CN106568978A - Serum amyloid protein A detection method and reagent - Google Patents
Serum amyloid protein A detection method and reagent Download PDFInfo
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- CN106568978A CN106568978A CN201610979683.9A CN201610979683A CN106568978A CN 106568978 A CN106568978 A CN 106568978A CN 201610979683 A CN201610979683 A CN 201610979683A CN 106568978 A CN106568978 A CN 106568978A
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- 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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Abstract
The invention discloses a serum amyloid protein A detection method. The serum amyloid protein A detection method comprises following steps: serum SAA antigen of a tested body is mixed with a latex cross-linked substance coated with SAA antibody, in vitro agglutination reaction is carried out, and quantitative determination of the content of SAA in serum is carried out. The invention also discloses a serum amyloid protein A detection reagent. The serum amyloid protein A detection method and the reagent possesses following advantages: linearity range is wide, detection is rapid, detection sensitivity is high, accuracy is high, cost is low, and the reagent is stable.
Description
Technical field
The present invention relates to detectable technical field, more particularly, to a kind of serum amyloid A protein detection method.This
It is bright to further relate to a kind of serum amyloid A protein detectable.
Background technology
Serum amyloid A protein (serum amyloid A protein, SAA) is the polymorphic of class polygenes coding
Protein family, organizes the precursor substance of amyloid A, belongs to Acute reaction protein.Inflammation or disease infection period SAA exist
Raise rapidly in 48～72h, 1000 times of initial concentration can be increased to, and decline (Urieli rapidly in Recovery Phase of diseases
Shoval S, et, al.Expression and function of serum amyloid A, a major acutephase
Protein, in normal and disease states [J] .Curr Opin Hematol, 2000,7 (1):64-69.).
SAA is raised in virus and bacterium infection, and CRP is hardly raised or raised not substantially in virus infection, and both combine inspection
Survey contributes to discriminating bacteria infection and virus infection.Yang Lanhui etc. research show that hand-foot-mouth disease infant SAA is significantly raised, and with disease
The order of severity of feelings is proportionate, and (Yang Lanhui waits the application of .C- reactive proteins and amyloid A in hand-foot-mouth disease diagnosis and treatment
Research, laboratory medicine and clinical, 2012,11 (22), 2838-2839).Additionally, monitoring SAA/CRP odds ratios individually detect SAA
Or CRP has bigger using value.SAA suffers from kinds of tumors such as pulmonary carcinoma, hepatocarcinoma, breast carcinoma, carcinoma of prostate, carcinomas of endometrium
There is rising in various degree in person's body, and its level has obvious related to the active stage of tumor, grade malignancy and Invasion and Metastasis
Property, reflect the curative effect and prognosis (Cocco E, et al.Serumamyloid A of tumor patient:a novel biomarker
For endometrial cancer [J] .Cancer, 2010,116 (4):843-851.).SAA levels also with amyloidosis
Disease, coronary heart disease, rejection (Muller T, et al.Prospective analysis of 10different
Parameters of acute renal allograft rejection [J] .Transplant Proc, 1992,24 (6):
2731-2734.) etc. disease (Chen Changqiang, etc. progress [J] of the serum amyloid A protein in disease application. inspection doctor
Learn, 2012,27 (9):It is 776-779.) closely related, provide more preferable reference information for clinical diagnosises.
Being presently used for the method for clinical serum SAA detections mainly includes that Colloidal Gold, latex strengthen rate scattering ratio
Turbid method, microsphere capture enzyme immunoassay (MEIA), elisa (ELISA), latex enhancing immune turbidimetry etc..Latex
Enhancing rate nephelometry can only be used on supporting specific protein analyser and testing cost is of a relatively high, immune colloid
Golden method can only be supported the use with the digital quantitative analysis instrument of gold mark, it is impossible to meet the requirement of automatization's rapid batch detection, nearly 2 years
Latex enhancing immune turbidimetry disclosed in Patents, using polyclonal antibody latex intensified method, it is adaptable to full-automatic biochemical point
Analyzer, but its range of linearity is narrower, it is cumbersome if high concentration SAA sample is run in clinical practice need to constantly dilute.
The content of the invention
It is an object of the invention to provide a kind of serum amyloid A protein detection method, it has can simultaneously meet linear
Wide and automatization's rapid batch detection requirement, with good accuracy, precision, anti-interference and sensitivity, Neng Gouman
The characteristics of sufficient Clinical Laboratory is required.The invention also discloses a kind of serum amyloid A protein detectable.
First technical scheme of the present invention be：
Serum amyloid A protein detection method, including：The serum S AA antigens of subject and SAA antibody will be coated with
Latex cross-linking agent mixes, and agglutination, SAA contents in quantitative determination serum are carried out in vitro.
The SAA antigens occur in the liquid phase to coagulate with the latex cross-linking agent of SAA antibody is coated with after corresponding dilution
Collection reaction, the liquid phase environment pH is 6.0-9.0, temperature is 15 DEG C -40 DEG C.
The detecting instrument that the detection method is adopted for：Automatic biochemistry analyzer；
Analysis method is：Two point end assay；
The Direction of Reaction is：Rise reaction；
Calibrating mode is：Spline；
Determining wavelength is：546nm；
Temperature of the measurement is：37℃；
Sample：R1：R2=8 μ l:250μl:50μl；
Step is：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagent R2 are added immediately,
The 1st reading point absorbance A 1 is read, 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing, calculate A2-A1 differences,
Draw serum amyloid A protein content in sample.
Second technical scheme of the present invention be：
Serum amyloid A protein detectable, including reagent R1, reagent R2,
Reagent R1 includes：The buffer that pH is 6.5-8.5, concentration is 10-100mmol/L, mass percent is 0.01%-
2.00% surfactant, mass percent is the reaction promoter of 0.01%-5.00%, and mass percent is 0.05%-
3.00% electrolyte, mass percent is the preservative of 0.01%-1.00%, and mass percent is 0.01%-
4.00% stabilizer；
Reagent R2 includes：Mass percent is the surfactant of 0.05%-3.00%, and mass percent is 0.02%-
12.00% stabilizer, mass percent is 0.01%-1.00% preservative, and mass percent is the electricity of 0.05%-3.00%
The buffer that Xie Zhi, pH are 6.5-8.5, concentration is 10-100mmol/L, latex cross-linking agent；And
The latex cross-linking agent is to be marked with the big latex particle of SAA polyclonal antibodies and be marked with SAA monoclonal antibodies
Little latex particle.
The SAA polyclonal antibodies are the complete antibody containing functional part or antibody fragment, selected from rabbit-anti people's polyclone
Antibody or sheep anti-human polyclonal antibody；The SAA monoclonal antibodies are the complete antibody containing functional part, antibody fragment or weight
Group antibody.
The big latex particle, its particle diameter is 110-380nm, and mass percent is 0.01%-0.06%, the little latex
Granule, its particle diameter is 20-120nm, and mass percent is 0.10%-0.50%, big latex particle in the reagent R2 and little
The mixed proportion of latex particle is 1:2～1:16.
The buffer is glycine buffer, Tris buffer, HEPES buffer solution, borate buffer solution, phosphoric acid buffer
One kind in liquid.
The surfactant be nonionic surfactant, the nonionic surfactant be Tween20,
At least one in Tween80, Emulgen B-66, Triton X-100.
The reaction promoter be Macrogol 2000, Macrogol 4000, Macrogol 8000, bromination second diformazan ammonia,
At least one in polybrene.
The electrolyte is one or more in positive valence metal ion, and the preservative is sodium azide, nitrine lithium, benzene first
At least one in acid and its esters, sorbic acid salt, sodium nitrite, PC300, the stabilizer is protein, metal complex
At least one in agent, suspending agent, the protein is at least one in bovine serum albumin or gelatin, the metal chelating agent
For at least one in HEDTA, CMOM, CMOS, the suspending agent includes at least in ethylene glycol, Lactose, sucrose, glycerol
Kind.
The serum amyloid A protein detection method and reagent place of the present invention has the advantage that as follows：
1st, range of linearity width：For 5-500mg/L, Healthy People term of reference is≤10mg/L, substantially meets clinical needs；
2nd, detect quick：Applicable automatic biochemistry analyzer, meets batch detection demand, detects fast and convenient；
3rd, detection sensitivity is high：The proportioning of the compositions such as reagent buffer, interfacial agent improves the sensitivity of reaction；
4th, accuracy is high：Using the reagent proportioning of optimization, and trace to the source to International Reference material NIBSC 92/680, it is ensured that survey
Determine the accuracy of result；
5th, low cost：0.9988 is reached with the dependency of import reagent, alternative import reagent can be reduced into after market
Use cost；
6th, stable reagent：The each composition proportion optimization of reagent, reagent can be stablized 18 months under 2-8 DEG C of airtight condition.
Description of the drawings
With reference to the accompanying drawings and examples the present invention is further described：
Fig. 1 is the operating procedure of the serum amyloid A protein detection method of the present invention；
Fig. 2 is SAA reagents of the present invention and Siemens's contrast agent/method dependency；
Fig. 3 is that the SAA reagents range of linearity of the present invention is determined；
Fig. 4 is the traceability of SAA of the present invention.
Specific embodiment
Embodiment 1
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 6.5, concentration be 100mmol/L Tris buffer, surfactant is 0.01% for mass percent
Tween20, reaction promoter is the Macrogol 2000 that mass percent is 5.00%, and electrolyte is for mass percent
1.20% Sodium Chloride, preservative is the sodium azide that mass percent is 0.20%, and it is 0.90% that stabilizer is mass percent
Bovine serum albumin.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:2, the percentage composition of big latex particle is 0.06%,
SAA rabbit anti-human polyclonal antibodies are coated with, size is 110nm, the percentage composition of little latex particle is 0.12%, is coated with
SAA monoclonal antibodies, size is 120nm；Surfactant is Tween20 that mass percent is 0.05%；Stabilizer
For the bovine serum albumin that mass percent is 0.20%；Preservative is sodium azide that mass percent is 0.90%；Electrolyte is
Mass percent is 1.20% Sodium Chloride；Buffer for pH value be 6.5, concentration be 100mmol/L Tris buffer.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 2
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 8.5, concentration be 100mmol/L glycine buffer, surfactant be mass percent be 2.00%
Tween80, reaction promoter is the Macrogol 4000 that mass percent is 0.01%, and electrolyte is for mass percent
0.01% magnesium chloride, preservative is the nitrine lithium that mass percent is 0.01%, and it is 0.01% that stabilizer is mass percent
Gelatin.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:16, the percentage composition of big latex particle is
0.01%, SAA rabbit anti-human polyclonal antibodies are coated with, size is 380nm, and the percentage composition of little latex particle is
0.16%, SAA monoclonal antibodies are coated with, size is 20nm；Surfactant is 3.00% for mass percent
Tween80；Stabilizer is gelatin that mass percent is 0.10%；Preservative is nitrine lithium that mass percent is 0.01%；
Electrolyte is potassium chloride that mass percent is 0.05%；Buffer is that the glycine that 8.5, concentration is 10mmol/L delays for pH value
Rush liquid.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 3
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 7.0, concentration be 50mmol/L HEPES buffer solution, surfactant is 0.50% for mass percent
Emulgen B-66, reaction promoter is the Macrogol 8000 that mass percent is 2.50%, and electrolyte is mass percent
For 1.50% potassium chloride, preservative is benzoic acid that mass percent is 1.00% and salt, and stabilizer is mass percent
Gelatin (2.00%) and bovine serum albumin (2.00%) mixture for 4.00%.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:8, the percentage composition of big latex particle is 0.03%,
SAA sheep anti-human polyclonal antibodies are coated with, size is 200nm, the percentage composition of little latex particle is 0.24%, is coated with
SAA monoclonal antibodies, size is 100nm；Surfactant is Emulgen B-66 that mass percent is 1.50%；Surely
Agent is determined for HEDTA that mass percent is 0.02%；Preservative is benzoic acid that mass percent is 0.50% and its esters；Electricity
Solution matter is magnesium chloride that mass percent is 1.50%；Buffer for pH value be 7.0, concentration be 50mmol/L borate buffer
Liquid.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 4
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 7.5, concentration be 60mmol/L borate buffer solution, surfactant is 1.00% for mass percent
Tritonx-100, reaction promoter is the bromination second diformazan ammonia that mass percent is 1.00%, and electrolyte is for mass percent
3.00% Sodium Chloride (1.50%) and magnesium chloride (1.50%) mixture, preservative is Pyrusussuriensiss that mass percent is 0.50%
Barbiturates, stabilizer is HEDTA that mass percent is 2.00%.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:5, the percentage composition of big latex particle is 0.02%,
SAA sheep anti-human polyclonal antibodies are coated with, size is 160nm, the percentage composition of little latex particle is 0.10%, is coated with
SAA monoclonal antibodies, size is 80nm；Surfactant is Tritonx-100 that mass percent is 2.00%；It is stable
Agent is CMOS that mass percent is 1.00%；Preservative is sorbic acid salt that mass percent is 1.00%；Electrolyte is
Mass percent is 0.10% Sodium Chloride；Buffer for pH value be 7.5, concentration be 20mmol/L HEPES buffer solution.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 5
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 8.0, concentration be 70mmol/L phosphate buffer, surfactant is 1.60% for mass percent
Tritonx-100 (0.80%) and Tween20 (0.80%) mixture, reaction promoter for mass percent be 3.00% it is poly-
Solidifying amine, electrolyte is the Sodium Chloride that mass percent is 2.00%, and preservative is the sodium nitrite that mass percent is 0.07%,
Stabilizer is CMOM that mass percent is 3.00%.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:10, the percentage composition of big latex particle is
0.05%, SAA sheep anti-human polyclonal antibodies are coated with, size is 320nm, and the percentage composition of little latex particle is
0.50%, SAA monoclonal antibodies are coated with, size is 40nm；Surfactant is 3.00% for mass percent
Tween20 (1.50%) and Tritonx-100 (1.50%)；Stabilizer is bovine serum albumin that mass percent is 2.00%
And gelatin mixture (1.00%) (1.00%)；Preservative is sodium nitrite that mass percent is 0.05%；Electrolyte is matter
Amount percentage ratio is 0.30% potassium chloride；Buffer for pH value be 7.0, concentration be 30mmol/L phosphate buffer.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 6
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 6.5, concentration be 80mmol/L Tris buffer, surfactant is 0.80% for mass percent
Tween80, reaction promoter is polybrene (1.00%) that mass percent is 2.00% and Macrogol 2000 (1.00%)
Mixture, electrolyte is the Sodium Chloride that mass percent is 3.00%, and preservative is the PC300 that mass percent is 1.00%,
Stabilizer is CMOS that mass percent is 1.50%.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:4, the percentage composition of big latex particle is 0.04%,
SAA rabbit anti-human polyclonal antibodies are coated with, size is 250nm, the percentage composition of little latex particle is 0.16%, is coated with
SAA monoclonal antibodies, size is 60nm；Surfactant is Tween20 that mass percent is 0.10%；Stabilizer is
Mass percent is 2.50% CMOM；Preservative is PC300 that mass percent is 0.10%；Electrolyte is mass percent
Sodium Chloride (0.20%) and magnesium chloride (1.20%) mixture for 0.40%；It is that 8.0, concentration is that buffer is pH value
The Tris buffer of 30mmol/L.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 7
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 7.0, concentration be 50mmol/L glycine buffer, surfactant is 1.20% for mass percent
Tween20, reaction promoter is Macrogol 4000 (1.00%), Macrogol 2000 that mass percent is 3.00%
(1.00%) and bromination second diformazan ammonia (1.00%) mixture, electrolyte is the Sodium Chloride that mass percent is 1.50%, anti-corrosion
Agent is PC300 (0.50%) that mass percent is 1.00% and nitrine lithium (0.50%), and stabilizer is for mass percent
2.00% ethylene glycol.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:6, the percentage composition of big latex particle is 0.02%,
SAA rabbit anti-human polyclonal antibodies are coated with, size is 180nm, the percentage composition of little latex particle is 0.12%, is coated with
SAA monoclonal antibodies, size is 110nm；Surfactant is Tween80 that mass percent is 1.00%；Stabilizer
For the ethylene glycol that mass percent is 12.00%；Preservative for sodium azide (0.10%) that mass percent is 0.20% and
PC300 (0.10%) mixture；Electrolyte is Sodium Chloride that mass percent is 1.00%；It is 6.5, concentration that buffer is pH value
For the glycine buffer of 60mmol/L.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 8
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 6.5, concentration be 10mmol/L Tris buffer, surfactant is 2.00% for mass percent
Tween80, reaction promoter is the Macrogol 4000 that mass percent is 4.00%, and electrolyte is for mass percent
2.00% Sodium Chloride, preservative is the sodium azide that mass percent is 1.00%, and it is 1.00% that stabilizer is mass percent
Lactose.
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:3, the percentage composition of big latex particle is 0.05%,
SAA sheep anti-human polyclonal antibodies are coated with, size is 150nm, the percentage composition of little latex particle is 0.15%, is coated with
SAA monoclonal antibodies, size is 90nm；Surfactant is Tween80 that mass percent is 1.20%；Stabilizer is
Mass percent is 3.00% Lactose；Preservative is sodium azide that mass percent is 0.30%；Electrolyte is quality percentage
Than for 2.00% potassium chloride；Buffer for pH value be 8.5, concentration be 70mmol/L Tris buffer.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 9
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 7.0, concentration be 20mmol/L Tris buffer, surfactant is 1.50% for mass percent
Tween20, reaction promoter is the bromination second diformazan ammonia that mass percent is 2.50%, and electrolyte is for mass percent
1.00% Sodium Chloride, preservative is the sodium azide that mass percent is 0.30%, and it is 4.00% that stabilizer is mass percent
Bovine serum albumin (2.00%) and sucrose mixture (2.00%).
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:12, the percentage composition of big latex particle is
0.03%, SAA sheep anti-human polyclonal antibodies are coated with, size is 120nm, and the percentage composition of little latex particle is
0.36%, SAA monoclonal antibodies are coated with, size is 100nm；Surfactant is 3.00% for mass percent
Tween20 (1.00%), Tween80 (1.00%) and Emulgen-66 (1.00%) mixture；Stabilizer is mass percent
For 4.00% sucrose, preservative is sodium azide that mass percent is 0.40%；It is 0.40% that electrolyte is mass percent
Sodium Chloride；Buffer for pH value be 7.0, concentration be 80mmol/L Tris buffer.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Embodiment 10
Serum amyloid A protein detectable, including reagent R1, reagent R2.Concretely：
Reagent R1 includes：Buffer, surfactant, reaction promoter, electrolyte, preservative, stabilizer.Wherein：It is slow
Rush liquid for pH value be 8.5, concentration be 30mmol/L HEPES buffer solution, surfactant is 0.60% for mass percent
Tween80, reaction promoter is the Macrogol 2000 that mass percent is 0.05%, and electrolyte is for mass percent
0.10% potassium chloride, preservative is the nitrine lithium that mass percent is 0.05%, and it is 3.00% that stabilizer is mass percent
Gelatin (1.00%), HEDTA (1.00%) and glycerol mixture (1.00%).
Reagent R2 includes：Latex cross-linking agent, surfactant, stabilizer, preservative, electrolyte, buffer.Wherein：Glue
The mixed proportion of big latex particle and little latex particle is 1 in newborn cross-linking agent:11, the percentage composition of big latex particle is
0.02%, SAA rabbit anti-human polyclonal antibodies are coated with, size is 300nm, and the percentage composition of little latex particle is
0.22%, SAA monoclonal antibodies are coated with, size is 30nm；Surfactant is 1.60% for mass percent
Tween80；Stabilizer is bovine serum albumin (2.00%), CMOS (2.00%) and glycerol that mass percent is 6.00%
(2.00%) mixture；Preservative is nitrine lithium that mass percent is 0.60%；It is 1.50% that electrolyte is mass percent
Sodium Chloride；Buffer for pH value be 8.5, concentration be 30mmol/L HEPES buffer solution.
As shown in Figure 1, the side of serum amyloid A protein detection is carried out using aforementioned serum amyloid A protein detectable
Method is：
Analysis method：Two point end assay；The Direction of Reaction：Rise reaction；Calibrating mode：Spline；Determine wavelength：546nm；
Temperature of the measurement：37℃；Sample：R1：R2=8 μ l:250μl:50μl.
Operational approach：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagents are added immediately
R2, reads the 1st reading point absorbance A 1, and 37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing.Calculate A2-A1 poor
Value, according to calibration curve serum amyloid A protein content in sample is calculated.
Anti-interference is tested
Using two people's whole blood samples that SAA contents are normal and abnormal, the interfering material of different content is separately added into, is adopted
Reagent R1, R2 of the proportioning of embodiment 3 is measured (remaining embodiment reagent equally has similar experimental result), interference effect
As a result it is as follows：
When SAA concentration be 10mg/L, bilirubin≤60mg/dL, fat milk≤500mg/dL, hemoglobin≤750mg/
dL。
When SAA concentration be 40mg/L, bilirubin≤60mg/dL, fat milk≤500mg/dL, hemoglobin≤750mg/
dL。
Effect example
1. reagent of the present invention compares with other brand reagents
Reagent of the present invention and certain producer's reagent carry out performance com-parison and analysis, are respectively compared its precision, stability, effectively
Phase, dependency etc..Precision is determined：(using reagent R1, R2 of the proportioning of embodiment 1, remaining embodiment reagent is same for reagent of the present invention
Sample has similar experimental result) serum-concentration normal (≤10mg/L) and two abnormal samples are determined respectively with certain producer's reagent
This, withinrun precision is by replication in 1h 20 times when result is calculated；Betweenrun precision by determining 1 time respectively daily, continuously
20d is determined, betweenrun precision is calculated.Relativity determination：Reagent of the present invention (using reagent R1, R2 of the proportioning of embodiment 2, remaining
Embodiment reagent equally has similar experimental result) and certain producer's reagent compare with Siemens SAA reagents respectively, as a result such as
Shown in table 1.
The reagent of the present invention of table 1 compares with other brand reagents
2. the inventive method compares with additive method
300 serum samples of certain hospital laboratory are randomly selected, the inventive method (latex enhancing immune ratio is respectively adopted
Turbid method, (using reagent R1, R2 of the proportioning of embodiment 4, remaining embodiment reagent equally has similar experimental result)) and immunity
Scattered light urbidmetry (Siemens Process), as a result such as following table, no significant difference (P ＞ 0.05) between two methods,
As shown in table 2.Two methods correlation coefficient (r)=0.998 can be obtained by Fig. 2.
The comparison (mg/L) of the two methods SAA testing result of table 2
As shown in Figure 3, Jing tests, the range of linearity width of SAA of the present invention, are 5-500mg/L, Healthy People term of reference for≤
10mg/L, substantially meets clinical needs.
Accuracy is tested
As shown in Figure 4, using the reagent proportioning of optimization, and trace to the source to International Reference material NIBSC 92/680, it is ensured that
The accuracy of measurement result.
The preferred embodiments of the present invention are the foregoing is only, the scope of the claims of the present invention, every utilization is not thereby limited
Equivalent structure or equivalent flow conversion that description of the invention and content are made, or directly or indirectly it is used in other related skills
Art field, is included within the scope of the present invention.
Claims (10)
1. serum amyloid A protein detection method, including：By the Serum SA Staphylococal Protein A of subject and the latex for being coated with SAA antibody
Cross-linking agent mixes, and agglutination, SAA contents in quantitative determination serum are carried out in vitro.
2. the detection method of serum amyloid A protein according to claim 1, it is characterised in that：The SAA antigens and bag
There is in the liquid phase agglutination in the latex cross-linking agent for being had SAA antibody, the liquid phase environment pH is after corresponding dilution
6.0-9.0, temperature are 15 DEG C -40 DEG C.
3. the detection method of serum amyloid A protein according to claim 1 and 2, it is characterised in that：
The detecting instrument that the detection method is adopted for：Automatic biochemistry analyzer；
Analysis method is：Two point end assay；
The Direction of Reaction is：Rise reaction；
Calibrating mode is：Spline；
Determining wavelength is：546nm；
Temperature of the measurement is：37℃；
Sample：R1：R2=8 μ l:250μl:50μl；
Step is：8 μ l samples and 250 μ l reagents R1 are mixed and are incubated 3-5min after 37 DEG C, and 50 μ l reagent R2 are added immediately, are read
1st reading point absorbance A 1,37 DEG C of incubation 5min, read the 2nd reading point absorbance A 2 after mixing, calculate A2-A1 differences, draw
Serum amyloid A protein content in sample.
4. serum amyloid A protein detectable, including reagent R1, reagent R2, it is characterised in that：
Reagent R1 includes：The buffer that pH is 6.5-8.5, concentration is 10-100mmol/L, mass percent is 0.01%-
2.00% surfactant, mass percent is the reaction promoter of 0.01%-5.00%, and mass percent is 0.05%-
3.00% electrolyte, mass percent is the preservative of 0.01%-1.00%, and mass percent is 0.01%-
4.00% stabilizer；
Reagent R2 includes：Mass percent is the surfactant of 0.05%-3.00%, and mass percent is 0.02%-
12.00% stabilizer, mass percent is 0.01%-1.00% preservative, and mass percent is the electricity of 0.05%-3.00%
The buffer that Xie Zhi, pH are 6.5-8.5, concentration is 10-100mmol/L, latex cross-linking agent；And
The latex cross-linking agent is the big latex particle for being marked with SAA polyclonal antibodies and the little glue for being marked with SAA monoclonal antibodies
The mixture of newborn granule.
5. serum amyloid A protein detectable according to claim 4, it is characterised in that：The SAA polyclonal antibodies
It is the complete antibody containing functional part or antibody fragment, selected from rabbit anti-human polyclonal antibody or sheep anti-human polyclonal antibody；Institute
It is the complete antibody containing functional part, antibody fragment or recombinant antibodies to state SAA monoclonal antibodies.
6. serum amyloid A protein detectable according to claim 4, it is characterised in that：The big latex particle, its
Particle diameter is 110-380nm, and mass percent is 0.01%-0.06%, the little latex particle, its particle diameter be 20-120nm, matter
Amount percentage ratio is 0.10%-0.50%, and the mixed proportion of big latex particle and little latex particle in the reagent R2 is 1:2～
1:16。
7. serum amyloid A protein detectable according to claim 4, it is characterised in that：The buffer is sweet ammonia
One kind in acid buffer, Tris buffer, HEPES buffer solution, borate buffer solution, phosphate buffer.
8. serum amyloid A protein detectable according to claim 4, it is characterised in that：The surfactant is
Nonionic surfactant, the nonionic surfactant be Tween20, Tween80, Emulgen B-66,
At least one in Triton X-100.
9. serum amyloid A protein detectable according to claim 4, it is characterised in that：The reaction promoter is
At least one in Macrogol 2000, Macrogol 4000, Macrogol 8000, bromination second diformazan ammonia, polybrene.
10. serum amyloid A protein detectable according to claim 4, it is characterised in that：The electrolyte is nominal price
One or more in metal ion, the preservative is sodium azide, nitrine lithium, benzoic acid and its esters, sorbic acid salt, Asia
At least one in sodium nitrate, PC300, the stabilizer is at least one in protein, metal chelating agent, suspending agent, institute
Protein is stated at least one in bovine serum albumin or gelatin, the metal chelating agent be in HEDTA, CMOM, CMOS extremely
Few one kind, the suspending agent includes at least one in ethylene glycol, Lactose, sucrose, glycerol.
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