Immune globulin D transmittance-scattering latex turbidimetry detection kit

Abstract

The invention discloses an immunoglobulin D transmittance-scattering latex turbidimetry detection kit, and belongs to the technical field of biological detection. The immunoglobulin D transmission-scattering latex turbidimetry detection kit disclosed by the invention comprises a first reagent R1, a second reagent R2, a calibration product and a quality control product. The kit disclosed by the invention has the advantages of universality (relatively low cost) and detection result reliability (good repeatability of a low value and no false negative leak detection of a high value).

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more particularly to an immunoglobulin D through-scattering latex turbidimetry detection kit. Background Art

[0002] Immunoglobulin D (IgD) is a membrane receptor on B lymphocytes and a marker on the surface of mature B lymphocytes. During B lymphocyte proliferation and differentiation, IgD plays a crucial role in triggering and directing the synthesis of IgG. Human lymphocyte membrane-bound IgD is closely associated with immunodeficiency and immunoproliferative diseases. IgD interacts with IgM receptors and is genetically related. Research on the relationship between cell surface IgD and B lymphocyte differentiation is deepening, making accurate IgD measurement essential in current immune research.

[0003] Multiple myeloma (MM) is a malignant plasma cell clonal proliferative disease characterized by a series of clinical symptoms such as bone destruction and kidney damage caused by an abnormal increase in monoclonal immunoglobulins or their fragments. According to the differences in the immunoglobulin heavy chains secreted by the proliferating plasma cells, MM can be divided into light chain type, IgG type, IgA type, IgM type, IgD type and IgE type. Among them, the IgD type accounts for 1% to 8%, is slightly more common in my country, with a young onset and severe onset. Quantitative detection of IGD is a basic examination item for the diagnosis and treatment of MM, and is written into the "Guidelines for the Diagnosis and Treatment of Multiple Myeloma in China (Revised in 2024)".

[0004] However, there are currently no domestically produced quantitative IgD test kits for clinical use in China, and internationally, there are only IgD test kits from companies such as The BindingSite (TBS). However, the TBS test kit is expensive and cannot be widely used. In addition, its linear range is only 7.0-210 mg / L, and the high recheck rate of super-linear samples further increases the cost of use. In addition, the antigen excess of the TBS test kit can only reach 1680 mg / L, and its reliability is worrying. For newly diagnosed IgD-type MM patients (usually IGD concentration is 1000-7000 mg / L), it is easy to cause false negatives due to antigen excess and missed detection and misdiagnosis, delaying patients from receiving timely and effective treatment.

[0005] The above reasons have led to the current identification of IGD-type MM patients mainly relying on the addition of IGD qualitative detection during immunofixation electrophoresis when other examinations suspect the light chain type.

[0006] Equally important, treatment monitoring is available for other MM types using immunoturbidimetric assays for IgA, IgM, IgG, and IgE quantification kits. However, IGD MM requires urine M-protein / light chain quantification and expensive serum free light chain (FLC) testing for efficacy assessment. Note: Immunoglobulin quantification typically costs 10-20 yuan per patient, while free light chain testing typically costs 120-300 yuan per patient.

[0007] The average IGD concentration in a normal individual is around 30 mg / L. However, in pathologically elevated conditions, such as IGD-type MM, the concentration can reach as high as 1000-7000 mg / L. In pathologically reduced conditions, such as those caused by other types of MM with immunoparesis and immunodeficiency disorders, the concentration is typically less than 7 mg / L or even as low as 0.5 mg / L. Therefore, the detection reagent must take into account sensitivity, linear range, and a safe zone of antigen excess.

[0008] In summary, immune research and clinical diagnosis and treatment urgently need IgD detection quantitative kits that are universal (relatively low cost), have reliable test results (good repeatability for low values ​​and no false negatives or missed detections for high values), and can be widely used for screening and treatment monitoring.

[0009] Transmitted-scatter latex turbidimetry: Based on an all-in-one instrument (such as the Hitachi 3500 fully automated analyzer) that integrates a fusion-enhanced transmitted-scatter method, the same sample is placed in the same reaction cuvette, consuming only one reagent. Transmitted and scattered signals are measured simultaneously in a very short time. Low-value samples output scattering results, while medium- and high-concentration samples output transmission results, achieving a balanced sensitivity and detection range. The kit for this test method was officially named "Transmitted-scatter latex immunoturbidimetry" by the National Medical Product Administration (NMPA) in 2023.

[0010] Therefore, providing an immunoglobulin D through-scattering latex turbidimetry detection kit is a problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0011] In view of this, the present invention provides an immunoglobulin D through-scattering latex turbidimetry detection kit.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] An immunoglobulin D through-scattering latex turbidimetric detection kit, comprising a first reagent R1, a second reagent R2, a calibrator, and a quality control product;

[0014] The first reagent R1 includes the following components at the following concentrations: 25-200 mM Hepes-NaOH buffer pH 6.8-7.5, 1000-2500 mM NaCl, polyethylene glycol 12000 10 g / L, Tween-20 0.05%-2%, 6-aminohexanoic acid 1%-5%, sodium periodate 30-200 mM, rabbit polyclonal antibody blocking agent 5-20 ml / L;

[0015] The second reagent R2 includes components with the following concentrations: 50-200 mM buffer pH 7.0-7.6, Tween-20 4-16 ml / L, 80 nm carboxyl latex 2.4-3.2 g / L, 200 nm carboxyl latex 1.6-2.4 g / L, 350 nm carboxyl latex 1.0-1.8 g / L, protective agent 10-90 g / L, and rabbit anti-human IgD polyclonal antibody 1.2-6.8 mg / L.

[0016] Furthermore, the first reagent R1 and the second reagent R2 further include a preservative, and the preservative is 1 g / L sodium azide or 0.5 ml / L PC300.

[0017] Furthermore, in the second reagent R2, the buffer is Hepes-NaOH buffer, phosphate buffer or glycine-Tris buffer.

[0018] Furthermore, in the second reagent R2, the protective agent is at least one of 10-30 g / L sucrose, 10-30 g / L trehalose, and 10-30 g / L fructose.

[0019] Furthermore, the calibrator includes components at the following concentrations: 50 mM Hepes-NaOH buffer pH 6.8-8.2, IgD antigen 10-800 mg / L, sodium chloride 900 mM, Tween-20 10 ml / L, preservative Proclin 3000.5 ml / L, oxidized glutathione 0.2-2.0 mM, and protein disulfide isomerase 0.5-5.0 μM.

[0020] Furthermore, the quality control product includes components with the following concentrations: 50mM Hepes-NaOH buffer pH 6.8-8.2, IgD antigen 20-160mg / L, sodium chloride 900mM, Tween-20 10ml / L, preservative Proclin3000.5ml / L, oxidized glutathione 0.2-2.0mM, and protein disulfide isomerase 0.5-5.0μM.

[0021] Furthermore, the immunoglobulin D through-scattering latex turbidimetry detection kit is used in detecting IgD.

[0022] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses an immunoglobulin D through-scattering latex turbidimetric detection kit. The IgD quantitative detection kit needs to take into account both detection performance and detection cost.

[0023] First of all, its detection reagent needs to take into account sensitivity, linear range and antigen excess safety zone, and the transscattered latex immunoturbidimetry method is the first choice.

[0024] Then, the detection cost is controllable. For turbidimetry, the main cost is the antibody cost, so the antibody dosage needs to be controlled. According to the principle of antigen-antibody proportionality, the common method of controlling antibody dosage in this field is pre-dilution detection, that is, using the instrument's automatic dilution function to first dilute the sample 5-100 times, and then perform the antigen-antibody reaction, which significantly improves the antigen / antibody ratio, improves the detection linear range, increases the antigen excess safety zone, and can also moderately reduce the antibody dosage (cost controllable) when the performance is moderate. However, pre-diluting the sample will significantly reduce the sensitivity of the reagent, and the reagent needs to have sufficient sensitivity support before it can be used.

[0025] In IgD testing, the detection antibody recognizes the heavy chain delta chain, primarily the Fc segment. The IgD heavy chain consists of 450-550 amino acid residues, while the light chain contains approximately 210 amino acid residues. The C region of the human IgD molecule contains three Cδ domains. Cδ1 and Cδ2 are similar to the constant regions of other Igs, but Cδ3 exhibits a proline deficiency and increased N-glycans. These structural changes in IgD result in the formation of a specific hinge region. Located between CH1 and CH2, the hinge region is rich in prolines, making it easily stretchable and bendable, and susceptible to hydrolysis by enzymes such as papain and pepsin. This region connects the antibody's Fab and Fc segments, allowing the two Fab segments to move and bend, thereby enabling binding to antigenic epitopes at varying distances. The IgD hinge region is longer than that of other Igs, which determines its functional specificity. Therefore, human IgD exists in a "T" shape, while other immunoglobulins have a "Y" shape. The IgD hinge adopts a semi-extended α-helical conformation in solution. The two Fab fragments are mainly located on both sides of the Fc fragment (see Figure 1 ), and due to the flexibility of the half-extended hinge, it can rotate around the Fc fragment. The Fc part of the IgD molecule is usually hidden by the movement of the Fab part.

[0026] Heavy O-glycosylation in the first subregion of the hinge increases hinge rigidity and restricts the mobility of the Fab portion, promoting exposure of the Fc portion. Therefore, researchers hypothesized that if serum IgD molecules in samples could be quickly and effectively hyperglycosylated, this would facilitate binding between serum IgD molecules and anti-IgD antibodies in the detection reagent, thereby improving the sensitivity of the IgD detection kit.

[0027] Combined with 350nm large particle size latex, the scattered light detection of low concentrations by the through-scattered latex turbidimetry method will likely increase the detection sensitivity by dozens of times, so that the pre-dilution method can be used for detection, taking into account both reagent performance and cost control.

[0028] At the same time, during the reagent development process, researchers also found that the preparation of IgD calibrators and quality control products is extremely unstable and prone to degradation. Inaccurate reagent calibration will cause the detection system to be unable to correctly identify the content of the target substance in the sample, and unstable quality control products cannot effectively verify the reliability of the detection system. The combined effect of the two may cause systematic deviations in the test results, such as false positives or false negatives, which directly affect the accuracy of clinical diagnosis. If the freeze-drying process is used to prepare calibrators and quality control products, it will lead to complicated operations and introduce manual operation errors during re-dissolution, and the freeze-drying process will significantly increase the cost of the test kit. Literature research found that disulfide bonds ( Figure 2 ), and groups such as sulfhydryls and methionine in IgD molecules may locally become reducing under certain conditions. During hinge movement and bending, the accompanying reducing environment in the solution can lead to disulfide bond cleavage, significantly altering the structure and function of the IgD molecule and causing instability in calibrators and quality controls. Therefore, technicians hypothesized that if the reducing environment in the solution could be quickly suppressed and the correct disulfide bond formation in serum IgD molecules could be catalyzed, the stability of IgD molecules in liquid calibrators and quality controls would be further enhanced.

[0029] Based on these studies, this kit innovatively incorporates 6-aminohexanoic acid and periodic acid into Reagent 1, rapidly exposing the FC end of IgD in serum samples during the R1 reaction phase. This enhances reagent sensitivity, further increasing the sensitivity of IgD detection by transmission and scattering by over 50%. The use of large-particle latex and pre-dilution in the reagent achieves a CV of ≤3% at low IgD values ​​of 2.5 mg / L, a common clinical finding in myeloma patients. This significantly improves the reproducibility and reliability of IgD detection. Furthermore, the upper limit of linearity is increased to 800 mg / L, compared to 230 mg / L in TBS, resulting in an antigen excess of 9000 mg / L, enabling accurate determination of IgD-type MM in clinical settings. Each L of Reagent 2 utilizes only 15-50 ml of rabbit anti-human IgD polyclonal antibody, ensuring optimal reagent performance while maintaining cost effectiveness.

[0030] Innovatively, oxidized glutathione (GSSG) and protein disulfide isomerase (PDI) are added to the diluents of calibrators and quality controls to improve their stability. No freeze-drying is used, keeping costs manageable.

[0031] The kit of the present invention has both universal benefits (relatively low cost) and reliability of detection results (good repeatability of low values ​​and no false negative omissions of high values). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0033] Figure 1 is the molecular structure of IgD;

[0034] Figure 2 is the disulfide bond in the hinge region of IgD;

[0035] Figure 3 This is the reaction flow chart of the IgD kit;

[0036] Figure 4 Antigen stability of Examples 1-3 and Comparative Examples 1-3 of the present invention;

[0037] Figure 5 The linear range of Examples 7-9 and Comparative Examples 4-6 of the present invention;

[0038] Figure 6 These are the antigen excess curves of Examples 7-9 of the present invention and Comparative Examples 4-6. DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] Example 1

[0041] The diluents for the IgD reagent calibrators and controls contain the following components at the following concentrations:

[0042] 50 mM Hepes-NaOH buffer pH 6.8

[0043] NaCl 900mM

[0044] Tween-20 10mL / L

[0045] Oxidized glutathione 0.2 mM

[0046] Protein disulfide isomerase 0.5 μM

[0047] Proclin3000.5mL / L

[0048] Preparation method: Weigh 11.915g Hepes, 52.596g NaCl, 0.1225g oxidized glutathione, and 0.0535g protein disulfide isomerase, respectively, and add them to 800mL purified water. Stir until completely dissolved, then add 20% NaOH dropwise to adjust the pH to 6.8. Then, add 10mL Tween-20 and 0.5mL Proclin 300, stir until completely dissolved, and adjust the volume to 1L. Filter through a 450nm filter membrane to prepare the dilution solution of IgD reagent calibrator and quality control.

[0049] Example 2

[0050] The diluents for the IgD reagent calibrators and controls contain the following components at the following concentrations:

[0051] 50 mM Hepes-NaOH buffer pH 7.5

[0052] NaCl 900mM

[0053] Tween-20 10mL / L

[0054] Oxidized glutathione 1.2 mM

[0055] Protein disulfide isomerase 3.0 μM

[0056] Proclin3000.5mL / L

[0057] Preparation method: Weigh 11.915g Hepes, 52.596g NaCl, 0.7352g oxidized glutathione, and 0.321g protein disulfide isomerase, respectively, and add them to 800mL purified water. Stir until completely dissolved, then add 20% NaOH dropwise to adjust the pH to 7.5. Then, add 10mL Tween-20 and 0.5mL Proclin 300, stir until completely dissolved, and adjust the volume to 1L. Filter through a 450nm filter membrane to prepare the dilution solution of IgD reagent calibrator and quality control.

[0058] Example 3

[0059] The diluents for the IgD reagent calibrators and controls contain the following components at the following concentrations:

[0060] 50 mM Hepes-NaOH buffer pH 8.2

[0061] NaCl 900mM

[0062] Tween-20 10mL / L

[0063] Oxidized glutathione 2.0 mM

[0064] Protein disulfide isomerase 5.0 μM

[0065] Proclin3000.5mL / L

[0066] Preparation method: Weigh 11.915g Hepes, 52.596g NaCl, 1.2253g oxidized glutathione, and 0.535g protein disulfide isomerase respectively, add them to 800mL purified water, stir until completely dissolved, add 20% NaOH dropwise to adjust the pH to 8.2, then add 10mL Tween-20 and 0.5mL Proclin300, stir until completely dissolved, and adjust the volume to 1L. Filter through a 450nm filter membrane to prepare the dilution solution of IgD reagent calibrator and quality control.

[0067] Example 4

[0068] IgD reagent R1 contains the following components at the following concentrations:

[0069] 25 mM Hepes-NaOH buffer pH 6.8

[0070] NaCl 1000mM

[0071] PEG 12000 10g / L

[0072] Tween-20 0.05%

[0073] 6-Aminohexanoic acid 1%

[0074] Sodium periodate 30 mM

[0075] Proclin3000.5mL / L

[0076] DAKO rabbit polyclonal antibody blocking agent 5mL / L

[0077] Preparation method: 5.958 g Hepes, 58.44 g NaCl, 10 g 6-aminohexanoic acid, and 6.417 g sodium periodate were weighed separately and added to 800 mL of purified water. After stirring until completely dissolved, 20% NaOH was added dropwise to adjust the pH to 6.8. 0.5 mL Tween-20, 0.5 mL Proclin 300, 10 g PEG 12000, and 5 mL DAKO rabbit polyclonal antibody blocking agent were then added. After stirring until completely dissolved, the volume was adjusted to 1 L and filtered through a 450 nm filter membrane to prepare IgD reagent R1.

[0078] Example 5

[0079] IgD reagent R1 contains the following components at the following concentrations:

[0080] 100 mM Hepes-NaOH buffer pH 7.2

[0081] NaCl 1800mM

[0082] PEG 12000 10g / L

[0083] Tween-20 1.2%

[0084] 6-Aminohexanoic acid 3%

[0085] Sodium periodate 120 mM

[0086] Proclin3000.5mL / L

[0087] DAKO rabbit polyclonal antibody blocking agent 12mL / L

[0088] Preparation method: Weigh 23.83 g Hepes, 105.192 g NaCl, 30 g 6-aminohexanoic acid, and 25.667 g sodium periodate, respectively, add them to 800 mL purified water, stir until completely dissolved, add 20% NaOH dropwise to adjust the pH to 7.2, then add 12 mL Tween-20, 0.5 mL Proclin 300, 10 g PEG 12000, and 12 mL DAKO rabbit polyclonal antibody blocking agent, stir until completely dissolved, adjust the volume to 1 L, and filter through a 450 nm filter to prepare IgD reagent R1.

[0089] Example 6

[0090] IgD reagent R1 contains the following components at the following concentrations:

[0091] 200 mM Hepes-NaOH buffer pH 7.5

[0092] NaCl 2500mM

[0093] PEG 12000 10g / L

[0094] Tween-20 2%

[0095] 6-Aminohexanoic acid 5%

[0096] Sodium periodate 200 mM

[0097] Sodium azide 1g / L

[0098] DAKO rabbit polyclonal antibody blocking agent 20mL / L

[0099] Preparation method: Weigh 47.66 g Hepes, 146.1 g NaCl, 50 g 6-aminohexanoic acid, and 42.778 g sodium periodate, respectively, add them to 800 mL of purified water, stir until completely dissolved, add 20% NaOH dropwise to adjust the pH to 7.5, then add 20 mL Tween-20, 1 g sodium azide, 10 g PEG 12000, and 20 mL DAKO rabbit polyclonal antibody blocking agent, stir until completely dissolved, adjust the volume to 1 L, and filter through a 450 nm filter to prepare IgD reagent R1.

[0100] Example 7

[0101] A kit for determining the content or level of immunoglobulin D (IgD) comprises an IgD reagent R1 and an IgD reagent R2. The formulation and preparation method of IgD reagent R1 are the same as those in Example 4. The corresponding IgD reagent R2 contains the following components at the following concentrations:

[0102] 50 mM Hepes-NaOH buffer pH 7.0

[0103] 80nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 2.4g / L

[0104] 200nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 1.6g / L

[0105] 350nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 1.0g / L

[0106] DAKO rabbit anti-human IgD polyclonal antibody 1.2mg / L

[0107] Tween-20 4mL / L

[0108] Sucrose 20g / L

[0109] The preparation method of IgD reagent R2 is as follows:

[0110] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, and centrifuge the latex microspheres twice; take 1 mL of 10% mass concentration of 80 nm, 200 nm, and 350 nm latex microspheres, that is, 1 g of each of the three latex microspheres, centrifuge at 22,000 rpm for 15 min, remove the supernatant, and re-dissolve with 10 mL of the above-mentioned MES-NaOH buffer, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning and use;

[0111] (2) Latex activation: Taking 10 mL of the cleaned latex as an example, 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were weighed and dissolved in 5 mL of the MES-NaOH buffer used for cleaning. 1 mL of each was added to 10 mL of the three types of cleaned microspheres and mixed under magnetic stirring at room temperature for 10 min.

[0112] (3) Post-activation cleaning: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 mL of 50 mM Hepes-NaOH buffer (pH 7.0) was added for redissolution;

[0113] (4) Antibody coupling: 2.0 mL, 1.2 mL, and 0.5 mL of 15 mg / L DAKO rabbit anti-human IgD polyclonal antibody were taken and added sequentially to the three activated latex solutions reconstituted with Hepes-NaOH buffer. The solution was magnetically stirred at room temperature for 10 hours to complete the antibody coupling.

[0114] (5) Blocking and washing: After centrifuging the three latexes at 22,000 rpm for 15 min, the supernatant was removed. 80 nm, 200 nm and 350 nm latexes were re-dissolved by adding 12.5 mL (final latex concentration of about 7.2 g / L), 18.5 mL (final latex concentration of about 4.8 g / L) and 30 mL (final latex concentration of about 3.0 g / L) of blocking and washing solution (50 mM pH 7.0 Hepes-NaOH buffer, 4.0 mL / L Tween-20 and 20 g / L sucrose), respectively. The three latexes were mixed at a volume ratio of 1:1:1 and then stirred at room temperature for 2 hours for use (final concentrations of 80 nm carboxyl latex 2.4 g / L, 200 nm carboxyl latex 1.6 g / L, 350 nm carboxyl latex 1.0 g / L, and rabbit anti-human IgD polyclonal antibody 1.2 mg / L), which was the finished product R2.

[0115] Example 8

[0116] A kit for determining the content or level of immunoglobulin D (IgD) comprises an IgD reagent R1 and an IgD reagent R2. The formulation and preparation method of IgD reagent R1 are the same as those in Example 5. The corresponding IgD reagent R2 contains the following components at the following concentrations:

[0117] 100 mM PBS buffer pH 7.3

[0118] 80nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 2.8g / L

[0119] 200nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 2.0g / L

[0120] 350nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 1.4g / L

[0121] DAKO rabbit anti-human IgD polyclonal antibody 2.9mg / L

[0122] Tween-20 10mL / L

[0123] Trehalose 20g / L

[0124] The preparation method of IgD reagent R2 is as follows:

[0125] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, and centrifuge the latex microspheres twice; take 1 mL of 10% mass concentration of 80 nm, 200 nm, and 350 nm latex microspheres, that is, 1 g of each of the three latex microspheres, centrifuge at 22,000 rpm for 15 min, remove the supernatant, and re-dissolve with 10 mL of the above-mentioned MES-NaOH buffer, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning and use;

[0126] (2) Latex activation: Taking 10 mL of the cleaned latex as an example, 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were weighed and dissolved in 5 mL of the MES-NaOH buffer used for cleaning. 1 mL of each was added to 10 mL of the three types of cleaned microspheres and mixed under magnetic stirring at room temperature for 10 min.

[0127] (3) Post-activation cleaning: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 mL of 100 mM pH 7.3 PBS buffer was added for re-dissolution;

[0128] (4) Antibody coupling: 2.0 mL, 1.2 mL, and 0.5 mL of 30 mg / L DAKO rabbit anti-human IgD polyclonal antibody were taken and added sequentially to the three activated latex solutions reconstituted with PBS buffer. The solution was magnetically stirred at room temperature for 10 hours to complete the antibody coupling.

[0129] (5) Blocking and washing: After centrifuging the three latexes at 22,000 rpm for 15 min, the supernatant was removed. 80 nm, 200 nm and 350 nm latexes were re-dissolved by adding 10.7 mL (final latex concentration of about 8.4 g / L), 15 mL (final latex concentration of about 6.0 g / L) and 21.5 mL (final latex concentration of about 4.2 g / L) of blocking and washing solution (100 mM PBS buffer at pH 7.3, 10.0 mL / L Tween-20 and 20 g / L trehalose), respectively. The three latexes were mixed at a volume ratio of 1:1:1 and then stirred at room temperature for 2 hours for use (final concentrations of 80 nm carboxyl latex 2.8 g / L, 200 nm carboxyl latex 2.0 g / L, 350 nm carboxyl latex 1.4 g / L, and rabbit anti-human IgD polyclonal antibody 2.9 mg / L), which was the finished product R2.

[0130] Example 9

[0131] A kit for determining the content or level of immunoglobulin D (IgD) comprises an IgD reagent R1 and an IgD reagent R2. The formulation and preparation method of IgD reagent R1 are the same as those in Example 6. The corresponding IgD reagent R2 contains the following components at the following concentrations:

[0132] 200 mM Tris-glycine buffer pH 7.6

[0133] 80nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 3.2g / L

[0134] 200nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 2.4g / L

[0135] 350nm carboxyl latex (Suzhou Weidu Biotechnology Co., Ltd.) 1.8g / L

[0136] DAKO rabbit anti-human IgD polyclonal antibody 6.8mg / L

[0137] Tween-20 16mL / L

[0138] Fructose 30g / L

[0139] The preparation method of IgD reagent R2 is as follows:

[0140] (1) Latex cleaning: Use 100 mM MES-NaOH buffer, pH 5.5, and centrifuge the latex microspheres twice; take 1 mL of 10% mass concentration of 80 nm, 200 nm, and 350 nm latex microspheres, that is, 1 g of each of the three latex microspheres, centrifuge at 22,000 rpm for 15 min, remove the supernatant, and re-dissolve with 10 mL of the above-mentioned MES-NaOH buffer, then centrifuge again, remove the supernatant and re-dissolve, and complete the cleaning and use;

[0141] (2) Latex activation: Taking 10 mL of the cleaned latex as an example, 50 mg of NHS (N-hydroxysuccinimide) and 30 mg of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) were weighed and dissolved in 5 mL of the MES-NaOH buffer used for cleaning. 1 mL of each was added to 10 mL of the three types of cleaned microspheres and mixed under magnetic stirring at room temperature for 10 min.

[0142] (3) Post-activation cleaning: The three latexes activated in step (2) were centrifuged at 22,000 rpm for 15 min, the supernatant was removed, and 20 mL of 100 mM PBS buffer (pH 7.3) was added for redissolution;

[0143] (4) Antibody coupling: 2.0 mL, 1.2 mL, and 0.5 mL of 60 mg / L DAKO rabbit anti-human IgD polyclonal antibody were taken and added sequentially to the three activated latex solutions reconstituted with PBS buffer. The solution was magnetically stirred at room temperature for 10 hours to complete the antibody coupling.

[0144] (5) Blocking and washing: After centrifuging the three latexes at 22,000 rpm for 15 min, the supernatant was removed. 80 nm, 200 nm and 350 nm latexes were re-dissolved in 9.4 mL (final latex concentration of about 9.6 g / L), 12.5 mL (final latex concentration of about 7.2 g / L) and 16.5 mL (final latex concentration of about 5.4 g / L) of blocking and washing solution (100 mM PBS buffer solution at pH 7.3, 10 mL / L Tween-20, 2 mL / L PC300 and 20 g / L trehalose), respectively. The three latexes were mixed at a volume ratio of 1:1:1 and then stirred at room temperature for 2 hours for use (final concentrations of 80 nm carboxyl latex 3.2 g / L, 200 nm carboxyl latex 2.4 g / L, 350 nm carboxyl latex 1.8 g / L, and rabbit anti-human IgD polyclonal antibody 6.8 mg / L), which was the finished product R2.

[0145] Comparative Example 1

[0146] The oxidized glutathione and protein disulfide isomerase in Example 2 were removed to form Comparative Example 1.

[0147] Comparative Example 2

[0148] The oxidized glutathione in Example 2 was removed to form Comparative Example 2.

[0149] Comparative Example 3

[0150] The protein disulfide isomerase in Example 2 was removed to form Comparative Example 3.

[0151] Comparative Example 4

[0152] The 6-aminocaproic acid and sodium periodate of R1 in Example 8 were removed, and R2 remained unchanged to form Comparative Example 4.

[0153] Comparative Example 5

[0154] The 6-aminohexanoic acid of R1 in Example 8 was removed, and R2 was unchanged to form Comparative Example 5.

[0155] Comparative Example 6

[0156] The sodium periodate of R1 in Example 8 is removed, and R2 remains unchanged to form Comparative Example 6.

[0157] Test Example 1: Stability Verification of IgD Calibrator and Quality Control Diluents

[0158] (1) Using the diluents prepared in Examples 1, 2, 3 and Comparative Examples 1, 2, and 3, ATHENS natural human IgD (Cat. No. 16-16-090704) was diluted to a low concentration (30 mg / L) and a high concentration (600 mg / L), respectively. These low-concentration and high-concentration antigens were then divided into a 2-8°C real-time group and a 37°C accelerated group, respectively, and stored in a 2-8°C refrigerator and a 37°C oven for incubation;

[0159] (2) After calibration using the same instrument, the same set of reagents (Example 8) and calibrators, the real-time group and the accelerated group in (1) were measured in the same time period for 14 consecutive days. The reaction flow is shown in Figure 3 The relative deviations of the values ​​of each group on each day compared to the 0-day control were calculated. The results are shown in Tables 1, 2, and Figure 4 .

[0160] Table 1 Stability of IgD antigen diluted with different diluents at 2-8°C

[0161]

[0162]

[0163] Table 2 Stability of IgD antigen diluted with different diluents at 37°C

[0164]

[0165] The results are shown in Tables 1, 2 and Figure 4As shown, the simultaneous addition of oxidized glutathione and protein disulfide isomerase to the diluents of IgD antigen calibrators and quality controls inhibits the reducing environment in the solution and catalyzes the correct formation of disulfide bonds in IgD molecules, further promoting the stability of IgD molecules in the liquid calibrators and quality controls. For both the real-time and accelerated groups, the stability of low and medium-high values ​​over 14 consecutive days was significantly better than that of the control group with no or partial addition, with relative deviations within 10%, meeting the requirements for preparing liquid calibrators and quality controls in test kits.

[0166] Experimental Example 2 IgD kit performance verification

[0167] (1) Calibration

[0168] According to the reaction process of the IgD kit, Examples 7-9 and Comparative Examples 4-6 were calibrated respectively.

[0169] The instrument used is a Hitachi transmissivity-scattering all-in-one instrument. Taking Hitachi 3500 as an example, the transmissivity-scattering all-in-one method is used. The instrument parameters are as follows: sample volume 3 μL, R1 addition volume 120 μL; R2 addition volume 80 μL; transmission wavelength 700 nm, scattering angle 20 degrees; two-point endpoint method 18-34 reading points; scattering calibration points 0.00, 10, 90, 200; transmission calibration points: 10, 90, 200, 450, 600, 800; the calibration results are shown in Tables 3 and 4.

[0170] Table 3 20° scattering calibration results of Examples 7-9 and Comparative Examples 4-6

[0171]

[0172] Table 4 Transmission calibration results of Examples 7-9 and Comparative Examples 4-6

[0173]

[0174] The results, shown in Tables 3 and 4, show that the addition of 6-aminohexanoic acid and sodium periodate to R1 rapidly and effectively hyperglycosylated serum IgD molecules in the sample, stabilizing the IgD antigen conformation and facilitating binding between the IgD molecules and the anti-IgD antibodies in the test reagent. Combined with the large 350nm particle size latex, the low-concentration scattered light detection method of the latex turbidimetry significantly improved low-end analytical sensitivity, enabling subsequent pre-dilution of samples for testing.

[0175] (2) Low value repeatability verification

[0176] After calibration, the low-value samples were measured 10 times in succession using Examples 7-9 and Comparative Examples 4-6 according to the reaction process of the IgD kit to compare the low-value precision.

[0177] The instrument used is a Hitachi transmissive scattering all-in-one instrument. Taking Hitachi3500 as an example, the transmissive scattering all-in-one method is used; the machine parameters are: sample volume 0.15μL (0.15μL is the use of the instrument pre-dilution function. The fully automatic biochemical analyzer automatically dilutes 6μL of sample to 120μL. After dilution, 3μL is taken for measurement, which is equivalent to 0.15μL), R1 addition amount 120μL; R2 addition amount 80μL; transmission wavelength 700nm, scattering angle 20 degrees; two-point endpoint method 18-34 reading points; transmissive scattering fusion area 10-50mg / L, results less than 50mg / L are reported as scattering results, and results greater than or equal to 50mg / L are reported as transmission results.

[0178] The results are shown in Table 5.

[0179] Table 5 Low value precision of Examples 7-9 and Comparative Examples 4-6

[0180]

[0181] The results are shown in Table 5. After adding 6-aminohexanoic acid and sodium periodate to R1, the serum IgD molecules in the sample were quickly and effectively highly glycosylated, making the IgD antigen conformation more stable and more conducive to the binding of the IgD molecules to the anti-IgD antibodies in the detection reagent. Combined with 350nm large-particle latex, the scattered light detection of low concentrations through the scattered latex turbidimetry method improves the sensitivity of the reagent. When the sample is tested in a 20-fold pre-dilution mode, the low-end precision requirements are met (when the IgD concentration is 2.5mg / L, CV≤3%). This takes into account both reagent performance and cost control.

[0182] (3) Linear range verification

[0183] After calibration, the linear range of Examples 7-9 was verified according to the IgD kit reaction process. A high-value serum sample (serum IgD concentration 800 mg / L) and a low-value serum sample (serum IgD concentration 2 mg / L) were mixed at six concentration ratios of 1:0, 0.8:0.2, 0.6:0.4, 0.4:0.6, 0.2:0.8, and 0:1. Each concentration was measured three times. Correlation analysis was performed between the theoretical concentrations and the measured values.

[0184] The instrument used is a Hitachi transmissive scattering all-in-one instrument. Taking Hitachi3500 as an example, the transmissive scattering all-in-one method is used; the machine parameters are: sample volume 0.15μL (0.15μL is the use of the instrument pre-dilution function. The fully automatic biochemical analyzer automatically dilutes 6μL of sample to 120μL. After dilution, 3μL is taken for measurement, which is equivalent to 0.15μL), R1 addition amount 120μL; R2 addition amount 80μL; transmission wavelength 700nm, scattering angle 20 degrees; two-point endpoint method 18-34 reading points; transmissive scattering fusion area 10-50mg / L, results less than 50mg / L are reported as scattering results, and results greater than or equal to 50mg / L are reported as transmission results.

[0185] The results are shown in Table 6.

[0186] Table 6 IgD linear range verification results

[0187]

[0188] The results are shown in Table 6. The relative deviations of the measured values ​​of Examples 7-9 from the theoretical concentrations are all <±10%, while the relative deviations of the measured values ​​of Comparative Examples 4-6 from the theoretical concentrations are >±20%, and the high values ​​of Comparative Examples 4-6 cannot be measured. Figure 5 As shown, the correlations of Examples 7-9 within the linear range are R 2 =0.9999, R 2 =1, R 2 =0.9989, meeting the requirements of the detection reagent; the correlations of comparative examples 4-6 in the linear range are R 2 =0.9833, R 2 =0.9731, R 2 =0.9606, which does not meet the requirements of the test reagent. In summary, adding 6-aminocaproic acid and sodium periodate to R1 of the IgD reagent can significantly improve the linear range of the reagent. High-value samples do not need to be diluted and retested, which reduces the department's reagent and labor costs and better meets the needs of clinical test reagents.

[0189] (4) Antigen excess

[0190] After calibration, the antigen excess safety zone of Examples 7-9 was verified according to the IgD kit reaction process.

[0191] The instrument used is a Hitachi transmissive scattering all-in-one instrument. Taking Hitachi3500 as an example, the transmissive scattering all-in-one method is used; the machine parameters are: sample volume 0.15μL (0.15μL is the use of the instrument pre-dilution function. The fully automatic biochemical analyzer automatically dilutes 6μL of sample to 120μL. After dilution, 3μL is taken for measurement, which is equivalent to 0.15μL), R1 addition amount 120μL; R2 addition amount 80μL; transmission wavelength 700nm, scattering angle 20 degrees; two-point endpoint method 18-34 reading points; transmissive scattering fusion area 10-50mg / L, results less than 50mg / L are reported as scattering results, and results greater than or equal to 50mg / L are reported as transmission results.

[0192] The results are shown in Table 7.

[0193] Table 7 IgD antigen excess test results

[0194]

[0195] The results are shown in Table 7. When the IgD concentration in the sample reached 9016 mg / L, the measured values ​​of Examples 7-9 were still greater than 800 mg / L (the upper limit of linearity), and the instrument displayed an alarm message of '>TEST', prompting the need for dilution and retesting. On the other hand, in Comparative Examples 4-6, when the IgD concentration in the sample was 4500 mg / L, its measured value was <800 mg / L (the upper limit of linearity), and the instrument did not display an alarm message of '>TEST'. At this time, the measured value of the extremely high value sample may be underestimated, thereby affecting clinical diagnosis. Figure 6 Among them, when linearity = 800 mg / L, Examples 7-9 can simultaneously meet the requirements, and the antigen excess is measured to be above 9000 mg / L; while the antigen excess of Comparative Examples 4-6 can only be measured to about 3000 mg / L, indicating that after adding 6-aminocaproic acid and sodium periodate to R1 of the IgD reagent, the antigen excess safety zone of the reagent is significantly increased from 3000 mg / L to 9000 mg / L.

[0196] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An immunoglobulin D through-scattering latex turbidimetry detection kit, characterized in that: including a first reagent R1 and a second reagent R2; The first reagent R1 includes the following components at the following concentrations: 25-200 mM Hepes-NaOH buffer pH 6.8-7.5, 1000-2500 mM NaCl, 10 g / L polyethylene glycol 12000, 0.05%-2% Tween-20, 1%-5% 6-aminohexanoic acid, 30-200 mM sodium periodate, and 5-20 ml / L rabbit polyclonal antibody blocking agent; The second reagent R2 includes components with the following concentrations: 50-200 mM buffer pH 7.0-7.6, Tween-20 4-16 ml / L, 80 nm carboxyl latex 2.4-3.2 g / L, 200 nm carboxyl latex 1.6-2.4 g / L, 350 nm carboxyl latex 1.0-1.8 g / L, protective agent 10-90 g / L, and rabbit anti-human IgD polyclonal antibody 1.2-6.8 mg / L.

2. The immunoglobulin D through-scattering latex turbidimetry detection kit according to claim 1, characterized in that: The first reagent R1 and the second reagent R2 further include a preservative, which is 1 g / L sodium azide or 0.5 ml / L PC300.

3. An immunoglobulin D through-scattering latex turbidimetry detection kit according to claim 1 or 2, characterized in that: In the second reagent R2, the buffer is Hepes-NaOH buffer, phosphate buffer or glycine-Tris buffer.

4. The immunoglobulin D through-scattering latex turbidimetry detection kit according to claim 3, characterized in that: In the second reagent R2, the protective agent is at least one of 10-30 g / L sucrose, 10-30 g / L trehalose, and 10-30 g / L fructose.

5. The immunoglobulin D through-scattering latex turbidimetry detection kit according to claim 4, characterized in that: Also includes calibrators and quality controls; The calibrator includes the following components at the following concentrations: 50 mM Hepes-NaOH buffer pH 6.8-8.2, IgD antigen 10-800 mg / L, sodium chloride 900 mM, Tween-20 10 ml / L, preservative Proclin 3000.5 ml / L, oxidized glutathione 0.2-2.0 mM, protein disulfide isomerase 0.5-5.0 μM. The quality control product includes the following components at the following concentrations: 50 mM Hepes-NaOH buffer pH 6.8-8.2, IgD antigen 20-160 mg / L, sodium chloride 900 mM, Tween-20 10 ml / L, preservative Proclin 3000.5 ml / L, oxidized glutathione 0.2-2.0 mM, protein disulfide isomerase 0.5-5.0 μM.

6. Use of the immunoglobulin D through-scattering latex turbidimetry detection kit according to claim 1 in detecting IgD.

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