Kit and method for detecting ratio of kappa-FLC to lambda-FLC through single channel

Abstract

The invention discloses a kit and a method for detecting the ratio of kappa-FLC to lambda-FLC through a single channel. The kit for detecting the ratio of kappa-FLC to lambda-FLC through the single channel comprises a reagent 1, a reagent 2 and a calibrator reagent, wherein the reagent 1 comprises latex microspheres coated with lambda-FLC and kappa-FLC polyclonal antibodies, and a first buffer solution; the reagent 2 comprises an FLC monoclonal antibody, a second buffer solution and 0.1-1wt% of a second surfactant; the calibrator reagent comprises a third buffer solution and a calibrator with different ratios of a kappa-FLC antigen to a lambda-FLC antigen. The kit for detecting the ratio of the kappa-FLC to the lambda-FLC in the serum through the single channel, provided by the invention, can accurately determine the ratio of the kappa-FLC to the lambda-FLC, and is low in required cost, high in detection efficiency, large in test flux, high in detection accuracy and good in specificity.

Description

Technical Field

[0001] The present application belongs to the field of in vitro immunodiagnosis, and specifically relates to a kit and method for single-channel detection of the ratio of serum κ-FLC and λ-FLC. Background Art

[0002] Multiple myeloma is a malignant disease characterized by abnormal proliferation of clonal plasma cells, accompanied by an increase in monoclonal immunoglobulin. It is the second most common malignant tumor in the blood system. Its clinical manifestations include increased blood calcium, renal damage, anemia, and osteolytic lesions.

[0003] Immunoglobulins are composed of two light chains and two heavy chains connected by disulfide bonds. The light chains are divided into two types, kappa and lambda, and the normal ratio of the two in the human body is 2:1. The molecular weight of the light chain is smaller than that of the heavy chain. Therefore, the synthesis rate of light chains in the human body is slightly faster than that of heavy chains. In addition to combining with heavy chains to form immunoglobulins, some light chains fail to combine with heavy chains and are in a free state, namely free light chains. Free light chains can be quickly cleared and metabolized by the kidneys. The concentration of free light chains depends on the dynamic balance between the synthesis and secretion of plasma cells and the clearance of the kidneys. The free light chain content in normal people is relatively low. However, in multiple myeloma, the level of free light chains in the body can be increased to 30-40 times that of normal people, exceeding the metabolic capacity of the kidneys, and the κ / λ ratio of free light chains is abnormal. Clinical data show that 96% of multiple myeloma patients have abnormal serum κ-FLC or λ-FLC, or abnormal FLCκ / λ ratio; the normal FLCκ / λ ratio is between 0.26 and 1.65. If FLCκ / λ is less than 0.26 or FLCκ / λ is greater than 1.65, it indicates that the patient produces excessive free light chains in the body.

[0004] Multiple myeloma is common in middle-aged and elderly people. With the advancement of aging, serum free light chain (FLC) detection will become a routine blood test item in the future. Serum free light chain (FLC) is an important indicator recommended for clinical diagnosis and treatment of multiple myeloma at home and abroad. It has high sensitivity and specificity and has been included in the national multiple myeloma diagnosis and treatment guidelines.

[0005] The prior art uses a two-step method - latex-enhanced immunoturbidimetry to detect κ-FLC and λ-FLC respectively, and then calculates the FLCκ / λ ratio. The existing two-step method - latex-enhanced immunoturbidimetry to detect the FLCκ / λ ratio has the following shortcomings: (1) High detection cost: It is equivalent to measuring two items, and the transportation, storage, and production costs are twice as high (2) Low detection throughput: The project needs to occupy 4 reagent positions, which is not conducive to hospital menu testing and efficient testing. Therefore, it is urgent to improve the two-step method - latex-enhanced immunoturbidimetry to detect free light chain projects. Summary of the invention

[0006] Aiming at the shortcomings of high cost and low throughput of the two-step method for detecting the ratio of κ-FLC and λ-FLC in the prior art, the present invention provides a single-channel kit for detecting the ratio of κ-FLC and λ-FLC.

[0007] The present invention selects a polyclonal antibody that can simultaneously recognize both λ-FLC and κ-FLC antigens and a monoclonal antibody that only recognizes one of the antigens to realize a one-step multiple myeloma detection technology, thereby achieving the goal of increasing throughput and reducing costs.

[0008] In a first aspect, the present invention provides a single-channel kit for detecting the ratio of κ-FLC to λ-FLC, the kit comprising reagent 1, reagent 2, and a calibrator reagent; wherein:

[0009] Reagent 1 includes latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies and a first buffer;

[0010] Reagent 2 comprises FLC monoclonal antibody, a second buffer and 0.1-1 wt % of a second surfactant;

[0011] The calibrator reagent includes a third buffer and calibrators having different ratios of κ-FLC antigen to λ-FLC antigen.

[0012] Preferably, in reagent 1, the λ-FLC and κ-FLC polyclonal antibodies are mouse anti-human λ-FLC and κ-FLC polyclonal antibodies;

[0013] and / or, the concentration of latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 0.01-1wt%, preferably 0.05-0.1wt%;

[0014] And / or, the particle size of the latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 80-120 nm, preferably 100 nm.

[0015] Preferably, the pH value of the first buffer in reagent 1, the second buffer in reagent 2, or the third buffer in the calibrator reagent is independently 6 to 9;

[0016] and / or, the concentration of the first buffer in reagent 1, the second buffer in reagent 2, or the third buffer in the calibrator reagent is independently 10 mmol / L to 100 mmol / L, preferably 20 mmol / L to 50 mmol / L;

[0017] And / or, the first buffer in reagent 1 or the second buffer in reagent 2 or the third buffer in the calibrator reagent is independently one or a combination of two or more of MES buffer, borate buffer, MOPS buffer, phosphate buffer or glycine buffer.

[0018] Preferably, reagent 1 further comprises 0.1 wt% to 5 wt% of a first stabilizer, and / or, 0.1 wt% to 1 wt% of a first surfactant, and / or, 0.1 wt% to 10 wt% of a first preservative;

[0019] Preferably, the first stabilizer in reagent 1 is one or a combination of two or more of bovine serum albumin, mannitol, magnesium chloride, and sodium bisulfate, preferably bovine serum albumin; and / or, the concentration of the first stabilizer in reagent 1 is 0.5wt% to 3wt%;

[0020] Preferably, the first surfactant in reagent 1 is selected from one or a combination of two or more of Tween, TX-100, Briji-35 and dodecyl glycoside; and / or, the concentration of the first surfactant in reagent 1 is 0.1wt% to 0.5wt%, preferably 0.3wt%;

[0021] Preferably, the first preservative in reagent 1 is selected from one or a combination of two or more of ammonium azide, phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, and Proclin 300, preferably 2-phenoxyethanol; and / or, the first preservative in reagent 1 is 0.1 wt% to 1 wt%.

[0022] Preferably, the second surfactant in reagent 2 is dodecyl glucoside, or a combination of dodecyl glucoside and one or more of the following substances: Tween, TX-100, Briji-35, mannitol;

[0023] and / or, the concentration of the second surfactant in reagent 2 is 0.1 wt% to 0.5 wt%, preferably 0.3 wt%;

[0024] Preferably, the mass ratio of dodecyl glucoside to mannitol in the second surfactant in reagent 2 is (1-3):1.

[0025] Preferably, reagent 2 further contains 0.05 wt% to 10 wt% of an electrolyte, and / or 0.01 wt% to 1 wt% of a reaction accelerator, and / or 0.1 wt% to 5 wt% of a second preservative;

[0026] Preferably, the electrolyte in reagent 2 is selected from one or a combination of two or more of sodium chloride, potassium chloride, magnesium chloride, and magnesium sulfate; and / or, the mass percentage of the electrolyte in reagent 2 is 0.05wt% to 1wt%;

[0027] Preferably, the reaction accelerator in reagent 2 is selected from one or a combination of two or more of polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 8000. Preferably, the reaction accelerator is polyethylene glycol 8000; and / or, the mass percentage of the reaction accelerator in reagent 2 is 0.01wt% to 0.5wt%.

[0028] Preferably, the second preservative in reagent 2 is selected from one or a combination of two or more of sodium azide, phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, and Proclin 300, preferably 2-phenoxyethanol;

[0029] And / or, the mass percentage of the second preservative in reagent 2 is 0.1 wt % to 3 wt %.

[0030] Preferably, the FLC monoclonal antibody in reagent 2 is a λ-FLC monoclonal antibody or a κ-FLC monoclonal antibody;

[0031] and / or, the FLC monoclonal antibody in reagent 2 is a mouse anti-human FLC monoclonal antibody;

[0032] And / or, the mass percentage of the FLC monoclonal antibody in reagent 2 is 0.0005wt% to 0.005wt%, preferably 0.0007wt%.

[0033] Preferably, the calibrator reagent further contains a third stabilizer and / or a third preservative;

[0034] Preferably, the third stabilizer is selected from one or a combination of two or more of bovine serum albumin, mannitol, magnesium chloride and sodium bisulfate, preferably bovine serum albumin; and / or, the mass percentage of the third stabilizer in the calibrator is 0.01wt% to 1wt%, preferably 0.01wt% to 0.5wt%;

[0035] Preferably, the third preservative is selected from one or a combination of two or more of sodium azide, phenol, p-hydroxybenzoic acid, 2-phenoxyethanol and Proclin300, preferably 2-phenoxyethanol; and / or, the mass percentage of the third preservative in the calibrator is 0.1wt% to 3wt%.

[0036] Preferably, the concentration of λ-FLC in the calibrator reagent is 8.0 to 200 mg / L, and the concentration of κ-FLC is 4.5 mg / L to 160 mg / L.

[0037] And / or, the ratio of κ-FLC to λ-FLC in the calibrator reagent is 0, 0.1-0.3, 0.5-1.5, 3-7 and 10-20.

[0038] Preferably, the preparation method of the latex microspheres and polyclonal antibody coupled particles in reagent 1 comprises the following steps: after mixing the latex microspheres with the fourth buffer, adding an activator to activate the latex microspheres; adding free light chain polyclonal antibodies to couple the activated latex microspheres with the free light chain polyclonal antibodies; and then blocking with BSA to obtain the latex microspheres and polyclonal antibody coupled particles;

[0039] Preferably, the fourth buffer is one or a combination of two or more of MES buffer, borate buffer, MOPS buffer, phosphate buffer or glycine buffer;

[0040] Preferably, the pH value of the fourth buffer is 6 to 9;

[0041] Preferably, the concentration of the fourth buffer is 10 mmol / L to 100 mmol / L;

[0042] Preferably, the mass ratio of the latex microspheres to the activator is 20:(1-3);

[0043] Preferably, the activator is one or both of EDC and NHS.

[0044] Preferably, the activators are EDC and NHS;

[0045] Preferably, in the activator, the mass ratio of EDC to NHS is 1:(1-3), preferably 1:2; preferably, the mass ratio of the free light chain polyclonal antibody to the activated latex microspheres is 50 μg / mg-200 μg / mg, preferably 100 μg / mg;

[0046] Preferably, the final concentration of BSA during the blocking process is 0.5-2 wt%, preferably 1 wt%.

[0047] In a second aspect, the present invention provides use of the kit in detecting the ratio of λ-FLC to κ-FLC.

[0048] In a third aspect, the present invention provides a method for detecting the ratio of λ-FLC to κ-FLC, the method comprising taking a sample to be tested, performing an immune reaction using the kit, and obtaining the ratio of κ-FLC to λ-FLC by performing turbidity detection on the reaction product.

[0049] The single-channel kit for detecting the ratio of serum κ-FLC to λ-FLC provided by the present invention can accurately determine the ratio of κ-FLC to λ-FLC, with R>0.99. The kit provided by the present invention has low cost, high detection efficiency, large test throughput, high detection accuracy and good specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Shown is a reaction process diagram of the kit provided by the present invention;

[0051] Figure 2 Shown is the linear regression equation of sample correlation obtained using the kit of Example 4;

[0052] Figure 3 Shown is the linear regression equation of sample correlation obtained using the kit of Comparative Example 1;

[0053] Figure 4 Shown is the linear regression equation of sample correlation obtained using the kit of Example 5;

[0054] Figure 5 Shown is the linear regression equation of sample correlation obtained using the kit of Example 7;

[0055] Figure 6 Shown is the linear regression equation of sample correlation obtained using the kit of Example 8;

[0056] Figure 7 Shown is the linear regression equation obtained using the kit of Comparative Example 1;

[0057] Figure 8 Shown is the linear regression equation obtained using the kit of Example 4. DETAILED DESCRIPTION

[0058] The present invention provides a single-channel kit and method for detecting the ratio of kappa-FLC to lambda-FLC. The kit provided by the present invention can be used to directly detect the ratio of serum kappa-FLC to lambda-FLC.

[0059] The kit provided by the invention contains a polyclonal antibody capable of simultaneously recognizing both λ-FLC and κ-FLC antigens and a monoclonal antibody capable of only recognizing λ-FLC antigen or only recognizing κ-FLC antigen.

[0060] Specifically, in one embodiment provided by the present invention, the kit contains reagent 1, reagent 2, and a calibrator reagent; wherein,

[0061] Reagent 1 includes latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies and a first buffer;

[0062] Reagent 2 comprises FLC monoclonal antibody, a second buffer and 0.1 wt % to 1 wt % of a second surfactant;

[0063] The calibrator reagent includes a third buffer and calibrators having different ratios of κ-FLC antigen to λ-FLC antigen.

[0064] In a specific embodiment of the present invention, the latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 are formed by coupling latex microspheres with λ-FLC and κ-FLC polyclonal antibodies.

[0065] In a specific embodiment of the present invention, the λ-FLC and κ-FLC polyclonal antibodies in reagent 1 can recognize λ-FLC and κ-FLC.

[0066] In a specific embodiment of the present invention, in reagent 1, the λ-FLC and κ-FLC polyclonal antibodies are mouse anti-human λ-FLC and κ-FLC polyclonal antibodies;

[0067] In a specific embodiment of the present invention, the concentration of latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 0.01wt% to 1wt%, for example, 0.01wt%, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt%, 0.65wt%, 0.7wt%, 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt% or a range composed thereof.

[0068] In a specific embodiment of the present invention, the concentration of latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 0.05wt% to 0.1wt%, for example, 0.05wt%, 0.06wt%, 0.07wt%, 0.08wt%, 0.09wt%, 0.1wt% or a range of their compositions.

[0069] In a specific embodiment of the present invention, the particle size of the latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 80nm to 120nm, for example, 80nm, 90nm, 100nm, 110nm, 120nm or a range thereof, preferably 100nm.

[0070] In a specific embodiment of the present invention, the pH value of the first buffer in reagent 1, the second buffer in reagent 2, or the third buffer in the calibrator reagent is independently 6 to 9, for example, 6, 7, 8, 9 or a range composed thereof.

[0071] In a specific embodiment of the present invention, the concentration of the first buffer in reagent 1 or the second buffer in reagent 2 or the third buffer in the calibrator reagent is independently 10mmol / L to 100mmol / L, and can also be independently 20mmol / L to 50mmol / L, for example, 10mmol / L, 20mmol / L, 25mmol / L, 30mmol / L, 35mmol / L, 40mmol / L, 45mmol / L, 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L or a range thereof.

[0072] In a specific embodiment of the present invention, the first buffer in reagent 1 or the second buffer in reagent 2 or the third buffer in the calibrator reagent is independently one or a combination of two or more of MES buffer, borate buffer, MOPS buffer, phosphate buffer or glycine buffer.

[0073] In a specific embodiment of the present invention, reagent 1 further comprises 0.1wt% to 5wt% of a first stabilizer, for example, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt% or a range thereof, and / or, 0.1wt% to 1wt% of a first surfactant, for example, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt%, 0.65wt%, 0.7wt%, 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt% or a range thereof, and / or, 0.1wt% to 10wt% of a first preservative, for example, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, 8wt%, 8.5wt%, 9wt%, 9.5wt%, 10wt% or a range thereof;

[0074] In a specific embodiment of the present invention, the first stabilizer in reagent 1 is one or a combination of two or more of bovine serum albumin, mannitol, magnesium chloride, and sodium bisulfate, preferably bovine serum albumin; preferably, the concentration of the first stabilizer in reagent 1 is 0.5wt% to 3wt%, for example, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt% or a range composed thereof.

[0075] In a specific embodiment of the present invention, the first surfactant in reagent 1 is selected from one or a combination of two or more of Tween, TX-100, Briji-35 and dodecyl glycoside; preferably, the concentration of the first surfactant in reagent 1 is 0.1wt% to 0.5wt%, for example, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt% or a range of their compositions, preferably 0.3wt%;

[0076] In one embodiment of the present invention, the first preservative in reagent 1 is selected from sodium azide 、 Phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, Proclin 300 or a combination of two or more thereof, preferably 2-phenoxyethanol; preferably, the first preservative in reagent 1 is 0.1wt%-1wt%, for example, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt%, 0.65wt%, 0.7wt%, 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt% or a range thereof.

[0077] In a specific embodiment of the present invention, the second surfactant in reagent 2 is dodecyl glucoside, or a combination of dodecyl glucoside and one or more of the following substances: Tween, TX-100, Briji-35, mannitol;

[0078] In a specific embodiment of the present invention, the concentration of the second surfactant in reagent 2 is 0.1wt% to 0.5wt%, for example, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt% or a range of their compositions, preferably 0.3wt%.

[0079] In a specific embodiment of the present invention, the mass ratio of dodecyl glucoside to mannitol in the second surfactant in reagent 2 is (1-3):1.

[0080] In a specific embodiment of the present invention, reagent 2 further contains 0.05wt% to 10wt% of an electrolyte, for example, 0.05wt%, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 5.5wt%, 6wt%, 6.5wt%, 7wt%, 7.5wt%, 8wt%, 8.5wt%, 9wt%, 9.5wt%, 10wt% or a range thereof; and / or, 0.01wt% to 1wt% of a reaction accelerator, for example, 0.01wt%, 0.05wt%, 0.1wt%, 0. 15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt%, 0.65wt%, 0.7wt%, 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt% or a range thereof, and / or, 0.1wt% to 5wt% of a second preservative, for example, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt% or a range thereof.

[0081] In a specific embodiment of the present invention, the electrolyte in reagent 2 is selected from one or a combination of more than one of sodium chloride, potassium chloride, magnesium chloride and magnesium sulfate; preferably, the mass percentage of the electrolyte in reagent 2 is 0.05wt% to 1wt%, for example, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt%, 0.65wt%, 0.7wt%, 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt% or a range thereof.

[0082] In a specific embodiment of the present invention, the reaction accelerator in reagent 2 is selected from one or a combination of two or more of polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 8000. Preferably, the reaction accelerator is polyethylene glycol 8000.

[0083] In a specific embodiment of the present invention, the mass percentage of the reaction accelerator in reagent 2 is 0.01wt% to 0.5wt%, for example, 0.01wt%, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt% or a range of their compositions.

[0084] In a specific embodiment of the present invention, the second preservative in reagent 2 is selected from one or a combination of two or more of sodium azide, phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, and Proclin 300, preferably 2-phenoxyethanol.

[0085] In a specific embodiment of the present invention, the mass percentage of the second preservative in reagent 2 is 0.1wt% to 3wt%, for example, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt% or a range of their compositions.

[0086] In a specific embodiment of the present invention, the FLC monoclonal antibody in reagent 2 is a λ-FLC monoclonal antibody or a κ-FLC monoclonal antibody.

[0087] In a specific embodiment of the present invention, the FLC monoclonal antibody in reagent 2 is a mouse anti-human FLC monoclonal antibody.

[0088] In a specific embodiment of the present invention, the mass percentage of FLC monoclonal antibody in reagent 2 is 0.0005wt% to 0.005wt%, for example, 0.0005wt%, 0.0007wt%, 0.001wt%, 0.0015wt%, 0.002wt%, 0.0025wt%, 0.003wt%, 0.0035wt%, 0.004wt%, 0.0045wt%, 0.005wt% or a range of their compositions, preferably 0.0007wt%.

[0089] In a specific embodiment of the present invention, the calibrator reagent further contains a third stabilizer and / or a third preservative;

[0090] In a specific embodiment of the present invention, the third stabilizer is selected from bovine serum albumin, mannitol, magnesium chloride, sodium bisulfate, or a combination of two or more thereof, preferably bovine serum albumin; preferably, the mass percentage of the third stabilizer in the calibrator is 0.01wt% to 1wt%, for example, 0.01wt%, 0.05wt%, 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, 0.55wt%, 0.6wt%, 0.65wt%, 0.7wt%, 0.75wt%, 0.8wt%, 0.85wt%, 0.9wt%, 0.95wt%, 1wt% or a range thereof, preferably 0.01-0.5wt%;

[0091] In a specific embodiment of the present invention, the third preservative is selected from one or a combination of two or more of phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, and Proclin 300, preferably 2-phenoxyethanol; preferably, the mass percentage of the third preservative in the calibrator is 0.1wt% to 3wt%, for example, 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt% or a range composed thereof.

[0092] In a specific embodiment of the present invention, the concentration of λ-FLC in the calibrator reagent is 8.0-200 mg / L, and the concentration of κ-FLC is 4.5-160 mg / L.

[0093] In a specific embodiment of the present invention, the ratio of κ-FLC to λ-FLC in the calibrator reagent is 0, 0.1-0.3, 0.5-1.5, 3-7 and 10-20.

[0094] In a specific embodiment of the present invention, the preparation method of latex microspheres and polyclonal antibody coupled particles in reagent 1 includes the following steps: after mixing the latex microspheres with the fourth buffer, adding an activator to activate the latex microspheres; adding free light chain polyclonal antibodies, coupling the activated latex microspheres with the free light chain polyclonal antibodies; and then using BSA for blocking to obtain the latex microspheres and polyclonal antibody coupled particles.

[0095] In a specific embodiment of the present invention, the fourth buffer is one or a combination of two or more of MES buffer, borate buffer, MOPS buffer, phosphate buffer or glycine buffer;

[0096] In a specific embodiment of the present invention, the pH value of the fourth buffer is 6 to 9, for example, 6, 7, 8, 9 or a range composed thereof.

[0097] In a specific embodiment of the present invention, the concentration of the fourth buffer is 10 to 100 mmol / L, for example, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, 60 mmol / L, 70 mmol / L, 80 mmol / L, 90 mmol / L, 100 mmol / L or a range thereof.

[0098] In a specific embodiment of the present invention, the mass ratio of the latex microspheres to the activator is 20:(1-3), for example, 20:1, 20:1.5, 20:2, 20:2.5, 20:3 or a range thereof.

[0099] In a specific embodiment of the present invention, the activator is one or both of EDC and NHS.

[0100] In a specific embodiment of the present invention, the activating agents are EDC and NHS.

[0101] In a specific embodiment of the present invention, in the activator, the mass ratio of EDC to NHS is 1:(1-3), for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3 or a range thereof, preferably 1:2.

[0102] The EDC in the present invention is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0103] NHS in the present invention is N-hydroxysuccinimide.

[0104] In a specific embodiment of the present invention, the mass ratio of the free light chain polyclonal antibody to the activated latex microspheres is 50 μg / mg to 200 μg / mg, for example, 50 μg / mg, 100 μg / mg, 150 μg / mg, 200 μg / mg or a range thereof, preferably 100 μg / mg.

[0105] In a specific embodiment of the present invention, when the latex microspheres are blocked, the final concentration of BSA is 0.5-2wt%, such as 0.5wt%, 0.6wt%, 1wt%, or a range thereof, preferably 1wt%.

[0106] In a specific embodiment of the present invention, there is provided a use of the kit in detecting the ratio of λ-FLC to κ-FLC.

[0107] In a specific embodiment of the present invention, a method for detecting the ratio of λ-FLC to κ-FLC is provided, the method comprising taking a sample to be tested, performing an immune reaction using the kit, and obtaining the ratio of κ-FLC to λ-FLC by performing turbidity detection on the reaction product.

[0108] In a specific embodiment of the present invention, the FLC monoclonal antibody in reagent 2 is a monoclonal antibody that only recognizes κ-FLC or a monoclonal antibody that only recognizes λ-FLC.

[0109] The principle of the single-channel method for determining the ratio of λ-FLC and κ-FLC provided by the present invention is as follows: Figure 1 As shown, latex microspheres coated with anti-human λ-FLC and κ-FLC antibodies specifically bind to the antigens in the sample to form a complex, which then binds to the mouse anti-human λ-FLC antibody to form a complex to produce turbidity, and the turbidity is related to the ratio of λ-FLC and κ-FLC.

[0110] Dodecyl glycoside fully exposes the antigen binding site without affecting the binding of the antigen to the polyclonal antibody, allowing the κ-FLC antigen to react with the κ-FLC antibody monoclonal antibody. The optimal concentration of dodecyl glycoside is 0.3%. It can also accelerate the reaction system to quickly reach the reaction endpoint.

[0111] The latex microspheres in reagent 1 are coupled with an antibody against λ-FLC and κ-FLC, which can recognize λ-FLC and κ-FLC. Under the action of the second surfactant in reagent 2, they are combined with the κ-FLC antibody in reagent 2 again to form a complex to produce an absorbance change, and the absorbance change is related to the ratio of κ-FLC to λ-FLC. Only one set of reagents and one set of channels are required, and the test throughput is twice that of the conventional two-step method; the cost of reagent preparation and transportation is lower than that of the two-step method; it is simpler and more convenient.

[0112] In order to achieve single-channel testing of the ratio of λ-FLC and κ-FLC in the present invention, 1) the interference of immunoglobulins in the reaction system on the free light chain and microsphere reaction needs to be resolved; 2) a multi-antibody that can achieve "one-to-many" needs to be screened out, that is, one FLC antibody can simultaneously recognize serum λ-FLC and κ-FLC antigens; 3) the hidden antigen binding sites of λ-FLC and κ-FLC need to be fully exposed.

[0113] Specifically, when the inventors of the present application selected mouse anti-human free light chain antibodies, they first selected antibodies that only recognized the binding site of the light chain and heavy chain to solve the interference of immunoglobulins on free light chains. They screened out polyclonal antibodies that could achieve "one-to-many", that is, one FLC antibody indiscriminately recognized λ-FLC and κ-FLC antigens in serum, and screened out monoclonal antibodies that only recognized λ-FLC or κ-FLC. At the same time, the inventors of the present application found that the use of surfactants containing dodecyl glycosides could expose specific detection sites and make the detection results more accurate.

[0114] Antibodies and latex microspheres are raw materials with a relatively large cost in latex immunoturbidimetry. In the conventional two-step method-latex immunoturbidimetry, microspheres and κ-FLC antibodies and λ-FLC antibodies must be used separately for coupling and then tested separately. Compared with the traditional two-step method, the present invention can measure λ-FLC and κ-FLC through a single channel, and only one set of reagents is needed to directly measure the ratio of κ-FLC and λ-FLC. In the present invention, only one coupling is required, and the use of antibodies and microspheres is greatly reduced. The cost is about half of the conventional two-step method-latex immunoturbidimetry, and the production, transportation, and storage costs of reagents are greatly reduced. When used, the test amount of the kit provided by the present invention is twice that of the conventional two-step method-latex immunoturbidimetry in the prior art. The ratio is directly measured without calculation, and the detection throughput is large and the efficiency is high. It has the advantages of being simple and convenient. The kit provided by the present invention can accurately measure the ratio of κ-FLC and λ-FLC, R>0.99.

[0115] The surfactant used in the kit provided by the present invention contains dodecyl glycoside, which can fully expose the antigen binding site, so that the antigen exposes other binding sites after reacting with the antibody coupled to the microsphere, and continues to react with κ-FLC or λ-FLC, thereby increasing the accuracy of the reaction.

[0116] The kit provided by the present invention can be applied to Hitachi 7180 fully automatic biochemical analyzer, Abbott series, Beckman series, Roche series and other fully automatic biochemical analyzers, and the specific parameters on each instrument can be appropriately adjusted.

[0117] In order to further illustrate the present application, the present application will be clearly and completely described below in conjunction with the embodiments. However, the present application can be implemented in a variety of different forms, and the embodiments are only a part. The present invention is not limited to the embodiments, and the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0118] It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention.

[0119] The FLC free light chain polyclonal antibody used in the example was purchased from Agilent Technologies (International) Private Company's mouse anti-human free light chain polyclonal antibody with catalog number A0117; the κ-FLC antibody was purchased from Agilent Technologies (International) Private Company's mouse anti-human free κ-FLC light chain monoclonal antibody with catalog number A0111; the κ-FLC antigen was purchased from Agilent Technologies (International) Private Company with catalog number A0120; and the λ-FLC antigen was purchased from Agilent Technologies (International) Private Company with catalog number A0121.

[0120] Example 1. Preparation of free light chain polyclonal antibody-coated latex microspheres in reagent 1

[0121] 1). Activation of latex microspheres

[0122] Take 100 μl of latex microspheres (particle size: 100 nm, concentration 10 wt%) and 1 ml of 2-(N-morpholine)-ethanesulfonic acid buffer (also known as MES, 20 mM pH 6.0) and shake for 10 min; add 50 μl and 100 μl of EDC and NHS respectively (both concentrations are 10 mg / ml), activate at 37°C for 20 min. Centrifuge at 15000 rpm / min for 30 minutes and remove the supernatant.

[0123] 2). Coupling

[0124] FLC free light chain polyclonal antibody was added, the mass ratio of FLC polyclonal antibody to latex microspheres was 100 μg / mg, and the final volume was 1100 μl with 2-(N-morpholine)-ethanesulfonic acid buffer (also known as MES: 20 mM pH 7.0), and coupled at 37°C for 2 h.

[0125] 3). Closed

[0126] 10 wt % BSA 110 μl buffer was added for blocking at 37° C. for 1 h, and centrifuged at 15000 rpm / min for 45 min, and the supernatant was removed.

[0127] 4). Cleaning

[0128] The cells were washed with a washing solution (20 mM MOPS + 0.5% BSA + 0.5% TX100 + 0.1% phenoxyethanol, pH 7.0) and the supernatant was removed by centrifugation.

[0129] 5). Resuspend

[0130] The precipitate was re-dissolved by adding 10 ml of latex diluent (20 mM MOPS + 0.5% BSA + 0.1% TWEEN + 0.5% TX100 + 0.1% phenoxyethanol, pH 7.0), and ultrasonically dispersed by cell disruptor for 8 min, ultrasonically for 0.6 s, with an interval of 0.4 s. The ultrasonicated latex reagent was stored at 2-8°C for future use.

[0131] Example 2: Preparation of free light chain polyclonal antibody-coated latex microspheres in reagent 1

[0132] 1). Activation of latex microspheres

[0133] Take 100 μl of latex microspheres (particle size: 120 nm, concentration 10 wt%) and shake with 1 ml of 2-(N-morpholine)-ethanesulfonic acid (also known as MES, 20 mM pH 6.0) for 10 min; add 50 μl and 100 μl of EDC and NHS respectively (both concentrations are 10 mg / ml), activate at 37°C for 20 min. Centrifuge at 15000 rpm / min for 30 minutes and remove the supernatant.

[0134] 2). Coupling

[0135] FLC free light chain polyclonal antibody was added, the mass ratio of FLC polyclonal antibody to latex microspheres was 100 μg / mg, and the mixture was coupled at 37°C for 2 h.

[0136] 3). Closed

[0137] 10 wt % BSA 110 μl buffer was added for blocking at 37° C. for 1 h, and centrifuged at 15000 rpm / min for 45 min, and the supernatant was removed.

[0138] 4). Cleaning

[0139] 10 wt % BSA 110 μl buffer was added for blocking at 37° C. for 1 h, and centrifuged at 15000 rpm / min for 45 min, and the supernatant was removed.

[0140] 5). Resuspend

[0141] Add 10 ml of latex diluent (20 mm MOPS + 0.5% BSA + 0.1%

[0142] TWEEN+0.5% TX100+0.1% phenoxyethanol, pH 7.0) was re-dissolved, and the cell disruptor was ultrasonically dispersed for 8 minutes, ultrasonically for 0.6 seconds, with an interval of 0.4 seconds. The ultrasonicated latex reagent was stored at 2-8°C for later use.

[0143] Example 3. Preparation of free light chain polyclonal antibody-coated latex microspheres in reagent 1

[0144] 1). Activation of latex microspheres

[0145] Take 100 μl of latex microspheres (particle size: 80 nm, concentration 10 wt%) and shake with 1 ml of 2-(N-morpholine)-ethanesulfonic acid (also known as MES, 20 mM pH 6.0) for 10 min; add 50 μl and 100 μl of EDC and NHS respectively (both concentrations are 10 mg / ml), activate at 37°C for 20 min. Centrifuge at 15000 rpm / min for 30 minutes and remove the supernatant.

[0146] 2). Coupling

[0147] FLC free light chain polyclonal antibody was added, the mass ratio of FLC polyclonal antibody to latex microspheres was 100 μg / mg, and the mixture was coupled at 37°C for 2 h.

[0148] 3). Closed

[0149] 10 wt % BSA 110 μl buffer was added for blocking at 37° C. for 1 h, and centrifuged at 15000 rpm / min for 45 min, and the supernatant was removed.

[0150] 4). Cleaning

[0151] The cells were washed with a washing solution (20 mm MOPS + 0.5% BSA + 0.5% TX100 + 0.1% phenoxyethanol, pH 7.0) and the supernatant was removed by centrifugation.

[0152] 5). Resuspend

[0153] Add 10 ml of latex diluent (20 mm MOPS + 0.5% BSA + 0.1%

[0154] TWEEN+0.5% TX100+0.1% phenoxyethanol, pH 7.0) was re-dissolved, and the cell disruptor was ultrasonically dispersed for 8 minutes, ultrasonically for 0.6 seconds, with an interval of 0.4 seconds. The ultrasonicated latex reagent was stored at 2-8°C for later use.

[0155] Example 4: A single-channel kit for detecting the ratio of κ-FLC to λ-FLC

[0156] Preparation of Reagent 1

[0157] Take the free light chain polyclonal antibody-coated latex microspheres obtained in Example 1 and prepare reagent 1 according to the following dosage:

[0158]

[0159] Reagent 2 preparation

[0160] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 7.50±0.05, make up to volume, and store at 2-8°C.

[0161]

[0162] Calibrator preparation

[0163] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 7.50±0.05, add different proportions of pure antigens, and store at 2-8°C.

[0164] MOPS buffer 20mM

[0165] Bovine serum albumin (stabilizer) 0.1g / L 2-Phenoxyethanol (preservative) 1g / L

[0166] The κ-FLC / λ-FLC values ​​were 0, 0.25, 1, 5, and 15, respectively. The specific concentrations of the κ-FLC antigen and the λ-FLC antigen are shown in Table 1 below.

[0167] Table 1

[0168] κ-FLC / λ-FLC value κ-FLC(mg / L) λ-FLC(mg / L) 0 0 10 0.25 2.5 10 1 10 10 5 50 10 15 150 10

[0169] Reagent 1 is a milky white solution, reagent 2 is a colorless or light yellow solution, and the calibrator is a colorless and transparent solution.

[0170] Example 5: A single-channel kit for detecting the ratio of κ-FLC to λ-FLC

[0171] Similar to Example 4, the difference is that the reaction accelerator used in reagent 2 is PEG2000. The specific preparation method of reagent 2 is as follows:

[0172] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 7.50±0.05, make up to volume, and store at 2-8°C.

[0173]

[0174]

[0175] Example 6: A single-channel kit for detecting the ratio of κ-FLC to λ-FLC

[0176] Similar to Example 4, the difference is that the free light chain polyclonal antibody-coated latex microspheres in Reagent 1 are the free light chain polyclonal antibody-coated latex microspheres prepared in Example 2, and the particle size of the free light chain polyclonal antibody-coated latex microspheres is 120 nm.

[0177] Example 7: A single-channel kit for detecting the ratio of κ-FLC to λ-FLC

[0178] Preparation of Reagent 1

[0179] Take the free light chain polyclonal antibody-coated latex microspheres obtained in Example 1 and prepare reagent 1 according to the following dosage:

[0180]

[0181] Reagent 2 preparation

[0182] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 9.0±0.05, make up to volume, and store at 2-8°C.

[0183]

[0184] Calibrator preparation

[0185] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 8.0±0.05, add different proportions of pure antigens, and store at 2-8°C.

[0186] MES buffer 50mM

[0187] Bovine serum albumin (stabilizer) 10g / L 2-Phenoxyethanol (preservative) 1g / L

[0188] The κ-FLC / λ-FLC values ​​were 0, 0.25, 1, 5, and 15, respectively. The specific concentrations of the κ-FLC antigen and the λ-FLC antigen are shown in Table 2 below.

[0189] Table 2

[0190] κ-FLC / λ-FLC value κ-FLC(mg / L) λ-FLC(mg / L) 0 0 10 0.25 2.5 10 1 10 10 5 50 10 15 150 10

[0191] Reagent 1 is a milky white solution, reagent 2 is a colorless or light yellow solution, and the calibrator is a colorless and transparent solution.

[0192] Example 8: A single-channel kit for detecting the ratio of κ-FLC to λ-FLC

[0193] Preparation of Reagent 1

[0194] Take the free light chain polyclonal antibody-coated latex microspheres obtained in Example 1 and prepare reagent 1 according to the following dosage:

[0195]

[0196] Reagent 2 preparation

[0197] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 7.50±0.05, make up to volume, and store at 2-8°C.

[0198]

[0199]

[0200] Calibrator preparation

[0201] Take 90% of the prepared volume of pure water, accurately weigh the materials as shown below, stir evenly, adjust the pH to 9.0±0.05, add different proportions of pure antigens, and store at 2-8°C.

[0202] MOPS buffer 20mM

[0203] Bovine serum albumin (stabilizer) 5g / L

[0204] Proclin 300 (preservative) 30g / L

[0205] The κ-FLC / λ-FLC values ​​were 0, 0.25, 1, 5, and 15, respectively. The specific concentrations of the κ-FLC antigen and the λ-FLC antigen are shown in Table 3 below.

[0206] Table 3

[0207] κ-FLC / λ-FLC value κ-FLC(mg / L) λ-FLC(mg / L) 0 0 10 0.25 2.5 10 1 10 10 5 50 10 15 150 10

[0208] The κ-FLC / λ-FLC values ​​are 0, 0.25, 1, 5, and 15, respectively. Reagent 1 is a milky white solution, reagent 2 is a colorless or light yellow solution, and the calibrator is a colorless and transparent solution.

[0209] Comparative Example 1

[0210] Similar to Example 4, the difference is that the surfactant used in Reagent 2 does not contain dodecyl glycoside. The specific preparation method of Reagent 2 is as follows:

[0211] Take 90% of the prepared volume of pure water, accurately weigh the materials in the order in the table below, stir evenly, adjust the pH to 7.50±0.05, make up to volume, and store at 2-8°C.

[0212]

[0213]

[0214] Performance Testing

[0215] The samples were tested using a Hitachi 7180 fully automatic biochemical analyzer at a wavelength of 570nm and a temperature of 37°C. The analysis method used was the two-point endpoint method, the reaction direction was the rising method, and the calibration method was the spline calibration method. The calibration method was five-point calibration. The detection method was to add 3ul of the calibration reagent or sample to 150ul of reagent 1, mix well, incubate at 37°C for 5min, and measure the absorbance A1. The k / λ values ​​in the calibration reagent were 0, 0.25, 1, 5, and 15, respectively. 50ul of reagent 2 was added, incubated at 37°C for 5min, and the absorbance A2 was measured to calculate △A.

[0216] 1 Sample Correlation

[0217] 40 clinical samples (including negative samples and positive samples) were selected, and the commercialized two-step latex immunoturbidimetric kit of Jiangxi Yingda Biotechnology Co., Ltd. was used as a comparison reagent. The parameters of the comparison reagent and the embodiment were set respectively, and the measurement was performed at the same time. The results are shown in Table 4 below. The linear regression equation of Example 4 (containing dodecyl glycoside and the reaction promoter is PEG8000) is: y=1.0018x+0.0046, R 2 =0.9997 (e.g. Figure 2 The linear regression equation of comparative example 1 (without dodecyl glycoside) is y=0.7901x-0.2383, R 2 =0.8405 (e.g. Figure 3 The correlation is poor, which indicates that the dodecyl glycoside in reagent 2 plays an important role in sample accuracy. The linear regression equation of Example 5 (the reaction promoter is PEG2000) is y=1.066x-0.1041, R 2 is 0.9908 (e.g. Figure 4 The linear regression equation of Example 7 (surfactant in reagent 2 is dodecyl glycoside) is: y=1.0857x-0.1228, R 2 =0.9886 (e.g. Figure 5 The linear regression equation of Example 8 (the surfactant in reagent 2 is dodecyl glucoside and mannitol in a mass ratio of 1:1) is: y=1.0439x-0.0792, R 2 =0.9912 (e.g. Figure 6 The correlation is slightly worse than that of Example 4, which indicates that the ratio of mannitol to dodecyl glucoside in reagent 2 plays an important role in sample accuracy.

[0218] Table 4 Correlation results between the reagents of the present invention and the comparison reagent samples

[0219]

[0220]

[0221] 2 Repeatability

[0222] Two samples with different κ / λ-FLC values ​​were tested ten times and their coefficients of variation were calculated. The results are shown in Table 5. As shown in Table 5, the coefficients of variation of Example 4, Example 5 and Example 6 are all less than 10%, which indicates that the reagent of the present invention has good repeatability.

[0223] Table 5 Example repeatability

[0224]

[0225] 3 Linear

[0226] The linear high value sample was diluted with normal saline and at least 7 dilution concentrations (xi) were mixed. Each dilution concentration was tested 3 times using the kit of the embodiment, and the mean (yi) of the test results of each dilution concentration was obtained respectively. The linear regression equation was obtained with xi as the independent variable and yi as the dependent variable. Figure 7 The linear regression equation obtained by using the kit of Example 4 is shown. Figure 8 Shown is the linear regression equation obtained using the kit of Example 6.

[0227] The results are shown in Tables 6 and 7. The linear sample deviations are all less than 10%, and the linear sample deviations of Example 4 (microsphere particle size 100nm) of the present invention are all less than 10%. The linear sample relative deviation of Example 6 (microsphere particle size 120nm) is close to 10%, which is slightly worse than Example 4. Therefore, the preferred latex microsphere particle size is 100nm.

[0228] Table 6 Example 4 Linear

[0229]

[0230] Table 7 Example 6 Linear

[0231]

[0232] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A single-channel kit for detecting the ratio of κ-FLC to λ-FLC, characterized in that: The kit includes reagent 1, reagent 2, and a calibrator reagent; wherein, Reagent 1 includes latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies and a first buffer; Reagent 2 comprises FLC monoclonal antibody, a second buffer and 0.1 wt% to 1 wt% of a second surfactant; The calibrator reagent includes a third buffer and calibrators having different ratios of κ-FLC antigen to λ-FLC antigen.

2. The kit according to claim 1, characterized in that In reagent 1, the λ-FLC and κ-FLC polyclonal antibodies are mouse anti-human λ-FLC and κ-FLC polyclonal antibodies; and / or, the concentration of latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 0.01 wt% to 1 wt%, preferably 0.05 wt% to 0.1 wt%; And / or, the particle size of the latex microspheres coated with λ-FLC and κ-FLC polyclonal antibodies in reagent 1 is 80 nm to 120 nm, preferably 100 nm.

3. The kit according to claim 1 or 2, characterized in that The pH value of the first buffer in reagent 1, the second buffer in reagent 2, or the third buffer in the calibrator reagent is independently 6 to 9; and / or, the concentration of the first buffer in reagent 1, the second buffer in reagent 2, or the third buffer in the calibrator reagent is independently 10 mmol / L to 100 mmol / L, preferably 20 mmol / L to 50 mmol / L; And / or, the first buffer in reagent 1 or the second buffer in reagent 2 or the third buffer in the calibrator reagent is independently one or a combination of two or more of MES buffer, borate buffer, MOPS buffer, phosphate buffer or glycine buffer.

4. The kit according to any one of claims 1 to 3, characterized in that Reagent 1 further comprises 0.1 wt% to 5 wt% of a first stabilizer, and / or, 0.1 wt% to 1 wt% of a first surfactant, and / or, 0.1 wt% to 10 wt% of a first preservative; Preferably, the first stabilizer in reagent 1 is selected from one or a combination of two or more of bovine serum albumin, mannitol, magnesium chloride, and sodium bisulfate, preferably bovine serum albumin; and / or, the concentration of the first stabilizer in reagent 1 is 0.5wt% to 3wt%; Preferably, the first surfactant in reagent 1 is selected from one or a combination of two or more of Tween, TX-100, Briji-35 and dodecyl glycoside; and / or, the concentration of the first surfactant in reagent 1 is 0.1wt% to 0.5wt%, preferably 0.3wt%; Preferably, the first preservative in reagent 1 is selected from sodium azide 、 Phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, Proclin300 or a combination of two or more thereof, preferably 2-phenoxyethanol; and / or, the first preservative in reagent 1 is 0.1 wt% to 1 wt%.

5. The kit according to any one of claims 1 to 4, characterized in that The second surfactant in reagent 2 is dodecyl glucoside, or a combination of dodecyl glucoside and one or more of the following substances: Tween, TX-100, Briji-35, mannitol; and / or, the concentration of the second surfactant in reagent 2 is 0.1 wt% to 0.5 wt%, preferably 0.3 wt%; Preferably, the mass ratio of dodecyl glucoside to mannitol in the second surfactant in reagent 2 is (1-3):

1.

6. The kit according to any one of claims 1 to 5, characterized in that Reagent 2 further contains 0.05 wt% to 10 wt% of an electrolyte, and / or 0.01 wt% to 1 wt% of a reaction accelerator, and / or 0.1 wt% to 5 wt% of a second preservative; Preferably, the electrolyte in reagent 2 is selected from one or a combination of two or more of sodium chloride, potassium chloride, magnesium chloride, and magnesium sulfate; and / or, the mass percentage of the electrolyte in reagent 2 is 0.05wt% to 1wt%; Preferably, the reaction accelerator in reagent 2 is selected from one or a combination of two or more of polyethylene glycol 2000, polyethylene glycol 4000, and polyethylene glycol 8000. Preferably, the reaction accelerator is polyethylene glycol 8000; and / or, the mass percentage of the reaction accelerator in reagent 2 is 0.01wt% to 0.5wt%; Preferably, the second preservative in reagent 2 is selected from one or a combination of two or more of sodium azide, phenol, p-hydroxybenzoic acid, 2-phenoxyethanol, and Proclin 300, preferably 2-phenoxyethanol; and / or, the mass percentage of the second preservative in reagent 2 is 0.1wt% to 3wt%.

7. The kit according to any one of claims 1 to 6, characterized in that The FLC monoclonal antibody in reagent 2 is a λ-FLC monoclonal antibody or a κ-FLC monoclonal antibody; and / or, the FLC monoclonal antibody in reagent 2 is a mouse anti-human FLC monoclonal antibody; And / or, the mass percentage of the FLC monoclonal antibody in reagent 2 is 0.0005wt% to 0.005wt%, preferably 0.0007wt%.

8. The kit according to any one of claims 1 to 7, characterized in that The calibrator reagent also contains a third stabilizer and / or a third preservative; Preferably, the third stabilizer is selected from one or a combination of two or more of bovine serum albumin, mannitol, magnesium chloride and sodium bisulfate, preferably bovine serum albumin; and / or, the mass percentage of the third stabilizer in the calibrator is 0.01wt% to 1wt%, preferably 0.01wt% to 0.5wt%; Preferably, the third preservative is selected from one or a combination of two or more of sodium azide, phenol, p-hydroxybenzoic acid, 2-phenoxyethanol and Proclin 300, preferably 2-phenoxyethanol; and / or, the mass percentage of the third preservative in the calibrator is 0.1wt% to 3wt%.

9. The kit according to any one of claims 1 to 8, characterized in that The concentration of λ-FLC in the calibration reagent is 8.0mg / L~200mg / L, and the concentration of κ-FLC is 4.5mg / L~160mg / L. And / or, the ratio of κ-FLC to λ-FLC in the calibrator reagent is 0, 0.1-0.3, 0.5-1.5, 3-7 and 10-20.

10. The kit according to any one of claims 1 to 9, characterized in that The preparation method of the latex microspheres and polyclonal antibody coupled particles in reagent 1 comprises the following steps: after mixing the latex microspheres with the fourth buffer, adding an activator to activate the latex microspheres; adding free light chain polyclonal antibodies to couple the activated latex microspheres with the free light chain polyclonal antibodies; and then blocking with BSA to obtain the latex microspheres and polyclonal antibody coupled particles; Preferably, the fourth buffer is one or a combination of two or more of MES buffer, borate buffer, MOPS buffer, phosphate buffer or glycine buffer; Preferably, the pH value of the fourth buffer is 6 to 9; Preferably, the concentration of the fourth buffer is 10 mmol / L to 100 mmol / L; Preferably, the mass ratio of the latex microspheres to the activator is 20:(1-3); Preferably, the activator is one or both of EDC and NHS; Preferably, the activators are EDC and NHS; Preferably, in the activator, the mass ratio of EDC to NHS is 1:(1-3), preferably 1:2; Preferably, the mass ratio of the free light chain polyclonal antibody to the activated latex microspheres is 50 μg / mg to 200 μg / mg, preferably 100 μg / mg; Preferably, the final concentration of BSA during the blocking process is 0.5-2 wt%, preferably 1 wt%.

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