CN105548573A - Kit and method for detecting content of KAPPA light chain and application - Google Patents
Kit and method for detecting content of KAPPA light chain and application Download PDFInfo
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- CN105548573A CN105548573A CN201610071361.4A CN201610071361A CN105548573A CN 105548573 A CN105548573 A CN 105548573A CN 201610071361 A CN201610071361 A CN 201610071361A CN 105548573 A CN105548573 A CN 105548573A
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Abstract
The invention discloses a kit for detecting the content of a KAPPA light chain. The kit comprises a reagent R1, a reagent R2 and a KAPPA light chain calibrator, wherein the reagent R1 comprises a first buffer solution, a first electrolyte, a surfactant, a first stabilizer, a high-molecular accelerator and a first preservative; the reagent R2 comprises a second buffer solution, an anti-human KAPPA light chain antibody, a second electrolyte and a second preservative; the KAPPA light chain calibrator comprises a third buffer solution, a third stabilizer, a third preservative, an antioxidant and a KAPPA light chain antigen. The invention further discloses an application of the kit for detecting the content of the KAPPA light chain to the detection of the content of the KAPPA light chain and a method for detecting the content of the KAPPA light chain by adopting the kit for detecting the content of the KAPPA light chain. By adopting the kit and the detection method disclosed by the invention, the content of the KAPPA light chain can be detected easily and rapidly.
Description
Technical field
The invention belongs to the technical field detecting KAPPA light chain content, be specifically related to a kind of kit, method and the purposes that detect KAPPA light chain content.
Background technology
Light chain (Lightchain, L) is approximately made up of 214 amino acid residues, usually not carbohydrate containing, and molecular weight is about 24kD.Every bar light chain contains two cyclic peptide be made up of intrachain disulfide bond.L chain has two types: KAPPA (κ) and Lambda (λ), on same native immunoglobulin molecule, the type of L chain is always identical.In normal human serum κ: λ is about 2:1.
The paraplasm of cell clone will cause the increase of monoclonal globulin or intracellular immunoglobulin fragment (free light chain), and this will cause the proportional imbalance of κ and λ chain, and the ratio of κ and λ is not normal predictive of monoclonal Y globulinemia.When generating increase, the filtration of free light chain also increases, and when exceeding the heavy receptivity of renal tubule, this test quantitatively can detect the light chain in combination and intracellular immunoglobulin.
Quantitative detection different types of light chain content contributes to the diseases such as diagnosis macroglobulinemia (huge globulin generates and increases) and connective tissue disease (CTD) (as rheumatoid arthritis, systemic loupus erythematosus).
In addition, in the blood such as infection, acute, chronic hepatitis, cirrhosis, light chain also can increase, but generally all shows as κ, λ and increase simultaneously; The Urinary such as ephrosis, diabetes also can occur that κ, λ increase simultaneously.
There is many deficiencies in existing KAPPA light chain detection technique: as needed special equipment, sample needs pre-service, can not go up automatic clinical chemistry analyzer and carry out batch detection analysis etc.Euzymelinked immunosorbent assay (ELISA) Application comparison is general, and its principle is connected with solid phase carrier by specific antibody, forms insolubilized antibody; The washing unconjugated antibody of removing and impurity; Add testing sample, make it to react a period of time with insolubilized antibody, allow the antibody of antigen on solid phase carrier in sample be combined; The unconjugated material of washing removing; Add enzyme labelled antibody, the antigen on solid-phase immunity compound is combined with enzyme labelled antibody; The unconjugated enzyme labelled antibody of washing removing; The now amount positive correlation of the enzyme amount on solid phase carrier and the test substance in sample; Add substrate reactions colour developing.Degree according to color reaction qualitatively or quantitatively determines mensuration material.The method is heterogeneous immune detection system, measures process comparatively loaded down with trivial details, length consuming time, lacks unit testing working time; Automaticity is not high, difference between batch and repeatability relatively large; Need to be equipped with multiple Special Equipment, adds somewhat to cost.
Along with the continuous increase of Sample quantity, carry out scale batch operation in the urgent need to the more simple, fast method of one clinically.Immunoturbidimetry detects KAPPA light chain concentration directly can complete batch sample analysis on automatic clinical chemistry analyzer, simple to operate, can realize detection that is quick, high flux sample.
Summary of the invention
The object of the embodiment of the present invention is the above-mentioned deficiency overcoming prior art, provides a kind of kit detecting KAPPA light chain content, succinctly can detect the content of KAPPA light chain in sample rapidly, have higher accuracy and good specificity.
Another object of the embodiment of the present invention is the above-mentioned deficiency overcoming prior art, a kind of kit of above-mentioned detection KAPPA light chain content is provided to detect the purposes in KAPPA light chain content, can be used for the succinct content detecting KAPPA light chain in sample rapidly, there is higher accuracy and good specificity.
An object again of the embodiment of the present invention is the above-mentioned deficiency overcoming prior art, a kind of method adopting the kit of above-mentioned detection KAPPA light chain content to detect KAPPA light chain content is provided, succinctly can detect the content of KAPPA light chain in sample rapidly, there is higher accuracy and good specificity.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
Detect a kit for KAPPA light chain content, comprising: reagent R1, reagent R2 and KAPPA light chain calibration object; Described reagent R1 comprises: first damping fluid 5 ~ 600mmol/L, first electrolyte 5 ~ 60g/L, surfactant 1 ~ 200g/L, first stabilizing agent 2 ~ 50g/L, macromolecule accelerator 1 ~ 100g/L and first antiseptic 0.5 ~ 15g/L; Described reagent R2 comprises: second damping fluid 5 ~ 600mmol/L, anti-human KAPPA light chain antibody 0.1 ~ 20%w/v, second electrolyte 5 ~ 60g/L and second antiseptic 0.5 ~ 15g/L; Described KAPPA light chain calibration object comprises: the 3rd damping fluid 10 ~ 600mmol/L, the 3rd stabilizing agent 2 ~ 50g/L, the 3rd antiseptic 0.5 ~ 15g/L, antioxidant 0.01 ~ 10g/L and KAPPA Light Chain Antigen.
Further: described first damping fluid, described second damping fluid and described 3rd damping fluid are all selected from one or more in phosphate PBS damping fluid, trishydroxymethylaminomethane TRIS damping fluid, borate buffer, glycine buffer, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid CAPSO damping fluid, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES damping fluid; And/or described first antiseptic, described second antiseptic and described 3rd antiseptic are all selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate and ethyl mercury sodium thiosulfate; And/or described first electrolyte and described second electrolyte are all selected from one or more in sodium chloride, magnesium sulfate, magnesium chloride and potassium chloride; And/or described first stabilizing agent and described 3rd stabilizing agent are all selected from one or more in disodium ethylene diamine tetraacetate, mannitol, glucose, trehalose and bovine serum albumin; And/or described surfactant is selected from one or more in Triton series of surfactants, Tween series of surfactants, bay ether series of surfactants, polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl ether and polyoxethylene octylphenyl phenylate; And/or described macromolecule accelerator is selected from one or more in Macrogol 2000, Macrogol 4000, Macrogol 6000 and PEG 8000; And/or described anti-human KAPPA light chain antibody is selected from that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human and one or more in the anti-human KAPPA light chain antibody of duck; Described antioxidant is selected from one or more in butylated hydroxy anisole, dibutyl hydroxy toluene, benzene polyphenol, Licorice root antioxidant and dilauryl thiodipropionate.
Further: described anti-human KAPPA light chain antibody is monoclonal antibody and/or polyclonal antibody.
Further: described first damping fluid, described second damping fluid and described 3rd damping fluid are phosphate buffer; And/or described first antiseptic, described second antiseptic and described 3rd antiseptic are Sodium azide; And/or described first electrolyte and described second electrolyte are sodium chloride; And/or described first stabilizing agent and described 3rd stabilizing agent are disodium ethylene diamine tetraacetate; And/or described surfactant is polyoxyethylene laurel ether; And/or described macromolecule accelerator is Macrogol 6000; And/or described anti-human KAPPA light chain antibody is goat-anti people KAPPA light chain monoclonal antibody; And/or described antioxidant is butylated hydroxy anisole.
Further, described reagent R1 comprises: first damping fluid 30 ~ 400mmol/L, first electrolyte 6 ~ 45g/L, surfactant 3 ~ 70g/L, first stabilizing agent 3 ~ 35g/L, macromolecule accelerator 3 ~ 70g/L and first antiseptic 1.5 ~ 9g/L; Described reagent R2 comprises: second damping fluid 20 ~ 450mmol/L, anti-human KAPPA light chain antibody 0.1 ~ 20%w/v, second electrolyte 6 ~ 50g/L and second antiseptic 1.5 ~ 9g/L; Described KAPPA light chain calibration object comprises: the 3rd damping fluid 15 ~ 100mmol/L, the 3rd stabilizing agent 3 ~ 50g/L, the 3rd antiseptic 4 ~ 9g/L, antioxidant 0.4 ~ 10g/L and KAPPA Light Chain Antigen.
Further, described reagent R1 comprises: first damping fluid 10 ~ 100mmol/L, first electrolyte 5 ~ 15g/L, surfactant 1 ~ 50g/L, first stabilizing agent 5 ~ 15g/L, macromolecule accelerator 50 ~ 100g/L and first antiseptic 1 ~ 5g/L; And/or described reagent R2 comprises: second damping fluid 10 ~ 100mmol/L, anti-human KAPPA light chain antibody 0.5 ~ 10%w/v, second electrolyte 5 ~ 15g/L and second antiseptic 1 ~ 5g/L; And/or described KAPPA light chain calibration object comprises: the 3rd damping fluid 10 ~ 100mmol/L, the 3rd stabilizing agent 5 ~ 15g/L, the 3rd antiseptic 1 ~ 5g/L, antioxidant 0.01 ~ 0.8g/L and KAPPA Light Chain Antigen.
Further, described KAPPA light chain calibration object comprises: the 3rd damping fluid 5 ~ 600mmol/L, the 3rd stabilizing agent 2 ~ 50g/L, the 3rd antiseptic 0.5 ~ 15g/L, antioxidant 0.01 ~ 10g/L and KAPPA Light Chain Antigen 0 ~ 1600mg/L.
Further: in described KAPPA light chain calibration object, the concentration of described KAPPA Light Chain Antigen is 0 ~ 1200mg/L.
A kind of kit of detection KAPPA light chain content as above is detecting the purposes in KAPPA light chain content.
And, a kind of method adopting the kit of the KAPPA of detection light chain content as above to detect KAPPA light chain content, comprise: in testing sample, add described reagent R1, the volume of described testing sample is 5 μ L, the volume of described reagent R1 is 250 μ L, obtain the first mixed liquor, and obtain the absorbance OD1 of described first mixed liquor; In described first mixed liquor, add described reagent R2, the volume of described reagent R2 is 50 μ L, obtains the second mixed liquor, and obtains the absorbance OD2 of described second mixed liquor; Use described KAPPA light chain calibration object to obtain typical curve, on described typical curve, obtain the content of KAPPA light chain in described testing sample according to described absorbance OD1 and described absorbance OD2.
The beneficial effect of the embodiment of the present invention is as follows:
1, the kit of the detection KAPPA light chain content of the embodiment of the present invention, simple to operate, fast easy to use, succinctly can detect the content of KAPPA light chain in sample rapidly.
2, the kit of the detection KAPPA light chain content of the embodiment of the present invention, can meet the requirement that clinical fast high-flux detects sample, detection efficiency significantly improves, and clinical practice has broad prospects.
3, the kit of the detection KAPPA light chain content of the embodiment of the present invention, also has high specificity, highly sensitive, reproducible, differences between batches are little, the advantage such as stable reagent.
3, the kit of the detection KAPPA light chain content of the embodiment of the present invention makes it have the purposes detecting KAPPA light chain content.
4, the kit of the detection KAPPA light chain content of the embodiment of the present invention detects the method for KAPPA light chain content, and succinctly detect the content of KAPPA light chain in sample rapidly, detection efficiency is higher, and clinical practice has broad prospects.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of the method for detection KAPPA light chain content of the present invention;
Fig. 2 is the schematic flow sheet of the detection method of the KAPPA light chain content of embodiments of the invention 3;
Fig. 3 is the typical curve of the detection KAPPA light chain content of embodiments of the invention 3;
Fig. 4 is the detection KAPPA light chain content kit of embodiments of the invention 4 and the comparative result schematic diagram of Correlation to That Abroad kit.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The invention provides a kind of kit detecting KAPPA light chain content.This kit comprises: reagent R1, reagent R2 and KAPPA light chain calibration object.
Wherein, reagent R1 comprises: first damping fluid 5 ~ 600mmol/L, first electrolyte 5 ~ 60g/L, surfactant 1 ~ 200g/L, first stabilizing agent 2 ~ 50g/L, macromolecule accelerator 1 ~ 100g/L and first antiseptic 0.5 ~ 15g/L.In reagent R1, all the other are purified water.
Reagent R2 comprises: second damping fluid 5 ~ 600mmol/L, anti-human KAPPA light chain antibody 0.1 ~ 20%w/v, second electrolyte 5 ~ 60g/L and second antiseptic 0.5 ~ 15g/L.In reagent R2, all the other are purified water.In the present invention, what w/v represented is meant to Solute mass contained in the solution of unit volume.
KAPPA light chain calibration object comprises: the 3rd damping fluid 10 ~ 600mmol/L, the 3rd stabilizing agent 2 ~ 50g/L, the 3rd antiseptic 0.5 ~ 15g/L, antioxidant 0.01 ~ 10g/L and KAPPA Light Chain Antigen.In KAPPA light chain calibration object, all the other are purified water.
Preferably, reagent R1 comprises: first damping fluid 30 ~ 400mmol/L, first electrolyte 6 ~ 45g/L, surfactant 3 ~ 70g/L, first stabilizing agent 3 ~ 35g/L, macromolecule accelerator 3 ~ 70g/L and first antiseptic 1.5 ~ 9g/L.Reagent R2 comprises: second damping fluid 20 ~ 450mmol/L, anti-human KAPPA light chain antibody 0.1 ~ 20%w/v, second electrolyte 6 ~ 50g/L and second antiseptic 1.5 ~ 9g/L.KAPPA light chain calibration object comprises: the 3rd damping fluid 15 ~ 100mmol/L, the 3rd stabilizing agent 3 ~ 50g/L, the 3rd antiseptic 4 ~ 9g/L, antioxidant 0.4 ~ 10g/L and KAPPA Light Chain Antigen.
Preferably, reagent R1 comprises: first damping fluid 10 ~ 100mmol/L, first electrolyte 5 ~ 15g/L, surfactant 1 ~ 50g/L, first stabilizing agent 5 ~ 15g/L, macromolecule accelerator 50 ~ 100g/L and first antiseptic 1 ~ 5g/L.Reagent R2 comprises: second damping fluid 10 ~ 100mmol/L, anti-human KAPPA light chain antibody 0.5 ~ 10%w/v, second electrolyte 5 ~ 15g/L and second antiseptic 1 ~ 5g/L.KAPPA light chain calibration object comprises: the 3rd damping fluid 10 ~ 100mmol/L, the 3rd stabilizing agent 5 ~ 15g/L, the 3rd antiseptic 1 ~ 5g/L, antioxidant 0.01 ~ 0.8g/L and KAPPA Light Chain Antigen.
Preferably, KAPPA light chain calibration object comprises: the 3rd damping fluid 5 ~ 600mmol/L, the 3rd stabilizing agent 2 ~ 50g/L, the 3rd antiseptic 0.5 ~ 15g/L, antioxidant 0.01 ~ 10g/L and KAPPA Light Chain Antigen 0 ~ 1600mg/L.
Preferably, in KAPPA light chain calibration object, the concentration of KAPPA Light Chain Antigen is 0 ~ 1200mg/L.
When applying this kit, this KAPPA light chain calibration object can be mixed with high concentration single-point calibration product, becomes the reference calibrations product of multiple variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of multiple variable concentrations.
Preferably, in reagent R1, the first damping fluid is selected from one or more in phosphate PBS damping fluid, trishydroxymethylaminomethane TRIS damping fluid, borate buffer, glycine buffer, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid CAPSO damping fluid, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES damping fluid.First electrolyte is selected from one or more in sodium chloride, magnesium sulfate, magnesium chloride and potassium chloride.Surfactant is selected from one or more in Triton series of surfactants, Tween series of surfactants, bay ether series of surfactants (such as polyoxyethylene laurel ether), polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl ether and polyoxethylene octylphenyl phenylate.First stabilizing agent is selected from one or more in disodium ethylene diamine tetraacetate, mannitol, glucose, trehalose and bovine serum albumin.Macromolecule accelerator is selected from one or more in Macrogol 2000, Macrogol 4000, Macrogol 6000 and PEG 8000.First antiseptic is selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate and ethyl mercury sodium thiosulfate.
Preferably, in reagent R2, the second damping fluid is selected from one or more in phosphate PBS damping fluid, trishydroxymethylaminomethane TRIS damping fluid, borate buffer, glycine buffer, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid CAPSO damping fluid, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES damping fluid.Anti-human KAPPA light chain antibody selects that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human and one or more in the anti-human KAPPA light chain antibody of duck.Preferred, anti-human KAPPA light chain antibody is monoclonal antibody and/or polyclonal antibody.Second electrolyte is selected from one or more in sodium chloride, magnesium sulfate, magnesium chloride and potassium chloride.Second antiseptic is selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate and ethyl mercury sodium thiosulfate.
Preferably, in KAPPA light chain calibration object, the 3rd damping fluid is selected from one or more in phosphate PBS damping fluid, trishydroxymethylaminomethane TRIS damping fluid, borate buffer, glycine buffer, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid CAPSO damping fluid, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES damping fluid.3rd stabilizing agent is selected from one or more in disodium ethylene diamine tetraacetate, mannitol, glucose, trehalose and bovine serum albumin.3rd antiseptic is selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate and ethyl mercury sodium thiosulfate.Antioxidant is selected from one or more in butylated hydroxy anisole, dibutyl hydroxy toluene, benzene polyphenol, Licorice root antioxidant and dilauryl thiodipropionate.
Preferred, in reagent R1, the first damping fluid is phosphate buffer.First electrolyte is sodium chloride.Surfactant is polyoxethylene octylphenyl phenylate.First stabilizing agent is disodium ethylene diamine tetraacetate.Macromolecule accelerator is Macrogol 6000.First antiseptic is Sodium azide.
Preferred, in reagent R2, the second damping fluid is phosphate buffer.Anti-human KAPPA light chain antibody is goat-anti people KAPPA light chain monoclonal antibody.Second electrolyte is sodium chloride.Second antiseptic is Sodium azide.
Preferred, in KAPPA light chain calibration object, the 3rd damping fluid is phosphate buffer.3rd stabilizing agent is disodium ethylene diamine tetraacetate.3rd antiseptic is Sodium azide.Antioxidant is butylated hydroxy anisole.
The kit of the detection KAPPA light chain content of the embodiment of the present invention, simple to operate, fast easy to use, succinctly can detect the content of KAPPA light chain in sample rapidly; Can meet the requirement that clinical fast high-flux detects sample, detection efficiency significantly improves and has broad prospects in clinical practice; Also there is high specificity, highly sensitive, reproducible, differences between batches are little, the advantage such as stable reagent.
The kit that the embodiment of the present invention additionally provides a kind of above-mentioned detection KAPPA light chain content is detecting the purposes in KAPPA light chain content, may be used for the succinct content detecting KAPPA light chain in sample rapidly, has higher accuracy and good specificity.This kit is particularly suitable for the content detecting KAPPA light chain in urine specimen.
The embodiment of the present invention additionally provides a kind of method adopting the kit of above-mentioned detection KAPPA light chain content to detect KAPPA light chain content.The inventive method is immunoturbidimetry, and it is that a kind of antigen-antibody is in conjunction with dynamic measurement method.Its ultimate principle is: corresponding antigen-antibody reaction occurs the anti-human KAPPA light chain antibody in the KAPPA Light Chain Antigen in sample and reagent R2, forms insoluble antigen-antibody complex, makes reactant liquor produce certain turbidity.In reactant liquor during anti-human KAPPA antibody excess, the amount of the immune complex of formation increases, the turbidity also corresponding increase of reactant liquor along with the increase of KAPPA Light Chain Antigen amount in sample.Measure the light absorption value of this compound at a particular wavelength, the calibration curve demarcated with calibration object compares, and can calculate the content of the KAPPA light chain in sample.As shown in Figure 1, be the process flow diagram of the method for detection KAPPA light chain content of the present invention.The method comprises following step:
Step S10: add reagent R1 in testing sample, the volume of testing sample is the volume of 5 μ L, reagent R1 is 250 μ L, obtains the first mixed liquor, and obtains the absorbance OD1 of the first mixed liquor.
The volume of step S20: add reagent R2 in the first mixed liquor, reagent R2 is 50 μ L, obtains the second mixed liquor, and obtains the absorbance OD2 of the second mixed liquor.
Step S30: use KAPPA light chain calibration object to obtain typical curve, obtain the content of KAPPA light chain in testing sample according to absorbance OD1 and absorbance OD2 on typical curve.
Wherein, the fitting mode of typical curve comprises: Spline, quadratic equation, logit-log3p, logit-log4p, logit-log5p or Polygonal.
With specific embodiment, technical scheme of the present invention is described further below.
Embodiment 1
The kit of this detection KAPPA light chain content comprises: reagent R1, reagent R2 and KAPPA light chain calibration object.
Wherein, reagent R1 comprises following composition:
Reagent R2 comprises following composition:
KAPPA light chain calibration object comprises following composition:
The KAPPA Light Chain Antigen of respective amount adds in the mixed liquor of other reagent above-mentioned by calibration object concentration as required, is mixed to get KAPPA light chain calibration object.This KAPPA light chain calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.The present embodiment prepares the KAPPA light chain calibration object of 5 variable concentrations, and the concentration of its KAPPA Light Chain Antigen is respectively: 0mg/L, 200mg/L, 400mg/L, 800mg/L and 1200mg/L.By degerming with the membrane filtration of 0.22 μm for KAPPA light chain calibration object, preserve at 2 ~ 8 DEG C.
Embodiment 2
The kit of this detection KAPPA light chain content comprises: reagent R1, reagent R2 and KAPPA light chain calibration object.
Wherein, reagent R1 comprises following composition:
Reagent R2 comprises following composition:
KAPPA light chain calibration object comprises following composition:
The KAPPA Light Chain Antigen of respective amount adds in the mixed liquor of other reagent above-mentioned by calibration object concentration as required, is mixed to get KAPPA light chain calibration object.This KAPPA light chain calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.What the present embodiment was selected is high concentration single-point reference calibrations product, and KAPPA Light Chain Antigen concentration is 1200mg/L, by degerming with the membrane filtration of 0.22 μm for KAPPA light chain calibration object, preserves at 2 ~ 8 DEG C.Become the KAPPA light chain calibration object of 5 variable concentrations with normal saline dilution during use.The concentration of KAPPA Light Chain Antigen is respectively: 0mg/L, 200mg/L, 400mg/L, 800mg/L and 1200mg/L.
Embodiment 3
1. kit preparation
The kit of this detection KAPPA light chain content comprises: reagent R1, reagent R2 and KAPPA light chain calibration object.
Wherein, reagent R1 comprises following composition:
Reagent R2 comprises following composition:
KAPPA light chain calibration object comprises following composition:
The KAPPA Light Chain Antigen of respective amount adds in the mixed liquor of other reagent above-mentioned by calibration object concentration as required, is mixed to get KAPPA light chain calibration object.This KAPPA light chain calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.The present embodiment prepares the KAPPA light chain calibration object of 5 variable concentrations, and the concentration of its KAPPA Light Chain Antigen is respectively: 0mg/L, 200mg/L, 400mg/L, 800mg/L and 1200mg/L.By degerming with the membrane filtration of 0.22 μm for KAPPA light chain calibration object, preserve at 2 ~ 8 DEG C.
2. detection method
Adopt the kit of above-described embodiment 3, carry out detection by automatic clinical chemistry analyzer to testing sample and analyze, analytical approach is Two point end assay.As shown in Figure 2, be the schematic flow sheet of the detection method of the KAPPA light chain content of embodiments of the invention 3.In testing sample, add reagent R1, the volume of testing sample is the volume of 5 μ L, reagent R1 is 250 μ L, is mixed to get the first mixed liquor, and 37 DEG C of insulations 5 minutes, then reads the absorbance OD1 of this first mixed liquor.In the first mixed liquor, add reagent R2 further again, reagent R2 volume is 50 μ L, obtains the second mixed liquor, and 37 DEG C of insulations 5 minutes, reads the absorbance OD2 of the second mixed liquor.According to the absorbance OD1 of the first mixed liquor and the absorbance OD2 of the second mixed liquor.The predominant wavelength detected is 340nm, and commplementary wave length is 700nm.
3. Specification Curve of Increasing
Use KAPPA light chain calibration object on automatic clinical chemistry analyzer, record the typical curve of variable concentrations KAPPA standard items.As shown in Figure 3, be the typical curve of the detection KAPPA light chain content of embodiments of the invention 3, X-axis represents KAPPA light chain content, and Y-axis represents absorbance.This typical curve may be used for the content detecting sample KAPPA light chain.
4. variable concentrations sample accuracy detects
Get 3 parts of clinical samples at random, adopt the kit of embodiment 3, by Hitachi's automatic clinical chemistry analyzer to 3 this difference of increment duplicate detection 10 times, testing result is as shown in table 1.
The KAPPA light chain content (10 times) of table 1 three increment basis and the coefficient of variation
Result shows, and the coefficient of variation that three increments originally record is respectively 1.8%, 2.1%, 2.2%, is all less than 3%, shows that kit of the present invention has comparatively high precision.
Embodiment 4
1. kit preparation
The kit of this detection KAPPA light chain content comprises: reagent R1, reagent R2 and KAPPA light chain calibration object.
Wherein, reagent R1 comprises following composition:
Reagent R2 comprises following composition:
KAPPA light chain calibration object comprises following composition:
The KAPPA Light Chain Antigen of respective amount adds in the mixed liquor of other reagent above-mentioned by calibration object concentration as required, is mixed to get KAPPA light chain calibration object.This KAPPA light chain calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.The present embodiment prepares the KAPPA light chain calibration object of 5 variable concentrations, and the concentration of its KAPPA Light Chain Antigen is respectively: 0mg/L, 200mg/L, 400mg/L, 800mg/L and 1200mg/L.By degerming with the membrane filtration of 0.22 μm for KAPPA light chain calibration object, preserve at 2 ~ 8 DEG C.
2. detection method
Adopt the kit of embodiment 4, carry out detection by automatic clinical chemistry analyzer to testing sample and analyze, analytical approach is Two point end assay.In testing sample, add reagent R1, the volume of testing sample is the volume of 5 μ L, reagent R1 is 250 μ L, is mixed to get the first mixed liquor, and 37 DEG C of insulations 5 minutes, then reads the absorbance OD1 of this first mixed liquor.In the first mixed liquor, add reagent R2 further again, reagent R2 volume is 50 μ L, obtains the second mixed liquor, and 37 DEG C of insulations 5 minutes, reads the absorbance OD2 of the second mixed liquor.According to the absorbance OD1 of the first mixed liquor and the absorbance OD2 of the second mixed liquor.The predominant wavelength detected is 340nm, and commplementary wave length is 700nm.
3. different reagent contrast
Adopt the kit drawing standard curve of embodiment 4, its method is identical with the method for embodiment 3 drawing standard curve.Adopt kit and the import contrast agent (ROCHE company reagent) of the embodiment of the present invention 4 respectively, by Hitachi's automatic clinical chemistry analyzer, 50 parts of clinical samples are measured by each autoregressive parameter simultaneously, linear regression is carried out to measured value.The regression equation that linear regression obtains is Y=0.9848X+3.5719, coefficient R
2=0.9975.As shown in Figure 4, be the detection KAPPA light chain content kit of embodiments of the invention 4 and the comparative result schematic diagram of Correlation to That Abroad kit.Fig. 4 illustrates correlativity.Wherein X is reagent of the present invention, and Y is contrast agent.Result shows kit of the present invention and contrast agent correlativity very well, illustrates that kit of the present invention has good specificity and accuracy.Kit of the present invention goes for the full-automatic of the series such as Hitachi, Olympus, Beckman and semi-automatic biochemical analyzer, and analytical approach and parameter suitably can adjust according to different type of machines.
Embodiment 5
1. kit preparation
The kit of this detection KAPPA light chain content comprises: reagent R1, reagent R2 and KAPPA light chain calibration object.
Wherein, reagent R1 comprises following composition:
Reagent R2 comprises following composition:
KAPPA light chain calibration object comprises following composition:
The KAPPA Light Chain Antigen of respective amount adds in the mixed liquor of other reagent above-mentioned by calibration object concentration as required, is mixed to get KAPPA light chain calibration object.This KAPPA light chain calibration object can be high concentration single-point calibration product, becomes the reference calibrations product of 5 variable concentrations in use with normal saline dilution, also directly can be prepared into the reference calibrations product of 5 variable concentrations.What the present embodiment was selected is high concentration single-point reference calibrations product, and KAPPA Light Chain Antigen concentration is 1200mg/L, by degerming with the membrane filtration of 0.22 μm for KAPPA light chain calibration object, preserves at 2 ~ 8 DEG C.Become the KAPPA light chain calibration object of 5 variable concentrations with normal saline dilution during use.The concentration of KAPPA Light Chain Antigen is respectively: 0mg/L, 200mg/L, 400mg/L, 800mg/L and 1200mg/L.
2. detection method
Adopt the kit of embodiment 5, carry out detection by automatic clinical chemistry analyzer to testing sample and analyze, analytical approach is Two point end assay.In testing sample, add reagent R1, the volume of testing sample is the volume of 5 μ L, reagent R1 is 250 μ L, is mixed to get the first mixed liquor, and 37 DEG C of insulations 5 minutes, then reads the absorbance OD1 of this first mixed liquor.In the first mixed liquor, add reagent R2 further again, reagent R2 volume is 50 μ L, obtains the second mixed liquor, and 37 DEG C of insulations 5 minutes, reads the absorbance OD2 of the second mixed liquor.According to the absorbance OD1 of the first mixed liquor and the absorbance OD2 of the second mixed liquor.The predominant wavelength detected is 340nm, and commplementary wave length is 700nm.
3. different embodiment kit testing result contrast
Drawing standard curve.Preparing a KAPPA light chain concentration is the sample of 517mg/L, and use the kit of embodiment 3,4,5 to sample duplicate detection 10 times respectively, testing result is as shown in table 2.
The KAPPA Concentration Testing Comparative result of table 2 embodiment 3 ~ 5 kit
From table 2, the KAPPA light chain content that three kinds of embodiment kits record is all close to actual value, and the coefficient of variation that three kinds of kits record all is less than 5%, kit stable performance of the present invention is described, measures accurately.
4. the range of linearity detects
By KAPPA light chain content normative reference product normal saline dilution, its concentration divides equally 10 concentration from 0 ~ 1200mg/L, adopt the kit of embodiment 5, according to the detection method of embodiment 5, each concentration samples replication 3 times, result is as shown in table 3, and the mean value and actual concentrations that measure concentration are carried out recovery analysis, result display deviation is all less than 10%, represents and linearly can reach 1200mg/L.
The table 3 kit range of linearity of the present invention testing result
5. precision measures
Kit of the present invention (adopting the kit of embodiment 5) withinrun precision and betweenrun precision is measured by the KAPPA light chain urine of 2 different contents, result is as shown in table 4 and table 5, kit withinrun precision of the present invention is 2.3% and 3.8%, and betweenrun precision is 5.4% and 6.6%.
Table 4 kit withinrun precision of the present invention
| Sample concentration | 80mg/L | 200mg/L |
| Measure average | 81.03mg/L | 209.44mg/L |
| Standard deviation | 1.9376 | 5.7176 |
| Withinrun precision | 2.39％ | 2.73％ |
Table 5 kit betweenrun precision of the present invention
| Sample concentration | 80mg/L | 200mg/L |
| Measure average | 81.88mg/L | 208.38mg/L |
| Standard deviation | 3.3951 | 7.0061 |
| Betweenrun precision | 4.15％ | 3.36％ |
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. detect a kit for KAPPA light chain content, it is characterized in that, comprising: reagent R1, reagent R2 and KAPPA light chain calibration object; Described reagent R1 comprises: first damping fluid 5 ~ 600mmol/L, first electrolyte 5 ~ 60g/L, surfactant 1 ~ 200g/L, first stabilizing agent 2 ~ 50g/L, macromolecule accelerator 1 ~ 100g/L and first antiseptic 0.5 ~ 15g/L; Described reagent R2 comprises: second damping fluid 5 ~ 600mmol/L, anti-human KAPPA light chain antibody 0.1 ~ 20%w/v, second electrolyte 5 ~ 60g/L and second antiseptic 0.5 ~ 15g/L; Described KAPPA light chain calibration object comprises: the 3rd damping fluid 10 ~ 600mmol/L, the 3rd stabilizing agent 2 ~ 50g/L, the 3rd antiseptic 0.5 ~ 15g/L, antioxidant 0.01 ~ 10g/L and KAPPA Light Chain Antigen.
2. the kit detecting KAPPA light chain content as claimed in claim 1, is characterized in that:
Described first damping fluid, described second damping fluid and described 3rd damping fluid are all selected from one or more in phosphate PBS damping fluid, trishydroxymethylaminomethane TRIS damping fluid, borate buffer, glycine buffer, 3-(cyclohexylamine)-2-hydroxyl-1-propane sulfonic acid CAPSO damping fluid, 4-hydroxyethyl piperazine ethanesulfonic acid HEPES damping fluid; And/or,
Described first antiseptic, described second antiseptic and described 3rd antiseptic are all selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate and ethyl mercury sodium thiosulfate; And/or,
Described first electrolyte and described second electrolyte are all selected from one or more in sodium chloride, magnesium sulfate, magnesium chloride and potassium chloride; And/or,
Described first stabilizing agent and described 3rd stabilizing agent are all selected from one or more in disodium ethylene diamine tetraacetate, mannitol, glucose, trehalose and bovine serum albumin; And/or,
Described surfactant is selected from one or more in Triton series of surfactants, Tween series of surfactants, bay ether series of surfactants, polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl ether and polyoxethylene octylphenyl phenylate; And/or,
Described macromolecule accelerator is selected from one or more in Macrogol 2000, Macrogol 4000, Macrogol 6000 and PEG 8000; And/or,
Described anti-human KAPPA light chain antibody is selected from that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human and one or several in the anti-human KAPPA light chain antibody of duck;
Described antioxidant is selected from one or more in butylated hydroxy anisole, dibutyl hydroxy toluene, benzene polyphenol, Licorice root antioxidant and dilauryl thiodipropionate.
3. the kit detecting KAPPA light chain content as claimed in claim 1, is characterized in that: described anti-human KAPPA light chain antibody is monoclonal antibody and/or polyclonal antibody.
4. the kit detecting KAPPA light chain content as claimed in claim 1, is characterized in that:
Described first damping fluid, described second damping fluid and described 3rd damping fluid are phosphate buffer; And/or,
Described first antiseptic, described second antiseptic and described 3rd antiseptic are Sodium azide; And/or,
Described first electrolyte and described second electrolyte are sodium chloride; And/or,
Described first stabilizing agent and described 3rd stabilizing agent are disodium ethylene diamine tetraacetate; And/or,
Described surfactant is polyoxyethylene laurel ether; And/or,
Described macromolecule accelerator is Macrogol 6000; And/or,
Described anti-human KAPPA light chain antibody is goat-anti people KAPPA light chain monoclonal antibody; And/or,
Described antioxidant is butylated hydroxy anisole.
5. the kit detecting KAPPA light chain content as claimed in claim 1, is characterized in that,
Described reagent R1 comprises: first damping fluid 30 ~ 400mmol/L, first electrolyte 6 ~ 45g/L, surfactant 3 ~ 70g/L, first stabilizing agent 3 ~ 35g/L, macromolecule accelerator 3 ~ 70g/L and first antiseptic 1.5 ~ 9g/L;
Described reagent R2 comprises: second damping fluid 20 ~ 450mmol/L, anti-human KAPPA light chain antibody 0.1 ~ 20%w/v, second electrolyte 6 ~ 50g/L and second antiseptic 1.5 ~ 9g/L;
Described KAPPA light chain calibration object comprises: the 3rd damping fluid 15 ~ 100mmol/L, the 3rd stabilizing agent 3 ~ 50g/L, the 3rd antiseptic 4 ~ 9g/L, antioxidant 0.4 ~ 10g/L and KAPPA Light Chain Antigen.
6. the kit detecting KAPPA light chain content as claimed in claim 1, is characterized in that,
Described reagent R1 comprises: first damping fluid 10 ~ 100mmol/L, first electrolyte 5 ~ 15g/L, surfactant 1 ~ 50g/L, first stabilizing agent 5 ~ 15g/L, macromolecule accelerator 50 ~ 100g/L and first antiseptic 1 ~ 5g/L; And/or,
Described reagent R2 comprises: second damping fluid 10 ~ 100mmol/L, anti-human KAPPA light chain antibody 0.5 ~ 10%w/v, second electrolyte 5 ~ 15g/L and second antiseptic 1 ~ 5g/L; And/or,
Described KAPPA light chain calibration object comprises: the 3rd damping fluid 10 ~ 100mmol/L, the 3rd stabilizing agent 5 ~ 15g/L, the 3rd antiseptic 1 ~ 5g/L, antioxidant 0.01 ~ 0.8g/L and KAPPA Light Chain Antigen.
7. the kit detecting KAPPA light chain content as claimed in claim 1, it is characterized in that, described KAPPA light chain calibration object comprises: the 3rd damping fluid 5 ~ 600mmol/L, the 3rd stabilizing agent 2 ~ 50g/L, the 3rd antiseptic 0.5 ~ 15g/L, antioxidant 0.01 ~ 10g/L and KAPPA Light Chain Antigen 0 ~ 1600mg/L.
8. the kit detecting KAPPA light chain content as claimed in claim 1, it is characterized in that: in described KAPPA light chain calibration object, the concentration of described KAPPA Light Chain Antigen is 0 ~ 1200mg/L.
9. the kit of the detection KAPPA light chain content as described in any one of claim 1 ~ 8 is detecting the purposes in KAPPA light chain content.
10. adopt the kit of the detection KAPPA light chain content as described in any one of claim 1 ~ 8 to detect a method for KAPPA light chain content, it is characterized in that, comprising:
In testing sample, add described reagent R1, the volume of described testing sample is 5 μ L, and the volume of described reagent R1 is 250 μ L, obtains the first mixed liquor, and obtains the absorbance OD1 of described first mixed liquor;
In described first mixed liquor, add described reagent R2, the volume of described reagent R2 is 50 μ L, obtains the second mixed liquor, and obtains the absorbance OD2 of described second mixed liquor;
Use described KAPPA light chain calibration object to obtain typical curve, on described typical curve, obtain the content of KAPPA light chain in described testing sample according to described absorbance OD1 and described absorbance OD2.
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