3 .

[0148] Table 6 Linear analysis

[0149]

[0150]

[0151] From Table 6 and Figure 2 、 3 It can be seen that both the experimental group and the control group meet the requirements, but the experimental group has better linearity.

[0152] In summary, the immunoglobulin E antibody of the present invention can bind to immunoglobulin E in serum, and after binding, it can increase the activity of immunoglobulin E, and can significantly improve various indicators of the reagent including sensitivity, repeatability and linearity in immunoglobulin E detection.

[0153] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An immunoglobulin E antibody, characterized in that It comprises a heavy chain and a light chain; the amino acid sequence of the heavy chain is shown in SEQ ID NO.1, and the amino acid sequence of the light chain is shown in SEQ ID NO.

2.

2. The immunoglobulin E antibody according to claim 1, characterized in that The immunoglobulin E antibody is capable of binding to immunoglobulin E in serum.

3. A kit for determining IgE by latex-enhanced immunoturbidimetry, characterized in that: The invention comprises the immunoglobulin E antibody according to claim 1 or 2.

4. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 3, wherein Specifically including R1 reagent and R2 reagent; The R1 reagent includes: 20-50 mmol / L R1 buffer, 0.2-1.5 g / L surfactant, 10-30 g / L stabilizer, and 0.5-1 mL / L preservative; The R2 reagent includes: 10-50 mmol / L HEPES buffer, 10-40 mg / L immunoglobulin E antibody, 1-15 mL / L 100-200 nm latex microsphere particles, 2-30 mL / L 200-300 nm latex microsphere particles, 5-30 g / L stabilizer, and 0.05-2 g / L preservative.

5. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 4, wherein The R1 buffer is one of boric acid buffer, sodium borate buffer and Tris buffer.

6. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 4, wherein The surfactant is Tween-20.

7. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 4, wherein The stabilizer is one or more of trehalose, NaCl, and BSA.

8. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 4, wherein The preservative is PC-300.

9. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 4, wherein In the R2 reagent, immunoglobulin E antibodies are coupled with 100-200 nm latex microsphere particles to form first latex particles coated with IgE antibodies; immunoglobulin E antibodies are coupled with 200-300 nm latex microsphere particles to form second latex particles coated with IgE antibodies.

10. The kit for determining IgE by latex-enhanced immunoturbidimetry according to claim 9, wherein The specific preparation method of the first latex particles coated with IgE antibodies and the second latex particles coated with IgE antibodies is as follows: (1) Add latex microspheres with a particle size of "100-200 nm" and latex microspheres with a particle size of "200-300 nm" to the activation buffer at a ratio of 1:2 and mix evenly; then, place in a constant temperature stirrer at 30-37°C and reheat for 20-30 minutes, with a temperature range of 30-37°C; (2) Add activator EDC solution, mix well, and react in a constant temperature stirrer for 0.5-1h; (3) Add 10-40 mg / L of IgE antibody to the coupling buffer, mix well, and place in a constant temperature water bath for 10-30 min; (4) Place the solution in step (3) in the activated latex reagent and couple for 1 to 2 hours; (5) Add blocking solution, mix well, and place in a constant temperature stirrer for 1-2 hours; (6) After the reaction is completed, ultrasonic treatment is performed to disperse the particles and gel storage buffer is added; (7) After adding the gel storage solution, ultrasonicate it and place it in a constant temperature stirrer for aging for 16 to 24 hours.

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