CN109633167A - A kind of human serum amyloid A assay kit of highly sensitive, wide detection range - Google Patents
A kind of human serum amyloid A assay kit of highly sensitive, wide detection range Download PDFInfo
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- CN109633167A CN109633167A CN201811577825.4A CN201811577825A CN109633167A CN 109633167 A CN109633167 A CN 109633167A CN 201811577825 A CN201811577825 A CN 201811577825A CN 109633167 A CN109633167 A CN 109633167A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/5432—Liposomes or microcapsules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/70—Mechanisms involved in disease identification
- G01N2800/7095—Inflammation
Abstract
The present embodiments relate to field of immunodetection, and in particular to a kind of human serum amyloid A assay kit of highly sensitive, wide detection range.The human serum amyloid A assay kit includes: the SAA calibration object of reagent 1, reagent 2 and various concentration gradient；Wherein, reagent 2 includes that only the carboxylated latex microballoon of label someone SAA monoclonal antibody and the carboxylated latex microballoon for only marking someone SAA polyclonal antibody, the average grain diameter > of the carboxylated latex microballoon of described label someone SAA monoclonal antibody only mark the average grain diameter of the carboxylated latex microballoon of someone's SAA polyclonal antibody.The kit can complete the detection of single sample in 10min, precision, accuracy, linear and anti-interference etc. indices are all excellent in, the Earlier period of inflammation of infectious diseases can be diagnosed well, SAA content in serum can be detected, SAA content in blood plasma can be detected again, it is highly suitable for Biochemical Analyzer clinically, greatly facilitates clinical examination.
Description
Technical field
The present invention relates to field of immunodetection, and in particular to a kind of human serum amyloid of highly sensitive, wide detection range
Albumin A assay kit.
Background technique
Serum amyloid A protein (serum smyloid A, SAA) is the heterogeneous proteinoid in apolipoprotein family, is
A kind of Acute reaction protein, relative molecular weight about 12KD, function is related to third ingredient (HDL3) in high-density lipoprotein,
Macrophage is to 2-3 times that the affinity of HDL-SAA conjugate is to independent HDL affinity.SAA has 4 kinds of hypotypes, wherein
SAA1, SAA2 are acute proteins, are risen rapidly in the Acute response stage of inflammation；SAA3 gene cannot express in the mankind；SAA4
Can continuous expression, but be not involved in acute inflammatory reaction.SAA is divided into acute stage SAA (acute according to expression in vivo
SAA, A-SAA) and composing type SAA (constitutive SAA, C-SAA).SAA is mainly derived from liver cell group in normal human
The C-SAA for forming expression generates a series of cell factors after the stimulations such as body is infected, inflammation, damage, in cell factor
Regulation under A-SAA is horizontal increases rapidly, become at this time main SAA in vivo.The synthesis of A-SAA is mainly by IL-1 and TNF-α
Adjusting, individual IL-6 influences the synthesis of A-SAA little.The SAA synthesized in liver be released into after blood rapidly with it is highly dense
It spends lipoprotein (HDL) to combine, and is degraded by serum, cell surface and intracellular protease in liver.In liver, IL-6
The macrophage and fibroblast for cooperateing with stimulation to activate with IL-1, TNF-α largely synthesize A-SAA.The SAA that the outer cell of liver generates
Then sticked and endocytosis is through proteasome degradation by intercellular.SAA content < 10mg/L, acute stage, are anti-in normal human serum
At once, plasma SAA concentration can rise rapidly 100-1000 times in 5-6 hours with the development of body inflammatory reaction.In addition, SAA
Half-life period only has 50 minutes, and after body inflammatory reaction controlling, SAA is rapidly decreased to normal level again.Therefore SAA can be used as it is a kind of quick
The acute phase inflammatory marker of sense.
Currently, mainly having chromatography (colloidal gold, fluorescent microsphere) and latex turbidimetry method for SAA detection.Chromatography examination
Agent systematic variation is larger, and quantitative not accurate enough, sensitivity is inadequate；Latex turbidimetry method reagent can realize full-automatic mould in biochemical instruments
Formula operation, systematic error is small, and precision is good, and reagent stability is good, but the detection sensitivity of available reagent is not good enough, the range of linearity
It is not wide enough.
For convenience of clinical detection, it is necessary to develop a kind of Gao Ling that can detect SAA content in serum and plasma sample simultaneously
The kit of sensitivity, wide detection range.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
Goal of the invention
In order to solve the above technical problems, the purpose of the present invention is to provide a kind of people's blood of highly sensitive, wide detection range
Clear amyloid A assay kit.It can be complete in human serum amyloid A assay kit 10min provided by the invention
At the detection of single sample, precision, accuracy, linear and anti-interference etc. indices are all excellent in, and can diagnose sense well
The Earlier period of inflammation of infectious diseases can detect SAA content in serum and detect SAA content in blood plasma, be highly suitable for clinic
On Biochemical Analyzer, greatly facilitate clinical examination.
Solution
Purpose to realize the present invention, the embodiment of the invention provides a kind of human serum amyloid A assay kit, packets
Include the SAA calibration object of reagent 1, reagent 2 and various concentration gradient；Wherein, reagent 2 includes only marking someone SAA monoclonal antibody
Carboxylated latex microballoon and only mark someone SAA polyclonal antibody a carboxylated latex microballoon, described label someone's SAA monoclonal
The average grain diameter > of the carboxylated latex microballoon of antibody only marks the average grain of the carboxylated latex microballoon of someone's SAA polyclonal antibody
Diameter.
Above-mentioned human serum amyloid A assay kit in one possible implementation, only marks someone SAA mono-
The Average Particle Diameters of the carboxylated latex microballoon of clonal antibody are 150-300nm, and final concentration of 0.005%-0.1% (w/v) (should
Final concentration refer to do not include carboxylated latex microballoon of antibody itself final concentration)；Only mark the carboxylic of someone SAA polyclonal antibody
The Average Particle Diameters of base latex microballoon are 60-140nm, and (final concentration refers to not final concentration of 0.1%-0.4% (w/v)
The final concentration of carboxylated latex microballoon including antibody itself)；The final concentration of 0.003%- of people's SAA monoclonal antibody of label
0.015% (w/v)；The final concentration of 0.025%-0.085% of people's SAA polyclonal antibody (w/v) of label.What the final concentration referred to
It is final concentration of each component in reagent 2.
Above-mentioned human serum amyloid A assay kit in one possible implementation, only marks someone SAA mono-
The Average Particle Diameters of the carboxylated latex microballoon of clonal antibody are 240nm, final concentration of 0.02% (w/v)；Only label someone
The Average Particle Diameters of the carboxylated latex microballoon of SAA polyclonal antibody are 123nm, final concentration of 0.2% (w/v)；Label
People's SAA monoclonal antibody final concentration of 0.0065% (w/v)；Final concentration of 0.04% (the w/ of people's SAA polyclonal antibody of label
v)；
Or only marking the Average Particle Diameters of the carboxylated latex microballoon of someone SAA monoclonal antibody is 273nm, final concentration
For 0.01% (w/v)；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 70nm, end
Concentration is 0.3% (w/v)；People's SAA monoclonal antibody final concentration of 0.005% (w/v) of label；The people SAA of label is polyclonal
Antibody final concentration of 0.065% (w/v)；
Or only marking the Average Particle Diameters of the carboxylated latex microballoon of someone SAA monoclonal antibody is 223nm, final concentration
For 0.015% (w/v)；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 83nm, end
Concentration is 0.25% (w/v)；People's SAA monoclonal antibody final concentration of 0.0072% (w/v) of label；More grams of the people SAA of label
Grand antibody final concentration of 0.055% (w/v)；
Or only marking the Average Particle Diameters of the carboxylated latex microballoon of someone SAA monoclonal antibody is 240nm, final concentration
For 0.015% (w/v)；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 70nm, end
Concentration is 0.3% (w/v)；People's SAA monoclonal antibody final concentration of 0.006% (w/v) of label；The people SAA of label is polyclonal
Antibody final concentration of 0.075% (w/v).
Above-mentioned human serum amyloid A assay kit in one possible implementation, only marks someone SAA mono-
The carboxylated latex microballoon of clonal antibody and the carboxylated latex microspheres quality ratio for only marking someone SAA polyclonal antibody are 0.03-
0.4:1。
Above-mentioned human serum amyloid A assay kit includes enhanced sensitivity in reagent 1 in one possible implementation
Agent；Optionally, the sensitizer includes: in glucan, polyvinylpyrrolidone (PVP), ficoll 400 or poly-D-lysine
It is at least one；Still optionally further, dextran molecule amount (Mw) include 5000,25000,40000,80000,250000,
500000, at least one of 2000000；The molecular weight (Mw) of polyvinylpyrrolidone include 8000,10000,24000,
At least one of 58000；Poly-D-lysine molecular weight (Mw) includes at least one of 2000 or 5000.
Above-mentioned human serum amyloid A assay kit includes promoting to coagulate in one possible implementation, in reagent 1
Agent；Optionally, the coagulant includes at least one of PEG (polyethylene glycol) 6000, PEG8000 or PEG20000.
In one possible implementation, reagent 2 further includes stablizing to above-mentioned human serum amyloid A assay kit
Agent；Optionally, the stabilizer includes at least one of protein, PEG or sugar；Still optionally further, the protein packet
Include at least one of BSA (bovine serum albumin(BSA)), gelatin or casein；The sugar includes in sucrose, trehalose or glucose
At least one.
In one possible implementation, calibration object is different ladders to above-mentioned human serum amyloid A assay kit
Spend the people SAA of concentration；Optionally, people SAA concentration gradient are as follows: 0.00,15.00,40.00,120.00,250.00,500.00mg/
L。
Above-mentioned human serum amyloid A assay kit in one possible implementation, the matrix liquid of calibration object
Including following components: phosphate buffer, EDTA (ethylenediamine tetra-acetic acid) disodium or dipotassium EDTA, sodium citrate, heparin sodium or
Heparin lithium, trehalose, BSA, sodium chloride, potassium chloride；Optionally, the matrix liquid of calibration object includes the component of following final concentrations: 10-
The phosphate buffer (pH 7.2-7.4) of 50mM, the citron of the EDETATE SODIUM or dipotassium EDTA of 2-10mM, 1%-5% (w/v)
Sour sodium, the heparin sodium of 0.02%-0.2% (w/v) or heparin lithium, the trehalose of 1%-5% (w/v), 0.2%-1% (w/v)
Mannitol, the sodium chloride of BSA, 100-300mM of 0.1%-2% (w/v), 50-150mM potassium chloride.It is i.e. provided by the invention
Contain anti-coagulants such as a certain amount of heparin sodium, heparin lithium, EDETATE SODIUM, dipotassium EDTA, sodium citrate, steady in calibration object matrix liquid
Determine agent and inorganic salts etc..Suitable for measuring the SAA content human serum and blood plasma.
In one possible implementation, reagent 2 includes following groups to above-mentioned human serum amyloid A assay kit
Point: only the carboxylated latex microballoon of label someone SAA monoclonal antibody, the carboxylated latex of label someone SAA monoclonal antibody are micro-
Ball, buffer, surfactant, inorganic salts and preservative.
In one possible implementation, reagent 2 includes following ends to above-mentioned human serum amyloid A assay kit
The component of concentration: 0.005%-0.1% (w/v) only marks the carboxylated latex microballoon of someone SAA monoclonal antibody, and (average grain diameter is
150-300nm), the people SAA monoclonal antibody of the label of 0.003%-0.015%, 0.1%-0.4% (w/v) only mark someone
The people SAA of the carboxylated latex microballoon (average grain diameter 60-140nm) of SAA polyclonal antibody, the label of 0.025%-0.085%
Polyclonal antibody, the stabilizer of 0.2%-5% (w/v), the buffer of 10-100mM, the surface of 0.05%-0.3% (w/v) are living
Property agent, the inorganic salts of 50-600mM and 0.05%-0.1% (w/v) preservative.
In one possible implementation, reagent 1 includes following groups to above-mentioned human serum amyloid A assay kit
Point: buffer, BSA, surfactant, EDTA, inorganic salts and preservative；It optionally, include the group of following final concentrations in reagent 1
Point: the buffer of 10-100mM, the coagulant of 1.0%-10% (w/v), 0.2%-8% (w/v) sensitizer, 0.5%-5%
(w/v) surfactant of BSA, 0.05%-0.3% (w/v), the inorganic salts of EDTA, 50-600mM of 1-10mM and
The preservative of 0.05%-0.1% (w/v).
Above-mentioned human serum amyloid A assay kit in one possible implementation, in reagent 1 or reagent 2
Buffer include: independently of each other pH value be 5.0-9.0 MES buffer, phosphate buffer, MOPS buffer, TAPS
At least one of buffer, borate buffer solution, Tris-Cl buffer and HEPES buffer solution.
Above-mentioned human serum amyloid A assay kit in one possible implementation, in reagent 1 or reagent 2
Surfactant independently of each other include: at least one of Tween-20, Tween-80 or Triton X-100.
Above-mentioned human serum amyloid A assay kit in one possible implementation, in reagent 1 or reagent 2
Preservative independently of each other include: in ProClin300, merthiolate, sodium benzoate, Krovin300 or Krovin 500
It is at least one.Preservative can prevent reagent by microbial contamination.
Above-mentioned human serum amyloid A assay kit in one possible implementation, the people SAA monoclonal
Antibody is the specific monoclonal antibody derived from mouse；The people SAA polyclonal antibody is to resist derived from the specificity of rabbit or sheep more.People SAA
Purity >=95% of monoclonal antibody, potency >=1:100000；Purity >=95% of people's SAA polyclonal antibody, potency >=1:
10000。
In one possible implementation, the carboxylated latex is micro- for above-mentioned human serum amyloid A assay kit
Ball is polystyrene carboxylated latex microballoon.
Above-mentioned human serum amyloid A assay kit in one possible implementation, the human serum starch
Sample albumin A assay kit is used for the detection of serum and blood plasma.
The embodiment of the invention also provides a kind of preparation methods of human serum amyloid A assay kit, including under
It states step: preparing human serum amyloid A assay kit using above-mentioned formula.
Above-mentioned preparation method in one possible implementation, includes the following steps:
Reagent preparation 1: in the buffer of Suitable ionic strengths be added sensitizer, coagulant, BSA, surfactant,
EDTA, inorganic salts, preservative mix well preparation；
Reagent preparation 2: the monoclonal for the carboxylated latex microballoon label people SAA for being respectively 150-300nm with average grain diameter is anti-
The polyclonal antibody of body, the carboxylated latex microballoon for being 60-140nm with average grain diameter label people SAA, adds comprising suitable concentration
Buffer, inorganic salts, stabilizer, surfactant and preservative；Wherein, the carboxyl of the monoclonal antibody of people SAA is only marked
Latex microsphere and only concentration of the carboxylated latex microballoon in 2 system of reagent of the polyclonal antibody of label people SAA is respectively
0.005%-0.1% (w/v) and 0.1%-0.4% (w/v), the final concentration of the people SAA monoclonal antibody of label, polyclonal antibody
Respectively 0.003%-0.015% (w/v) and 0.025%-0.085% (w/v)；
It prepares calibration object: being diluted people SAA with the matrix liquid prepared, assignment obtains the calibration of series of concentrations gradient after tracing to the source
Product.
In one possible implementation, the preparation step of reagent 2 includes: to dilute phase with activating solution to above-mentioned preparation method
Answering the carboxylated latex microballoon of partial size to its mass concentration is 0.5%-3%, and the activation that mass concentration is 0.01%-0.1% is added
Agent EDC (1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride), is stirred at room temperature, activated carboxyl；After activation,
Centrifugation, washing remove extra EDC；Label buffer is added, ultrasound disperses latex microsphere, is separately added into SAA monoclonal
Antibody, SAA polyclonal antibody separate marking, are stirred at room temperature, and add terminate liquid and continue to stir；Final reaction solution is centrifuged,
The latex microsphere marked is resuspended with liquid is saved in washing respectively, the greater particle size microballoon of monoclonal antibody will be marked to resist with label smaller more
Partial size microballoon mixes to obtain reagent 2 in proportion；
Wherein, in reagent 2, the carboxylated latex microballoon and the more anti-carboxylated latex microspheres quality ratios of mark for marking monoclonal antibody are 0.03-
0.4:1。
In one possible implementation, the activating solution includes: that pH value is 4.0-6.0 to above-mentioned preparation method
MES buffer, phosphate buffer or the HEPES of 0.01-0.2M slowly at least one of fliud flushing.
In one possible implementation, the label buffer includes that pH value is 7.0-8.4 to above-mentioned preparation method
In the phosphate buffer of 0.01-0.2M, MOPS buffer, TAPS buffer, borate buffer solution or Tris-Cl buffer
It is at least one.
The embodiment of the invention also provides the application methods of above-mentioned human serum amyloid A assay kit, including under
State step: dominant wavelength 570nm, after 2 μ L samples, 150 μ L reagents 1 are added, 37 DEG C of water-bath 5min add 50 μ L reagents 2,
Absorbance A 1 is read after 30s, then reads absorbance A 2 after reacting 4min, calculates the Cha Zhi ⊿ A=A2-A1 of the two absorbance；With
Matched 6 calibration objects of above-mentioned human serum amyloid A assay kit first carry out multiple spot calibration (Spline or Logit-
Log (4P) model of fit)；The absorbance change when concentration (unit mg/L) of sample can be by its detection is on calibration curve
It is calculated.
Beneficial effect
1, at present on the market most emulsion reagents be only with a kind of partial size latex microsphere separate marking it is corresponding
Rabbit is mostly anti-, sheep is mostly anti-or pairing mouse monoclonal antibody, cannot take into account the sensitivity and detection high level of reagent well.And the present invention is implemented
The human serum amyloid A assay kit that example provides is simultaneously using more anti-, monoclonal antibodies, and the two separately marks, and monoclonal antibody marks
To guarantee the sensitivity of kit, how anti-label guarantees reagent in Spring layer compared with small particle microballoon and has wider greater particle size microballoon
Detection range.
The present invention mainly utilize the specific reaction of antigen and antibody, by the carboxylated latex microballoon of different-grain diameter anti-
It answers and is condensed into space (cuvette etc.) compared with large complex (light is stopped to pass through), measure that the increase of reaction solution turbidity in sample
The concentration of SAA.By on people SAA monoclonal antibody, the how anti-carboxylated latex microballoon for being tagged to different-grain diameter respectively of SAA, when in sample to be tested
People SAA and reagent in antibody specific reaction occurs, after generating latticed larger antigen-antibody complex, can make anti-
The turbidity in liquid is answered to increase.Within the scope of a certain concentration, the turbidity of reaction solution increases the amount with antigen-antibody complex at just
Than that is, directly proportional to the concentration of SAA in sample.The calibration of reagent is made under matched calibrating system, suitable model of fit
Curve, the light intensity variation by measuring sample can converse the concentration of SAA to be measured.
2, SAA monoclonal antibody, the average grain diameter microballoon label for the 60-140nm that arranges in pairs or groups are marked with the microballoon of 150-300nm average grain diameter
SAA is mostly anti-, can further improve the sensitivity and linear measurement range of kit of the present invention.The too small microballoon of partial size cannot be put well
The signal being immunoreacted greatly；And partial size is excessive, microballoon can then settle after long term storage due to gravity, influence measurement knot
Fruit.
3, microballoon marks SAA monoclonal antibody, SAA mostly anti-respectively, by adjusting the two concentration and ratio, can make monoclonal antibody, mostly anti-
Labeling effciency respectively realizes optimum state, to realize having preferable immunological response amplified signal in wide detection range, thus
The sensitivity and detection range of reagent can be taken into account well.When reacting with sample to be tested, the antibody of coupling can be with the people in sample
SAA specific binding, by the antigenic determinant of SAA itself multiplicity and reagent 2 specific antibody it is rich, can react
It is generated in system and occupies larger space, multiple antigenic-antibody complex, suitable reaction site distribution is more advantageous to immune multiple
It closes object and generates turbidity in reaction solution and changes, effectively improving the sensitivity of reagent, (Functional Sensitivity can achieve 0.20mg/
L), it and can keep linear well in the wide concentration range that SAA is 0.00-500.00mg/L, expand the detection of kit
Range.
4, buffer, coagulant, sensitizer etc. can provide suitable reaction condition, certain for immune response in reagent 1
The sensitivity for controlling reaction speed in degree, improving reagent, the especially coagulant in reagent 1 and sensitizer utilize its own
Different macromolecule advantages can preferably improve the joint efficiency and combination stability of antigen-antibody in local space, be conducive to
Improve the sensitivity of reagent；Be added in calibration object matrix liquid anti-coagulants (heparin sodium, heparin lithium, EDETATE SODIUM, dipotassium EDTA,
Sodium citrate etc.) detection that is conducive to plasma sample, the sample type that the reagent is suitable for detection is expanded to a certain extent.
5, the human serum amyloid A measurement reagent of the high sensitivity, wide detection range that are provided in the embodiment of the present invention
Box, polystyrene latex microspheres used are carboxyl modified, covalent peptide bond can be formed with antibody amino groups after activation, antibody can
It is securely joined in latex microsphere surface；Suitable buffer system and stabilizer etc. then further ensure that people's blood in reagent 2
The stability of clear amyloid A assay kit.
6, the human serum amyloid A measurement reagent of the high sensitivity, wide detection range that are provided in the embodiment of the present invention
The detection of single sample can be completed in 10min for box, have that high sensitivity (up to 0.20mg/L), detection range is wide (is in SAA
Good linear, linearly dependent coefficient R >=0.9900 is kept within the scope of the gradient concentration of 0.00-500.00mg/L), precision
The features such as good, accuracy is good and strong antijamming capability, it is dense can to measure SAA in serum and plasma sample under same set of calibrating system
Degree can diagnose the Earlier period of inflammation of infectious diseases applied to clinic well.The high sensitivity that is there is provided in the embodiment of the present invention, width
The human serum amyloid A assay kit of detection range does not generate precipitating, does not adhere to after reaction, be suitable for clinic
Biochemical Analyzer cleans convenient for Biochemical Analyzer.
Detailed description of the invention
One or more embodiments are illustrated by the picture in corresponding attached drawing, these exemplary theorys
The bright restriction not constituted to embodiment.Dedicated word " exemplary " means " being used as example, embodiment or illustrative " herein.
Here as any embodiment illustrated by " exemplary " should not necessarily be construed as preferred or advantageous over other embodiments.
Figure 1A is the calibration curve of the human serum amyloid A assay kit of the embodiment of the present invention 1 and its linearly divides
Analyse result.
Figure 1B is the calibration curve of the human serum amyloid A assay kit of the embodiment of the present invention 2 and its linearly divides
Analyse result.
Fig. 1 C is the calibration curve of the human serum amyloid A assay kit of the embodiment of the present invention 3 and its linearly divides
Analyse result.
Fig. 1 D is the calibration curve of the human serum amyloid A assay kit of the embodiment of the present invention 4 and its linearly divides
Analyse result.
Fig. 2 is that the human serum amyloid A assay kit of the embodiment of the present invention 4 and contrast agents detect clinic simultaneously
The correlation of 40 serum samples.
Fig. 3 is that the human serum amyloid A assay kit of the embodiment of the present invention 4 and contrast agents detect clinic simultaneously
The correlation of 40 Heparin plasma samples.
Fig. 4 be the human serum amyloid A assay kit of the embodiment of the present invention 4 and meanwhile detect 40 clinical serums and
The correlation of its corresponding anticoagulant plasma sample of EDTA.
Fig. 5 be the human serum amyloid A assay kit of the embodiment of the present invention 4 and meanwhile detect 40 clinical serums and
The correlation of its corresponding anticoagulant plasma sample of sodium citrate.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described, it is clear that described embodiments are some of the embodiments of the present invention, rather than
Whole embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.Unless otherwise explicitly stated, otherwise whole
In a specification and claims, it is to include that the term " include " or its transformations will be understood as
The element or component stated, and do not exclude other elements or other components.
In addition, in order to better illustrate the present invention, numerous details is given in specific embodiment below.
It will be appreciated by those skilled in the art that without certain details, the present invention equally be can be implemented.In some embodiments, right
It is not described in detail in raw material well known to those skilled in the art, element, method, means etc., in order to highlight master of the invention
Purport.
People SAA monoclonal antibody used in embodiment 1-4 is purchased from Medix Biochemica (its purity >=95%, effect
Valence >=1:100000), polyclonal antibody purchased from Beijing Apis Biotechnology Co., Ltd. (its purity >=95%, potency >=1:
10000), polystyrene carboxylated latex microballoon is purchased from Japan JSR.
Embodiment 1
A kind of human serum amyloid A assay kit, the school SAA including reagent 1, reagent 2 and various concentration gradient
Quasi- product；Wherein,
Component of the reagent 1 including following final concentrations: 20mM HEPES buffer solution (pH7.2), the BSA of 0.5% (w/v), 3%
(w/v) glucan that the Mw of PEG6000,1% (w/v) are 40000, the KCl of 150mM, the Tween-20 of 0.05% (w/v),
The ProClin300 of 0.1% (w/v), the EDTA of 2mM；
Reagent 2 includes the component of following final concentrations: only the carboxylated latex microballoon of label someone SAA monoclonal antibody is (average
Partial size is 240nm, microballoon final concentration of 0.02% (w/v)), the people SAA monoclonal antibody of the label of 0.0065% (w/v), only
The carboxylated latex microballoon (average grain diameter 123nm, microballoon final concentration of 0.2% (w/v)) of label someone SAA polyclonal antibody,
People SAA polyclonal antibody, the 50mM MOPS buffer (pH7.4), 100mM potassium chloride, 0.3% of the label of 0.04% (w/v)
(w/v) gelatin, 1% (w/v) BSA, 2% (w/v) sucrose, 0.2% (w/v) trehalose, 0.05% (w/v) Triton X-100,
The ProClin300 of 0.1% (w/v)；
Calibration object: containing 0.00,15.00,40.00,120.00,250.00, the people of six concentration gradients of 500.00mg/L
SAA, matrix liquid include the component of following final concentrations: the phosphate buffer (pH7.2) of 30mM, 2mM EDETATE SODIUM,
The sodium citrate of 1.5% (w/v), the heparin lithium of 0.5% (w/v), 1% (w/v) trehalose, 0.5% (w/v) mannitol, 1%
(w/v) BSA, 150mM sodium chloride, 50mM potassium chloride.
The preparation method of above-mentioned human serum amyloid A assay kit, includes the following steps:
Reagent preparation 1: dense to BSA, the end that final concentration of 0.5% (w/v) is added in 20mM HEPES buffer solution (pH7.2)
Degree is KCl, the final concentration of the PEG6000 of 3% (w/v), the Gentran 40 000 of final concentration of 1% (w/v), final concentration of 150mM
For the EDTA of the Tween-20 of 0.05% (w/v), the ProClin300 of final concentration of 0.1% (w/v), final concentration of 2mM, use
Reagent 1 is obtained after 0.22 μm of membrane filtration；
Reagent preparation 2: with 50mM MES buffer (pH5.0) dilute corresponding partial size carboxylated latex microballoon (240nm ,-
COOH amount 0.11-0.16mmol/g；123nm ,-COOH measure 0.15-0.22mmol/g) to its mass concentration be 1%, be separately added into
The activator EDC that mass concentration is 0.02%, 0.05%, is stirred at room temperature 20min activated carboxyl；After activation, 15000g
Centrifugation, washing remove extra EDC；Label buffer (50mM PBS, pH7.4) is added, ultrasound disperses latex microsphere, point
Not Jia Ru SAA monoclonal antibody (mark 240nm microballoon), SAA polyclonal antibody (mark 123nm microballoon) separate marking, be stirred at room temperature
1h adds terminate liquid and continues to stir 10min；Final reaction solution is centrifuged with 15000g, with 50mM PBS buffer solution
(pH7.4) it washs 1 time, is resuspended respectively with the preservation liquid of respective volume and has marked the latex (final concentration of 0.02% (w/ of 240nm microballoon
V), 123nm microballoon final concentration of 0.2% (w/v), people's SAA monoclonal antibody final concentration of 0.0065% (w/v) of label, mark
People's SAA polyclonal antibody final concentration of 0.04% (w/v) of note), it stirs and evenly mixs, saves the component of liquid are as follows: 50mM MOPS buffering
Liquid (pH7.4), 100mM potassium chloride, 0.3% (w/v) gelatin, 1% (w/v) BSA, 2% (w/v) sucrose, 0.2% (w/v) seaweed
The ProClin300 of sugar, 0.05% (w/v) Triton X-100,0.1% (w/v), obtains reagent 2；
It prepares calibration object: the SAA of vivoexpression being made of the calibration object matrix liquid of mentioned component and is diluted, is assigned after being traced to the source
Value obtain series of concentrations calibration object (series of concentrations of SAA be 0.00,15.00,40.00,120.00,250.00,
500.00mg/L)。
Embodiment 2
A kind of human serum amyloid A assay kit, the school SAA including reagent 1, reagent 2 and various concentration gradient
Quasi- product；Wherein,
Component of the reagent 1 including following final concentrations: 50mM MOPS buffer (pH7.4), the BSA of 1.5% (w/v), 2%
(w/v) poly-D-lysine that the Mw of PEG8000,5% (w/v) are 5000, the NaCl of 300mM, the Tween- of 0.05% (w/v)
80, the ProClin300 of 0.1% (w/v), the EDTA of 2mM；
Reagent 2 includes the component of following final concentrations: only the carboxylated latex microballoon of label someone SAA monoclonal antibody is (average
Partial size is 273nm, microballoon final concentration of 0.01% (w/v)), the people SAA monoclonal antibody of the label of 0.005% (w/v), only mark
The carboxylated latex microballoon (average grain diameter 70nm, microballoon final concentration of 0.3% (w/v)) of note someone SAA polyclonal antibody,
The people SAA polyclonal antibody of the label of 0.065% (w/v), 50mM HEPES buffer solution (pH7.2), 100mM ammonium chloride, 0.2%
(w/v) gelatin, 0.5% (w/v) BSA, 3% (w/v) sucrose, 0.5% (w/v) trehalose, 0.05% (w/v) Triton X-
100, the ProClin300 of 0.1% (w/v)；
Calibration object: containing 0.00,15.00,40.00,120.00,250.00, the people of six concentration gradients of 500.00mg/L
SAA, matrix liquid include the component of following final concentrations: the phosphate buffer (pH7.4) of 50mM, the EDETATE SODIUM of 5mM, 2%
(w/v) heparin lithium, 1.5% (w/v) trehalose, 0.3% (w/v) mannitol, 0.5% (w/ of sodium citrate, 0.3% (w/v)
V) BSA, 300mM sodium chloride, 50mM potassium chloride.
The preparation method of above-mentioned human serum amyloid A assay kit, includes the following steps:
Reagent preparation 1: dense to BSA, the end that final concentration of 1.5% (w/v) is added in 50mM MOPS buffer (pH7.4)
Degree is the PEG8000 of 2% (w/v), the poly-D-lysine (Mw5000) of final concentration of 5% (w/v), final concentration of 300mM
ProClin300, the final concentration of 5mM of NaCl, the Tween-80 of final concentration of 0.05% (w/v), final concentration of 0.1% (w/v)
EDTA, with after 0.22 μm of membrane filtration reagent 1；
Reagent preparation 2: with 50mM MES buffer (pH6.0) dilute corresponding partial size carboxylated latex microballoon (273nm ,-
COOH amount 0.05-0.08mmol/g；70nm ,-COOH measure 0.15-0.22mmol/g) to its mass concentration be 1%, be separately added into
The activator EDC that mass concentration is 0.02%, 0.06%, is stirred at room temperature 20min activated carboxyl；After activation, 15000g
Centrifugation, washing remove extra EDC；Label buffer (50mM MOPS, pH7.4) is added, ultrasound disperses latex microsphere,
It is separately added into SAA monoclonal antibody (mark 273nm microballoon), SAA polyclonal antibody (mark 70nm microballoon) separate marking, is stirred at room temperature
1h adds terminate liquid and continues to stir 10min；Final reaction solution is centrifuged with 15000g, with 50mM MOPS buffer
(pH7.4) it washs 1 time, is resuspended respectively with the preservation liquid of respective volume and has marked the latex (final concentration of 0.01% (w/ of 273nm microballoon
V), 70nm microballoon final concentration of 0.3% (w/v), people's SAA monoclonal antibody final concentration of 0.005% (w/v), people SAA are polyclonal
Antibody final concentration of 0.065% (w/v)), it stirs and evenly mixs, saves the component of liquid are as follows: 50mM HEPES buffer solution (pH7.2),
100mM ammonium chloride, 0.2% (w/v) gelatin, 0.5% (w/v) BSA, 3% (w/v) sucrose, 0.5% (w/v) trehalose, 0.05%
(w/v) ProClin300 of Triton X-100,0.1% (w/v), obtains reagent 2；
It prepares calibration object: the SAA of vivoexpression being made of the calibration object matrix liquid of mentioned component and is diluted, is assigned after being traced to the source
Value obtain series of concentrations calibration object (series of concentrations of SAA be 0.00,15.00,40.00,120.00,250.00,
500.00mg/L)。
Embodiment 3
A kind of human serum amyloid A assay kit, the school SAA including reagent 1, reagent 2 and various concentration gradient
Quasi- product；；Wherein,
Reagent 1 includes the component of following final concentrations: 50mM PBS buffer solution (pH7.4), the BSA of 2% (w/v), 1% (w/
V) PVP, the NH of 100mM that the Mw of PEG20000,2% (w/v) are 100004Cl, the Tween-80 of 0.1% (w/v), 0.1%
(w/v) EDTA of ProClin300,10mM；
Reagent 2 includes the component of following final concentrations: only the carboxylated latex microballoon of label someone SAA monoclonal antibody is (average
Partial size is 223nm, microballoon final concentration of 0.015% (w/v)), the people SAA monoclonal antibody of the label of 0.0072% (w/v), only
The carboxylated latex microballoon (average grain diameter 83nm, microballoon final concentration of 0.25% (w/v)) of label someone SAA polyclonal antibody,
People SAA polyclonal antibody, the 50mM TAPS buffer (pH8.0), 50mM magnesium chloride, 0.2% of the label of 0.055% (w/v)
(w/v) gelatin, 0.2% (w/v) BSA, 3% (w/v) sucrose, 1% (w/v) glucose, 0.05% (w/v) Tween-20,0.1%
(w/v) Krovin300；
Calibration object: containing 0.00,15.00,40.00,120.00,250.00, the people of six concentration gradients of 500.00mg/L
SAA, matrix liquid include the component of following final concentrations: the phosphate buffer (pH7.4) of 20mM, 3mM dipotassium EDTA,
The sodium citrate of 2.5% (w/v), the heparin lithium of 0.3% (w/v), 1.5% (w/v) trehalose, 0.2% (w/v) mannitol, 1%
(w/v) BSA, 200mM sodium chloride, 100mM potassium chloride.
The preparation method of above-mentioned human serum amyloid A assay kit, includes the following steps:
Reagent preparation 1: to the BSA, final concentration of that final concentration of 2% (w/v) is added in 50mM PBS buffer solution (pH7.4)
The NH of the PEG20000 of 1% (w/v), the PVP (Mw10000) of final concentration of 2% (w/v), final concentration of 100mM4Cl, final concentration
For the EDTA of the Tween-80 of 0.1% (w/v), the ProClin300 of final concentration of 0.1% (w/v), final concentration of 10mM, use
Reagent 1 is obtained after 0.22 μm of membrane filtration；
Reagent preparation 2: with 100mM MES buffer (pH6.0) dilute corresponding partial size carboxylated latex microballoon (223nm ,-
COOH amount 0.06-0.10mmol/g；83nm ,-COOH measure 0.25-0.38mmol/g) to its mass concentration be 1%, be separately added into
The activator EDC that mass concentration is 0.03%, 0.06%, is stirred at room temperature 20min activated carboxyl；After activation, 15000g
Centrifugation, washing remove extra EDC；Label buffer (50mM HEPES, pH7.4) is added, ultrasound disperses latex microsphere,
It is separately added into SAA monoclonal antibody (mark 223nm microballoon), SAA polyclonal antibody (mark 83nm microballoon) separate marking, is stirred at room temperature
1h adds terminate liquid and continues to stir 10min；Final reaction solution is centrifuged with 15000g, with 50mM HEPES buffer solution
(pH7.4) it washs 1 time, is resuspended respectively with the preservation liquid of respective volume and has marked the latex (final concentration of 0.015% (w/ of 223nm microballoon
V), 83nm microballoon final concentration of 0.25% (w/v), final concentration of 0.0072% (w/v) of people's SAA monoclonal antibody of label,
Final concentration of 0.055% (w/v) of people's SAA polyclonal antibody of label), it stirs and evenly mixs, saves the component of liquid are as follows: 50mM
TAPS buffer (pH8.0), 50mM magnesium chloride, 0.2% (w/v) gelatin, 0.2% (w/v) BSA, 3% (w/v) sucrose, 1% (w/
V) Krovin300 of glucose, 0.05% (w/v) Tween-20,0.1% (w/v), obtains reagent 2；
It prepares calibration object: the SAA of vivoexpression being made of the calibration object matrix liquid of mentioned component and is diluted, is assigned after being traced to the source
Value obtain series of concentrations calibration object (series of concentrations of SAA be 0.00,15.00,40.00,120.00,250.00,
500.00mg/L)。
Embodiment 4
A kind of human serum amyloid A assay kit, the school SAA including reagent 1, reagent 2 and various concentration gradient
Quasi- product；Wherein,
Component of the reagent 1 including following final concentrations: 50mM Tris-Cl buffer (pH8.0), the BSA of 2% (w/v), 3%
(w/v) glucan that the Mw of PEG8000,0.2% (w/v) are 500000, the MgCl of 50mM2, the Triton of 0.05% (w/v)
X-100, the ProClin300 of 0.1% (w/v), the EDTA of 5mM；
Reagent 2 includes the component of following final concentrations: only the carboxylated latex microballoon of label someone SAA monoclonal antibody is (average
Partial size is 240nm, microballoon final concentration of 0.015% (w/v)), the people SAA monoclonal antibody of the label of 0.006% (w/v), only
The carboxylated latex microballoon (average grain diameter 70nm, microballoon final concentration of 0.3% (w/v)) of label someone SAA polyclonal antibody,
People SAA polyclonal antibody, the 50mM MOPS buffer (pH7.2), 150mM ammonium chloride, 0.2% of the label of 0.075% (w/v)
(w/v) gelatin, 0.5% (w/v) BSA, 3% (w/v) sucrose, 1% (w/v) glucose, 0.5% (w/v) trehalose, 0.05%
(w/v) Krovin500 of Tween-80,0.1% (w/v)；
Calibration object: containing 0.00,15.00,40.00,120.00,250.00, the people of six concentration gradients of 500.00mg/L
SAA, matrix liquid include the component of following final concentrations: the phosphate buffer (pH7.3) of 20mM, the EDETATE SODIUM of 5mM, 3%
(w/v) heparin lithium, 5% (w/v) trehalose, 0.2% (w/v) mannitol, 0.5% (w/ of sodium citrate, 0.05% (w/v)
V) BSA, 150mM sodium chloride, 50mM potassium chloride.
The preparation method of above-mentioned human serum amyloid A assay kit, includes the following steps:
Reagent preparation 1: dense to BSA, the end that final concentration of 2% (w/v) is added in 50mM Tris-Cl buffer (pH8.0)
Degree is the PEG8000 of 3% (w/v), the glucan (Mw500000) of final concentration of 0.2% (w/v), final concentration of 50mM
MgCl2, Trinton X-100 of final concentration of 0.05% (w/v), final concentration of 0.1% (w/v) ProClin300, dense eventually
Degree be 5mM EDTA, with after 0.22 μm of membrane filtration reagent 1；
Reagent preparation 2: with 50mM MES buffer (pH6.0) dilute corresponding partial size carboxylated latex microballoon (240nm ,-
COOH amount 0.11-0.16mmol/g；70nm ,-COOH measure 0.15-0.22mmol/g) to its mass concentration be 1%, be separately added into
The activator EDC that mass concentration is 0.02%, 0.06%, is stirred at room temperature 20min activated carboxyl；After activation, 15000g
Centrifugation, washing remove extra EDC；Label buffer (50mM HEPES, pH7.4) is added, ultrasound disperses latex microsphere,
It is separately added into SAA monoclonal antibody (mark 240nm microballoon), SAA polyclonal antibody (mark 70nm microballoon) separate marking, is stirred at room temperature
1h adds terminate liquid and continues to stir 10min；Final reaction solution is centrifuged with 15000g, with 50mM PBS buffer solution
(pH7.4) it washs 1 time, is resuspended respectively with the preservation liquid of respective volume and has marked the latex (final concentration of 0.015% (w/ of 240nm microballoon
V), 70nm microballoon final concentration of 0.3% (w/v), people's SAA monoclonal antibody final concentration of 0.006% (w/v) of label, label
People's SAA polyclonal antibody final concentration of 0.075% (w/v)), stir and evenly mix, save the component of liquid are as follows: 50mM MOPS buffering
Liquid (pH7.2), 150mM ammonium chloride, 0.2% (w/v) gelatin, 0.5% (w/v) BSA, 3% (w/v) sucrose, 1% (w/v) grape
Sugar, 0.5% (w/v) trehalose, 0.05% (w/v) Tween-80,0.1% (w/v) Krovin500, obtain reagent 2；
It prepares calibration object: the SAA of vivoexpression being made of the calibration object matrix liquid of mentioned component and is diluted, is assigned after being traced to the source
Value obtain series of concentrations calibration object (series of concentrations of SAA be 0.00,15.00,40.00,120.00,250.00,
500.00mg/L)。
Effect example 1
To the human serum amyloid A assay kit that embodiment 1-4 is prepared, with 7180 full-automatic biochemical of Hitachi
Instrument is tested, parameter and steps are as follows: test dominant wavelength 570nm is first added 2 μ L of sample, adds 150 μ L reagents 1,
After 37 DEG C of incubation about 5min, 50 μ L reagents 2 are added, read absorbance A 1 after about 30s, then read absorbance A 2 after reacting about 4min
(calculate the Cha Zhi ⊿ A=A2-A1 of the two absorbance；
Multiple spot is carried out using matched 6 calibration objects to the human serum amyloid A assay kit of embodiment 1-4 to determine
Mark, obtains calibration curve and carries out linear analysis, as a result respectively as shown in figures 1A-d (Figure 1A, 1B, 1C, 1D).Wherein, horizontal
Coordinate is calibration solution concentration, and ordinate is the corresponding absorbance difference of each concentration calibration solution.The concentration (unit mg/L) of sample
Absorbance change when can be by its detection is calculated on calibration curve.
Performance evaluating test (application method is carried out to the human serum amyloid A assay kit of above-mentioned 4 embodiments
With above-mentioned test method, 7180 type full automatic biochemical apparatus of Hitachi):
(1) blank detection limit: absorbance value 3 times at wavelength 570nm of kit measurement zero people SAA sterling take it
Mean value the results are shown in Table 1；
(2) Functional Sensitivity: kit retest people's SAA content is serum sample 20 times of 0.20mg/L, is counted respectively
The average and standard deviation of measurement concentration is calculated, then calculates the coefficient of variation (CV), the results are shown in Table 1；Employment SAA content is 0.20mg/L
Plasma sample or people's SAA sterling carry out sensitivity test, the coefficient of variation CV value measured is within the acceptable range；
(3) high level people SAA sterling (300mg/L) accuracy: is added to low value people's SAA sterling by the volume ratio of 1:9
In (20mg/L), is tested with kit and sample after high level people SAA sterling, low value people SAA sterling and mixing is respectively repeated to examine
It surveys 3 times, takes its concentration mean value, calculate the rate of recovery, the results are shown in Table 1；
(4) precision: kit retest people's SAA content is serum sample 10 times of (80.00 ± 10.00) mg/L,
The average and standard deviation of measurement concentration is calculated separately, then calculates the coefficient of variation (CV), the results are shown in Table 1；Employment SAA content is
The plasma sample or people's SAA sterling of (80.00 ± 10.00) mg/L carry out precision test, and the coefficient of variation CV value measured is can
In the range of receiving；
(5) linear: with calibration object matrix liquid by linear high level people SAA sterling (600.00 ± 60.00) mg/L in [0.20-
500.00] 6 concentration samples out are diluted within the scope of mg/L, and (this 6 concentration are respectively 0.20mg/L, 4.00mg/L, 20.00mg/
L, 100.00mg/L, 250.00mg/L, 500.00mg/L), each sample replication 3 times takes its mean value.With diluted sample
Theoretical concentration value is x-axis, and measured concentration value is y-axis, establishes linear equation, calculates its linearly dependent coefficient R, the results are shown in Table 1；
(6) anti-interference: kit detects the glycerol of the hemoglobin of 500mg/dL, the bilirubin of 30mg/L, 3000mg/dL
The resisting rheumatoid disease factor (RF) sample each 3 times of three esters and 500IU/mL take it to measure the mean value of concentration, the results are shown in Table 1.
As can be known from Table 1, the kit prepared in parameter area of the present invention is provided with preferable blank detection and limits, is sensitive
Degree, accuracy, precision, linear and anti-interference ability.
Table 1
Effect example 2
Detect the performance evaluation of clinical sample
The reagent (application method of kit is with above-mentioned test method) and the contrast agents (serum of Siemens of embodiment 4
Amyloid A scattered light urbidmetry assay kit, application method is referring to its specification) 40 clinical serum samples are detected simultaneously
This, evaluates the correlation of the two, as a result (is wherein greater than the measured value of 200mg/L, contrast agents are first that sample is dilute as shown in Figure 2
It is detected after releasing 10 times, then is back-calculated and obtains).Wherein, the detection knot of the reagent horizontal, ordinate is respectively contrast agents, embodiment 4
Fruit, unit are mg/L.
The reagent and contrast agents of embodiment 4 detect 40 clinical Heparin plasma samples simultaneously, evaluate the phase of the two
Guan Xing, as a result as shown in Figure 3.Wherein, horizontal, ordinate is respectively contrast agents, the reagent testing result of embodiment 4, and unit is equal
For mg/L.
The reagent of embodiment 4 detects both 40 clinical serums and its anticoagulant plasma sample of corresponding EDTA, evaluation simultaneously
Correlation, as a result as shown in Figure 4.Wherein, the testing result horizontal, ordinate is respectively serum, the anticoagulant plasma sample of EDTA, it is single
Position is mg/L.
The reagent of embodiment 4 detects 40 clinical serums and its anticoagulant plasma sample of corresponding sodium citrate, evaluation simultaneously
The correlation of the two, as a result as shown in Figure 5.Wherein, the inspection horizontal, ordinate is respectively serum, the anticoagulant plasma sample of sodium citrate
It surveys as a result, unit is mg/L.
Clinical sample correlation the result shows that, human serum amyloid A assay kit provided by the invention can be very
Meet the detection of clinical serum sample and Heparin plasma sample well；And by the comparison with serum sample, reagent is developed
It can also detect that EDTA is anticoagulant and the anticoagulant plasma sample of sodium citrate well；And human serum amyloid protein provided by the invention
A assay kit has higher sensitivity and broader detection range, can preferably meet clinical demand.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (10)
1. a kind of human serum amyloid A assay kit, the SAA calibration including reagent 1, reagent 2 and various concentration gradient
Product；Wherein, reagent 2 includes only the carboxylated latex microballoon of label someone SAA monoclonal antibody and only label someone SAA Anti-TNF-α
The average grain diameter > of the carboxylated latex microballoon of body, the carboxylated latex microballoon of described label someone SAA monoclonal antibody is only marked
The average grain diameter of the carboxylated latex microballoon of someone's SAA polyclonal antibody.
2. human serum amyloid A assay kit according to claim 1, it is characterised in that: only label someone SAA
The Average Particle Diameters of the carboxylated latex microballoon of monoclonal antibody are 150-300nm, final concentration of 0.005%-0.1%；Only mark
The Average Particle Diameters for remembering the carboxylated latex microballoon of someone SAA polyclonal antibody are 60-140nm, final concentration of 0.1%-
0.4%；The final concentration of 0.003%-0.015% of people's SAA monoclonal antibody of label；People's SAA polyclonal antibody of label is dense eventually
Degree is 0.025%-0.085%；
Optionally, only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA monoclonal antibody are 240nm, dense eventually
Degree is 0.02%；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 123nm, dense eventually
Degree is 0.2%；People's SAA monoclonal antibody final concentration of 0.0065% of label；People's SAA polyclonal antibody of label is final concentration of
0.04%；
Or only marking the Average Particle Diameters of the carboxylated latex microballoon of someone SAA monoclonal antibody is 273nm, it is final concentration of
0.01%；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 70nm, final concentration of
0.3%；People's SAA monoclonal antibody final concentration of 0.005% of label；People's SAA polyclonal antibody of label is final concentration of
0.065%；
Or only marking the Average Particle Diameters of the carboxylated latex microballoon of someone SAA monoclonal antibody is 223nm, it is final concentration of
0.015%；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 83nm, final concentration of
0.25%；People's SAA monoclonal antibody final concentration of 0.0072% of label；People's SAA polyclonal antibody of label is final concentration of
0.055%；
Or only marking the Average Particle Diameters of the carboxylated latex microballoon of someone SAA monoclonal antibody is 240nm, it is final concentration of
0.015%；Only the Average Particle Diameters of the carboxylated latex microballoon of label someone SAA polyclonal antibody are 70nm, final concentration of
0.3%；People's SAA monoclonal antibody final concentration of 0.006% of label；People's SAA polyclonal antibody of label is final concentration of
0.075%.
3. human serum amyloid A assay kit according to claim 1, it is characterised in that: only label someone SAA
The carboxylated latex microballoon of monoclonal antibody and the carboxylated latex microspheres quality ratio for only marking someone SAA polyclonal antibody are 0.03-
0.4:1。
4. human serum amyloid A assay kit according to claim 1, it is characterised in that: include increasing in reagent 1
Quick dose；Optionally, the sensitizer include: in glucan, polyvinylpyrrolidone, ficoll 400 or poly-D-lysine extremely
Few one kind；Still optionally further, dextran molecule amount Mw include 5000,25000,40000,80000,250000,500000,
At least one of 2000000；The molecular weight Mw of polyvinylpyrrolidone include in 8000,10000,24000,58000 extremely
Few one kind；Poly-D-lysine molecular weight Mw includes at least one of 2000 or 5000；
It and/or include coagulant in reagent 1；Optionally, the coagulant includes in PEG6000, PEG8000 or PEG20000
At least one；
And/or reagent 2 further includes stabilizer；Optionally, the stabilizer includes at least one of protein, PEG or sugar；
Still optionally further, the protein includes at least one of BSA, gelatin or casein；The sugar includes sucrose, trehalose
Or at least one of glucose；
And/or people SAA concentration gradient in calibration object are as follows: 0.00,15.00,40.00,120.00,250.00,500.00mg/L
mg/L；
And/or the matrix liquid of calibration object includes following components: phosphate buffer, EDETATE SODIUM or dipotassium EDTA, citric acid
Sodium, heparin sodium or heparin lithium, trehalose, BSA, sodium chloride, potassium chloride；Optionally, the matrix liquid of calibration object include following ends it is dense
The component of degree: the pH value of 10-50mM is the phosphate buffer of 7.2-7.4, EDETATE SODIUM or dipotassium EDTA, the 1%- of 2-10mM
5% sodium citrate, the heparin sodium of 0.02%-0.2% or heparin lithium, the trehalose of 1%-5%, 0.2%-1% mannitol,
The potassium chloride of the sodium chloride of BSA, 100-300mM of 0.1%-2%, 50-150mM.
5. human serum amyloid A assay kit according to claim 3, it is characterised in that:
Reagent 2 includes following components: only the carboxylated latex microballoon of label someone SAA monoclonal antibody, only label someone SAA Dan Ke
Carboxylated latex microballoon, buffer, surfactant, inorganic salts and the preservative of grand antibody；Optionally, reagent 2 includes following ends
The component of concentration: carboxyl glue that 0.005%-0.1% only marks someone's SAA monoclonal antibody, that average grain diameter is 150-300nm
Newborn microballoon, people's SAA monoclonal antibody of the label of 0.003%-0.015%, 0.1%-0.4% only mark someone SAA polyclonal
Antibody, average grain diameter be 60-140nm carboxylated latex microballoon, people's SAA Anti-TNF-α of the label of 0.025%-0.085%
Body, the stabilizer of 0.2%-5%, the buffer of 10-100mM, the surfactant of 0.05%-0.3%, 50-600mM's is inorganic
The preservative of salt and 0.05%-0.1%；
And/or reagent 1 includes following components: buffer, BSA, surfactant, EDTA, inorganic salts and preservative；Optionally,
It include the component of following final concentrations: the increasing of the buffer of 10-100mM, the coagulant of 1.0%-10%, 0.2%-8% in reagent 1
Quick dose, the surfactant of BSA, 0.05%-0.3% of 0.5%-5%, 1-10mM EDTA, 50-600mM inorganic salts and
The preservative of 0.05%-0.1%.
6. human serum amyloid A assay kit according to claim 5, it is characterised in that: reagent 1 or reagent 2
In buffer include: independently of each other pH value be the MES buffer of 5.0-9.0, phosphate buffer, MOPS buffer,
At least one of TAPS buffer, borate buffer solution, Tris-Cl buffer and HEPES buffer solution；
And/or the surfactant in reagent 1 or reagent 2 includes: Tween-20, Tween-80 or Triton independently of each other
At least one of X-100；
And/or the preservative in reagent 1 or reagent 2 include: independently of each other ProClin300, merthiolate, sodium benzoate,
At least one of Krovin300 or Krovin 500.
7. human serum amyloid A assay kit according to claim 1, it is characterised in that: the people SAA Dan Ke
Grand antibody is the specific monoclonal antibody derived from mouse；The people SAA polyclonal antibody is to resist derived from the specificity of rabbit or sheep more；
And/or the carboxylated latex microballoon is polystyrene carboxylated latex microballoon.
8. human serum amyloid A assay kit according to claim 1, it is characterised in that: the human serum forms sediment
Powder sample albumin A assay kit is used for the detection of serum and blood plasma.
9. a kind of preparation method of human serum amyloid A assay kit, includes the following steps: using claim 1-8
The formula of described in any item human serum amyloid A assay kits prepares human serum amyloid A assay kit.
10. a kind of preparation method of human serum amyloid A assay kit, including using people's blood described in claim 5
The step of formula of clear amyloid A assay kit prepares human serum amyloid A assay kit, the step packet
It includes:
Reagent preparation 1: in the buffer of Suitable ionic strengths be added sensitizer, coagulant, BSA, surfactant, EDTA,
Inorganic salts, preservative mix well preparation；
Reagent preparation 2: people SAA is marked with the monoclonal antibody of carboxylated latex microballoon label people SAA, with carboxylated latex microballoon respectively
Polyclonal antibody, add the buffer comprising suitable concentration, inorganic salts, stabilizer, surfactant and preservative；
It prepares calibration object: being diluted people SAA with the matrix liquid prepared, assignment obtains the calibration object of series of concentrations gradient after tracing to the source；
Optionally, the preparation step of reagent 2 includes: to dilute the carboxylated latex microballoon of corresponding partial size to its mass concentration with activating solution
For 0.5%-3%, activator EDC (1- (3- dimethylamino-propyl) -3- ethyl carbon that mass concentration is 0.01%-0.1% is added
Diimmonium salt hydrochlorate), it is stirred at room temperature, activated carboxyl；After activation, centrifugation, washing remove extra EDC；Add label
Buffer, ultrasound disperse latex microsphere, are separately added into SAA monoclonal antibody, SAA polyclonal antibody separate marking, room temperature is stirred
It mixes, adds terminate liquid and continue to stir；By final reaction solution centrifugation, washing, it is micro- that the latex marked is resuspended respectively with preservation liquid
The greater particle size microballoon for marking monoclonal antibody is mixed to obtain reagent 2 with the more anti-relatively small particle microballoons of label by ball in proportion；The activation
Liquid include: the 0.01-0.2M that pH value is 4.0-6.0 MES buffer, phosphate buffer or HEPES slowly in fliud flushing extremely
Few one kind；The label buffer include the phosphate buffer of 0.01-0.2M that pH value is 7.0-8.4, MOPS buffer,
At least one of TAPS buffer, borate buffer solution or Tris-Cl buffer.
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Application publication date: 20190416 |