CN106353507A - Kit for detecting serum amyloid protein and application thereof - Google Patents
Kit for detecting serum amyloid protein and application thereof Download PDFInfo
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- CN106353507A CN106353507A CN201610711795.6A CN201610711795A CN106353507A CN 106353507 A CN106353507 A CN 106353507A CN 201610711795 A CN201610711795 A CN 201610711795A CN 106353507 A CN106353507 A CN 106353507A
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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 provides a kit for detecting serum amyloid protein and application thereof. The kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is prepared from a buffer solution, inorganic salt, a surfactant, a preservative, a stabilizer and interference elimination protein; the reagent R2 is prepared from the buffer solution, the inorganic salt, the surfactant, the preservative, the stabilizer and a polystyrene latex particle mixture; the polystyrene latex particle mixture is cross-linked with an SAA (Serum Amyloid A) antibody; the polystyrene latex particle mixture is a mixture of large-diameter polystyrene latex particles and small-diameter polystyrene latex particles. The kit disclosed by the invention is based on PETIA (Particle-enhanced Turbidimetric Immunoassay) and can be generally used for analysis of all kinds of full-automatic biochemical analyzer; during use, the required determining time is short, the specificity is high, the precision degree is high, and the accuracy degree is high.
Description
Technical field
The invention belongs to immunologic diagnosises field is and in particular to a kind of test kit of detection serum amyloid protein and its use
On the way.
Background technology
Serum amyloid protein a (serum amyloid a, saa) is a kind of Acute reaction protein, belongs to load fat egg
Heterogeneous proteinoid in white family, relative molecular mass is about 12000.In acute-phase response, through interleukin
(interleukin, il) and tumor necrosis factor (tumor necrosis factors, tnf) stimulate, saa in liver by
The macrophage being activated and fibroblast synthesis, can be increased to 100~1000 times of initial concentration.With c reactive protein (c-
Reactive protein, crp) it is similar to, saa is the sensitive indicator of reflection infectious disease Earlier period of inflammation, contributes to diagnosis scorching
Disease, assess its activity, monitor its activity and treatment.
At present, existing many methods are applied to the detection of serum saa, inhale including radioimmunoassay detection method, enzyme linked immunological
Adhesion test, immune rate nephelometry and microparticle capture enzyme immunoassay etc..These methods have preferable sensitivity and special
Property, can be used for automated analysiss.But, the saa test kit examined by Chinese food Drug Administration (cfda) at present
Only minority producer, and detection range, sensitivity, the standard such as specificity not high it is difficult to meet the market demand.
Content of the invention
For the problems referred to above, present invention is primarily targeted at provide a kind of test kit of detection serum amyloid protein and
Its purposes, based on Latex-enhanced immunoturbidimetric assay (petia), can be commonly used to all kinds of automatic clinical chemistry analyzer analyses,
During use, required minute is short, and specificity is high, and precision is good, and accuracy is high.
In order to achieve the above object, the present invention adopts the following technical scheme that a kind of reagent of detection serum amyloid protein
Box, including reagent r1 and reagent r2, described reagent r1 includes buffer, inorganic salt, surfactant, preservative, stabilizer and
Interference eliminates albumen;Described reagent r2 includes buffer, inorganic salt, surfactant, preservative, stabilizer and polystyrene colloidal
Newborn granulate mixture;Described polystyrene latex particles mixture is crosslinked with antiserum amyloid saa antibody;
Described polystyrene latex particles mixture is major diameter polystyrene latex particles and minor diameter polystyrene colloidal
The mixture of newborn granule, described major diameter polystyrene latex particles mean diameter is 180nm~500nm, and described minor diameter gathers
Styrene latex mean diameter is 32nm~82nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles
=1:(2-4).
As further preferably, described major diameter polystyrene latex particles mean diameter is 180nm, described minor diameter
Polystyrene latex mean diameter is 82nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=1:
2.
As further preferably, described polystyrene latex particles mixture crosslinking antiserum amyloid saa resists
The method adopting during body comprises the steps:
(1) carboxylated major diameter polystyrene latex particles and minor diameter polystyrene latex are cleaned in buffer
Grain, obtains present latex particulate with described buffer is resuspended;
(2) add 1- ethyl-(3- dimethylamino-propyl) phosphinylidyne diimmonium salt hydrochlorate edc and n- in described present latex particulate
Hydroxy thiosuccinimide sulfo-nhs, mixing makes dissolving, room temperature reaction, with the reacted described microgranule of buffer solution for cleaning,
Resuspended with described buffer;
(3) adopt buffer solution antiserum amyloid saa antibody;
(4) mixed by the described microgranule obtaining through step (2) with through the described antibody that step (3) obtains, room temperature is anti-
Should;
(5) use described microgranule after step (4) process for the buffer solution for cleaning, and be suspended in the coupling containing hydrophilic quenching molecules
Buffer is to close excessive reaction site;
(6) the described microgranule after cleaning is processed through step (5), resuspended in buffer, obtain the latex being coupled antibody
Granule.
As further preferred, in described step (1), add dispersant when described resuspended to prevent agglomerate, described
Dispersant is anion surfactant.
As further preferably, described dispersant is 0.005%sds (sodium lauryl sulphate) or las (straight chained alkyl
Benzene sulfonic acid sodium salt).
As further, preferably, in described step (2), the concentration of described edc and sulfo-nhs is respectively
20mg/ml and 15mg/ml-18mg/ml.
As further preferred, in described step (2), room temperature reaction 10-20min, in described step (4), room temperature is anti-
Answer 2-4 hour.
As further preferably, in described step (4), described hydrophilic quenching molecules are ethanolamine or tris, and concentration is
100mm.
As further, preferably, described buffer is selected from 3- morpholine -2-hydroxy-propanesulfonic acid, 2- (n- morpholine) second sulphur
Acid, 4- hydroxyethyl piperazine ethanesulfonic acid and 3- [double (2- ethoxy) amino of n-n-] -2- hydroxy-propanesulfonic acid;Described surfactant choosing
From TritonX x-100, lauric acid polyoxyethylene ester, polyoxyethylene oleic acid ester and lauryl amine polyoxyethylene ether;Described stabilizer choosing
From sucrose, metal chelating agent and antioxidant;Described interference eliminates the hbr blocker that albumen is scantibodies company;Institute
State inorganic salt and be selected from sodium chloride, potassium chloride and magnesium sulfate;Described preservative is selected from sodium azide, thimerosal and proclin 300.
As further, preferably, in every 1 liter of reagent r1, the consumption of each component is: buffer 7-15g, surfactant 1-
5ml, preservative 0.1-0.4ml, stabilizer 8-12g, interference eliminates albumen 1-5ml, inorganic salt 5-15g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: it is coated the latex particle 0.5-5g of saa antibody, buffer 5-20g,
Surfactant 1-4ml, preservative 0.1-0.4ml, stabilizer 5-20g, inorganic salt 8-12g.
As further, preferably, in every 1 liter of reagent r1, the consumption of each component is: buffer 9.76g, surfactant
3.6ml, preservative 0.2ml, stabilizer 10g, interference eliminates albumen 4ml, inorganic salt 9g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 2.2g of saa antibody, buffer 9.76g, table
Face activating agent 2ml, preservative 0.2ml, stabilizer 10g, inorganic salt 9g, balance of water.
A kind of purposes of the test kit of detection serum amyloid protein, for detecting serum amyloid protein.
The invention has the beneficial effects as follows:
(1) test kit of the present invention utilizes Latex-enhanced immunoturbidimetric assay to measure serum amyloid protein, is exaggerated inspection
Survey signal, improve detection sensitivity, shorten detection time.
(2) present invention is big using the emulsion reagent of mixing particle diameter, wherein the latex particle ratio of small particle, improves line
Property;Contain the latex particle of a certain proportion of big particle diameter, sensitivity is also higher simultaneously.
(3) test kit of the present invention has been specifically added the material preventing disturbing, good in anti-interference performance.Meanwhile, surfactant
Interpolation also improve precision.
(4) use automatic clinical chemistry analyzer to measure serum amyloid protein, not only make operation more easy, improve automatically
Change degree, and also a saving the Financial cost of a small amount of sample detection experiment.Automatic clinical chemistry analyzer is multiple batches of to a small amount of
Sample is detected, almost identical to the cost of a large amount of sample detection with single batch;Therefore, automatic clinical chemistry analyzer can be fitted
For the detection to a small amount of sample, and do not improve Financial cost.
Brief description
Fig. 1 is that the test kit of embodiment of the present invention 1-5 detects the absorbance change curve synoptic diagram obtaining during saa sample.
Specific embodiment
The embodiment of the present invention is passed through to provide a kind of test kit of detection serum amyloid protein and application thereof, solves existing
Technology pilot agent box detection range, sensitivity, the not high defect of the standard such as specificity, test kit of the present invention is based on latex intensified
Immunity transmission turbidity (petia), can be commonly used to all kinds of automatic clinical chemistry analyzer analyses, required minute during use
Short, specificity is high, and precision is good, and accuracy is high.
In order to solve drawbacks described above, the main thought of the embodiment of the present invention is as follows:
The embodiment of the present invention detects the test kit of serum amyloid protein, including reagent r1 and reagent r2, described reagent r1
Including buffer, inorganic salt, surfactant, preservative, stabilizer and interference elimination albumen;Described reagent r2 includes buffering
Liquid, inorganic salt, surfactant, preservative, stabilizer and polystyrene latex particles mixture;Described polystyrene latex
Grain mixture is crosslinked with antiserum amyloid saa antibody;
Described polystyrene latex particles mixture is major diameter polystyrene latex particles and minor diameter polystyrene colloidal
The mixture of newborn granule, described major diameter polystyrene latex particles mean diameter is 180nm~500nm, and described minor diameter gathers
Styrene latex mean diameter is 32nm~82nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles
=1:(2-4).
The method adopting during described polystyrene latex particles mixture crosslinking antiserum amyloid saa antibody includes
Following steps:
(1) carboxylated major diameter polystyrene latex particles and minor diameter polystyrene latex are cleaned in buffer
Grain, obtains present latex particulate with described buffer is resuspended;
(2) add edc and sulfo-nhs in described present latex particulate, mix and make dissolving, room temperature reaction is clear with buffer
Wash reacted described microgranule, resuspended with described buffer;
(3) adopt buffer solution antiserum amyloid saa antibody;
(4) mixed by the described microgranule obtaining through step (2) with through the described antibody that step (3) obtains, room temperature is anti-
Should;
(5) use described microgranule after step (4) process for the buffer solution for cleaning, and be suspended in the coupling containing hydrophilic quenching molecules
Buffer is to close excessive reaction site;
(6) the described microgranule after cleaning is processed through step (5), resuspended in buffer, obtain the latex being coupled antibody
Granule.
In order to above and other purpose, feature and the advantage of the present invention can be become apparent, number cited below particularly is implemented
Example, to illustrate test kit of detection serum amyloid protein of the present invention and application thereof.
Embodiment 1
Reagent r1 consists of the following composition: buffer (mes), inorganic salt (sodium chloride), surfactant (TritonX x-
100), preservative (proclin 300), stabilizer (sucrose) and interference eliminate albumen (hbr blocker).
Reagent r2 consists of the following composition: buffer (mes), inorganic salt (sodium chloride), surfactant (TritonX x-
100), preservative (proclin 300), stabilizer (sucrose) and polystyrene latex particles mixture, described polystyrene colloidal
Newborn granulate mixture is crosslinked with antiserum amyloid saa antibody, and the latex preparation process of coupled antibody includes activating, even
Connection, blend step, specifically comprise the following steps that
1. it is respectively washed two kinds of carboxylated mean diameters of each 100mg in coupling buffer (50mm mes, ph 6.0)
For 82nm and 180nm present latex particulate, then according to the following steps coupled antibody reaction is carried out to two kinds of latex, be coupled buffering with 5ml
Liquid is resuspended, is simultaneously introduced 0.005%sds to prevent agglomerate, avoids adding containing carboxyl, the change of sulfydryl and amino in coupling buffer
Compound.
2. add the sulfo-nhs of edc and 75mg of 100mg in above-mentioned microgranule, mixing makes dissolving.
3. room temperature reaction 15 minutes.
4. use coupling buffer centrifugal method Rapid Cleaning microgranule 2 times, use 5ml coupling buffer ultrasonic resuspended afterwards.
5., in the coupling buffer of 5ml, antibody protein concentration is micro- to provide for dissolving antiserum amyloid saa antibody
The aglucon of 1 to 10 times of excess that grain monolayer surface amasss group is defined.
6. immediately antibody protein and particulate particles are mixed.
7. room temperature reaction 2-4 hour after mixing.
8. clean microgranule with coupling buffer, and be resuspended in containing 100mm contain hydrophilic quenching molecules (i.e. ethanolamine or
Tris coupling buffer) is to close excessive reaction site.
9. clean microgranule, and resuspended in suitable buffer.
10. mix, two kinds of 82nm and 180nm latex particles of antibody will be coupled, according to the mixing of 1:2 ratio;Add one
Quantitative inorganic salt, buffer, surfactant, preservative, stabilizer, obtain reagent r2.
In every 1 liter of reagent r1, the consumption of each component is: buffer 9.76g, surfactant 3.6ml, preservative 0.2ml,
Stabilizer 10g, interference eliminates albumen 4ml, inorganic salt 9g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 2.2g of saa antibody, buffer 9.76g, table
Face activating agent 2ml, preservative 0.2ml, stabilizer 10g, inorganic salt 9g, balance of water.
Embodiment 2
Reagent r1 consists of the following composition: buffer (3- morpholine -2-hydroxy-propanesulfonic acid), inorganic salt (potassium chloride), surface
Activating agent (lauric acid polyoxyethylene ester), preservative (proclin 300), stabilizer (metal chelating agent) and interference eliminate albumen
(hbr blocker).
Reagent r2 consists of the following composition: buffer (3- morpholine -2-hydroxy-propanesulfonic acid), inorganic salt (potassium chloride), surface
Activating agent (lauric acid polyoxyethylene ester), preservative (proclin 300), stabilizer (metal chelating agent) and polystyrene latex
Granulate mixture, described polystyrene latex particles mixture is crosslinked with antiserum amyloid saa antibody, coupled antibody
Latex preparation process includes activating, and is coupled, blend step, specifically comprises the following steps that
1. being respectively washed two kinds of carboxylated mean diameters of each 100mg in coupling buffer (50mm, ph 6.0) is
Then two kinds of latex are carried out coupled antibody reaction, use 5ml coupling buffer by 82nm and 180nm present latex particulate according to the following steps
Resuspended, it is simultaneously introduced 0.005%sds to prevent agglomerate, avoid in coupling buffer adding containing carboxyl, the chemical combination of sulfydryl and amino
Thing.
2. add the sulfo-nhs of edc and 90mg of 100mg in above-mentioned microgranule, mixing makes dissolving.
3. room temperature reaction 15 minutes.
4. use coupling buffer centrifugal method Rapid Cleaning microgranule 2 times, use 5ml coupling buffer ultrasonic resuspended afterwards.
5., in the coupling buffer of 5ml, antibody protein concentration is micro- to provide for dissolving antiserum amyloid saa antibody
The aglucon of 1 to 10 times of excess that grain monolayer surface amasss group is defined.
6. immediately antibody protein and particulate particles are mixed.
7. room temperature reaction 2-4 hour after mixing.
8. clean microgranule with coupling buffer, and be resuspended in containing 100mm contain hydrophilic quenching molecules (i.e. ethanolamine or
Tris coupling buffer) is to close excessive reaction site
9. clean microgranule, and resuspended in suitable buffer.
10. mix, two kinds of 82nm and 180nm latex particles of antibody will be coupled, according to the mixing of 1:2 ratio;Add one
Quantitative inorganic salt, buffer, surfactant, preservative, stabilizer, obtain reagent r2.
In every 1 liter of reagent r1, the consumption of each component is: biological buffer 7g, surfactant 1ml, preservative 0.1ml, surely
Determine agent 8g, interference eliminates albumen 1ml, inorganic salt 5g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 0.5g of saa antibody, biological buffer 5g, table
Face activating agent 1ml, preservative 0.1ml, stabilizer 5g, inorganic salt 8g, balance of water.
Embodiment 3
Reagent r1 consists of the following composition: buffer (4- hydroxyethyl piperazine ethanesulfonic acid), inorganic salt (magnesium sulfate), lives in surface
Property agent (polyoxyethylene oleic acid ester), preservative (thimerosal), stabilizer (antioxidant) and interference eliminate albumen (hbr block
Agent).
Reagent r2 consists of the following composition: buffer (4- hydroxyethyl piperazine ethanesulfonic acid), inorganic salt (magnesium sulfate), lives in surface
Property agent (polyoxyethylene oleic acid ester), preservative (thimerosal), stabilizer (antioxidant) and polystyrene latex particles mixture,
Described polystyrene latex particles mixture is crosslinked with antiserum amyloid saa antibody, the latex preparation step of coupled antibody
Rapid inclusion activation, is coupled, blend step, specifically comprises the following steps that
1. being respectively washed two kinds of carboxylated mean diameters of each 100mg in coupling buffer (50mm, ph 6.0) is
Then two kinds of latex are carried out coupled antibody reaction, use 5ml coupling buffer by 82nm and 500nm present latex particulate according to the following steps
Resuspended, it is simultaneously introduced 0.005%sds to prevent agglomerate, avoid in coupling buffer adding containing carboxyl, the chemical combination of sulfydryl and amino
Thing.
2. add the sulfo-nhs of edc and 90mg of 100mg in above-mentioned microgranule, mixing makes dissolving.
3. room temperature reaction 10 minutes.
4. use coupling buffer centrifugal method Rapid Cleaning microgranule 2 times, use 5ml coupling buffer ultrasonic resuspended afterwards.
5., in the coupling buffer of 5ml, antibody protein concentration is micro- to provide for dissolving antiserum amyloid saa antibody
The aglucon of 1 to 10 times of excess that grain monolayer surface amasss group is defined.
6. immediately antibody protein and particulate particles are mixed.
7. room temperature reaction 2-4 hour after mixing.
8. clean microgranule with coupling buffer, and be resuspended in containing 100mm contain hydrophilic quenching molecules (i.e. ethanolamine or
Tris coupling buffer) is to close excessive reaction site
9. clean microgranule, and resuspended in suitable buffer.
10. mix, two kinds of 82nm and 500nm latex particles of antibody will be coupled, according to the mixing of 2:1 ratio;Add one
Quantitative inorganic salt, buffer, surfactant, preservative, stabilizer, obtain reagent r2.
In every 1 liter of reagent r1, the consumption of each component is: buffer 15g, surfactant 5ml, preservative 0.4ml, stable
Agent 12g, interference eliminates albumen 5ml, inorganic salt 15g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 5g of saa antibody, buffer 20g, live in surface
Property agent 4ml, preservative 0.4ml, stabilizer 20g, inorganic salt 12g, balance of water.
Embodiment 4
Reagent r1 consists of the following composition: buffer (3- [double (2- ethoxy) amino of n-n-] -2- hydroxy-propanesulfonic acid), no
Machine salt (sodium chloride), surfactant (lauryl amine polyoxyethylene ether), preservative (sodium azide), stabilizer (antioxidant) and dry
Disturb elimination albumen (hbr blocker).
Reagent r2 consists of the following composition: buffer (3- [double (2- ethoxy) amino of n-n-] -2- hydroxy-propanesulfonic acid), no
Machine salt (sodium chloride), surfactant (lauryl amine polyoxyethylene ether), preservative (sodium azide), stabilizer (antioxidant) and poly-
Styrene latex granulate mixture, described polystyrene latex particles mixture is crosslinked with antiserum amyloid saa antibody,
The latex preparation process of coupled antibody includes activating, and is coupled, blend step, specifically comprises the following steps that
1. being respectively washed two kinds of carboxylated mean diameters of each 100mg in coupling buffer (50mm, ph 6.0) is
Then two kinds of latex are carried out coupled antibody reaction, use 5ml coupling buffer by 32nm and 180nm present latex particulate according to the following steps
Resuspended, it is simultaneously introduced 0.005%sds to prevent agglomerate, avoid in coupling buffer adding containing carboxyl, the chemical combination of sulfydryl and amino
Thing.
2. add the sulfo-nhs of edc and 95mg of 100mg in above-mentioned microgranule, mixing makes dissolving.
3. room temperature reaction 20 minutes.
4. use coupling buffer centrifugal method Rapid Cleaning microgranule 2 times, use 5ml coupling buffer ultrasonic resuspended afterwards.
5., in the coupling buffer of 5ml, antibody protein concentration is micro- to provide for dissolving antiserum amyloid saa antibody
The aglucon of 1 to 10 times of excess that grain monolayer surface amasss group is defined.
6. immediately antibody protein and particulate particles are mixed.
7. room temperature reaction 2-4 hour after mixing.
8. clean microgranule with coupling buffer, and be resuspended in containing 100mm contain hydrophilic quenching molecules (i.e. ethanolamine or
Tris coupling buffer) is to close excessive reaction site.
9. clean microgranule, and resuspended in suitable buffer.
10. mix, two kinds of 32nm and 180nm latex particles of antibody will be coupled, according to the mixing of 1:3 ratio;Add one
Quantitative inorganic salt, buffer, surfactant, preservative, stabilizer, obtain reagent r2.
In every 1 liter of reagent r1, the consumption of each component is: buffer 12g, surfactant 3ml, preservative 0.3ml, stable
Agent 10g, interference eliminates albumen 4ml, inorganic salt 12g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 3g of saa antibody, buffer 15g, live in surface
Property agent 3ml, preservative 0.3ml, stabilizer 15g, inorganic salt 10g, balance of water.
Embodiment 5
Reagent r1 consists of the following composition: buffer (mes), inorganic salt (sodium chloride), surfactant (TritonX x-
100), preservative (proclin 300), stabilizer (sucrose) and interference eliminate albumen (hbr blocker).
Reagent r2 consists of the following composition: buffer (mes), inorganic salt (sodium chloride), surfactant (TritonX x-
100), preservative (proclin 300), stabilizer (sucrose) and polystyrene latex particles mixture, described polystyrene colloidal
Newborn granulate mixture is crosslinked with antiserum amyloid saa antibody, and the latex preparation process of coupled antibody includes activating, even
Connection, blend step, step is specific as follows:
1. it is respectively washed two kinds of carboxylated mean diameters of each 100mg in coupling buffer (50mm mes, ph 6.0)
For 130nm and 150nm present latex particulate, then according to the following steps coupled antibody reaction is carried out to two kinds of latex, be coupled buffering with 5ml
Liquid is resuspended, is simultaneously introduced 0.005%sds to prevent agglomerate, avoids adding containing carboxyl, the change of sulfydryl and amino in coupling buffer
Compound.
2. add the sulfo-nhs of edc and 100mg of 100mg in above-mentioned microgranule, mixing makes dissolving.
3. room temperature reaction 15 minutes.
4. use coupling buffer centrifugal method Rapid Cleaning microgranule 2 times, use 5ml coupling buffer ultrasonic resuspended afterwards.
5., in the coupling buffer of 5ml, antibody protein concentration is micro- to provide for dissolving antiserum amyloid saa antibody
The aglucon of 1 to 10 times of excess that grain monolayer surface amasss group is defined.
6. immediately antibody protein and particulate particles are mixed.
7. room temperature reaction 2-4 hour after mixing.
8. clean microgranule with coupling buffer, and be resuspended in containing 100mm contain hydrophilic quenching molecules (i.e. ethanolamine or
Tris coupling buffer) is to close excessive reaction site.
9. clean microgranule, and resuspended in suitable buffer.
10. mix, two kinds of 130nm and 150nm latex particles of antibody will be coupled, according to the mixing of 1:4 ratio;Add one
Quantitative inorganic salt, buffer, surfactant, preservative, stabilizer, obtain reagent r2.
In every 1 liter of reagent r1, the consumption of each component is: buffer 9.76g, surfactant 3.6ml, preservative 0.2ml,
Stabilizer 10g, interference eliminates albumen 4ml, inorganic salt 9g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 2.2g of saa antibody, biological buffer
9.76g, surfactant 2ml, preservative 0.2ml, stabilizer 10g, inorganic salt 9g, balance of water.
There is provided following experimental examples in order to confirm property indices and the Detection results of embodiment of the present invention test kit.
Experimental example 1 standard curve
Saa standard concentration is as follows: 0,50,100,150,200,300mg/l.
Take high standard product (300mg/l) and low concentration standard substance (50mg/l), gradient dilution creates 3 concentration values
Sample, add blank.In biochemical instruments, sample parallel assay 7 times respectively to 6 variable concentrations, unite to data
Meter is processed, and obtains the range of linearity, obtains during the test kit detection saa sample giving embodiment of the present invention 1-4 as shown in Figure 1
Absorbance change curve, wherein, x-axis represents concentration of specimens, and y-axis represents absorbance change value.
Method of testing: take above-mentioned sample 6 μ l, add reagent r1 about 162 μ l, mix latter 5 minutes, 37 DEG C of reaction temperature, so
Add reagent r2 about 30 μ l afterwards, mixing detects 700nm wavelength absorbance after 18 seconds, detects a 700nm wavelength again after spending 5 minutes
Absorbance, seeks the difference of absorbance twice.This law range of linearity is 1~300mg/l, and the intercept of its regression equation and slope are respectively
A=50.857, b=503.57, no significant difference (p > 0.05), (y=50.857x+503.57, r2=0.9911.
This curve is standard curve that saa concentration is during 1~300mg/l;As sample saa > 300mg/l, need to by after diluted sample again
Survey.
Experimental example 2 sensitivity test
Reference U.S. clinical Laboratory Standard association (clinical&laboratory standards institute:
Clsi, ep17a) file, measure transmission value changes 10 times with zero reference standard product (0mg/l), calculate its averageAnd standard deviation
(s).With transmission valueThe value calculating substitutes into calibration curve equation, and the concentration value of gained is the sensitivity of detection.
As seen from Table 1, the sensitivity of the embodiment of the present invention 1 detection kit is 1mg/l.
Table 1
saa(mg/l) | Measure number of times | Average | 2sd | m+2sd |
0 | 10 | 533 | 17.4 | 550.4 |
Experimental example 3 high level linear determination
Measure the sample of 10 kinds of different saa contents, each sample surveys 2 times, as seen from Table 2, the embodiment of the present invention 1 detection examination
The highest detection scope of agent box can reach 300mg/l.
Table 2
Saa theoretical value (mg/l) | Saa actual value (mg/l) | |
1 | 0 | 0.1 |
2 | 10 | 9.8 |
3 | 50 | 48.9 |
4 | 100 | 102.3 |
5 | 150 | 148.6 |
6 | 200 | 204.5 |
7 | 250 | 250.1 |
8 | 300 | 306.8 |
9 | 350 | 288.3 |
10 | 400 | 253.9 |
Experimental example 4 precision test
The requirement evaluated for precision according to (clsi) ep5-a2 file, measures basic, normal, high value saa concentration, 2h respectively
Interior each horizontal continuity measures 20 times, calculatesS, coefficient of variation coefficient of variation (cv).Daily mensure 1
Secondary, METHOD FOR CONTINUOUS DETERMINATION 20d.CalculateS and cv.Using the human serum sample of 2 kinds of different saa contents, measure the embodiment of the present invention 1 and examine
Batch interior and betweenrun precision of test agent box.Result shows, the withinrun precision of the embodiment of the present invention 1 detection kit is
4.58%, 3.13% and 2.37% (being shown in Table 3), and betweenrun precision is then 7.32%, 5.77% and 4.76% (being shown in Table 4).
Table 3
Withinrun precision | Low | In | High |
Measure number of times | 20 | 20 | 20 |
Meansigma methodss (mg/l) | 10.7 | 51.4 | 102.1 |
Minima (mg/l) | 8.5 | 47.3 | 98.9 |
Maximum (mg/l) | 13.6 | 56.5 | 112.3 |
Standard deviation (sd) | 4.12% | 3.08% | 2.13% |
The coefficient of variation (cv) | 4.58% | 3.13% | 2.37% |
Table 4
Betweenrun precision | Low | In | High |
Measure number of times | 20 | 20 | 20 |
Meansigma methodss (mg/l) | 11.3 | 50.5 | 117.6 |
Minima (mg/l) | 5.7 | 43.1 | 88.7 |
Maximum (mg/l) | 16.9 | 59.8 | 112.3 |
Standard deviation (sd) | 7.25% | 5.34% | 4.12% |
The coefficient of variation (cv) | 7.32% | 5.77% | 4.76% |
Experimental example 5 interference test
The measured value after interfering material is added to be contributive rate, result of the test table divided by the measured value before addition interfering material
The concentration of bright unconjugated bilirubin, conjugated bilirubin, hemoglobin and Chylomicron respectively in 200mg/dl, 200mg/dl,
During 5000mg/dl and below 21000ftu, they (are shown in Table 5) to the interference of measurement result all below 2%.
Table 5
Technical scheme in above-mentioned the embodiment of the present application, at least has the following technical effect that or advantage:
(1) test kit of the present invention utilizes Latex-enhanced immunoturbidimetric assay to measure serum amyloid protein, is exaggerated inspection
Survey signal, improve detection sensitivity, shorten detection time.
(2) present invention is big using the emulsion reagent of mixing particle diameter, wherein the latex particle ratio of small particle, improves line
Property;Contain the latex particle of a certain proportion of big particle diameter, sensitivity is also higher simultaneously.
(3) test kit of the present invention has been specifically added the material preventing disturbing, good in anti-interference performance.Meanwhile, surfactant
Interpolation also improve precision.
(4) use automatic clinical chemistry analyzer to measure serum amyloid protein, not only make operation more easy, improve automatically
Change degree, and also a saving the Financial cost of a small amount of sample detection experiment.Automatic clinical chemistry analyzer is multiple batches of to a small amount of
Sample is detected, almost identical to the cost of a large amount of sample detection with single batch;Therefore, automatic clinical chemistry analyzer can be fitted
For the detection to a small amount of sample, and do not improve Financial cost.
Although preferred embodiments of the present invention have been described, but those skilled in the art once know basic creation
Property concept, then can make other change and modification to these embodiments.So, claims are intended to be construed to including excellent
Select embodiment and fall into being had altered and changing of the scope of the invention.Obviously, those skilled in the art can be to the present invention
Carry out various change with modification without departing from the spirit and scope of the present invention.So, if these modifications of the present invention and modification
Belong within the scope of the claims in the present invention and its equivalent technologies, then the present invention is also intended to comprise these changes and modification exists
Interior.
Claims (10)
1. a kind of detection serum amyloid protein test kit, including reagent r1 and reagent r2 it is characterised in that: described reagent r1
Including buffer, inorganic salt, surfactant, preservative, stabilizer and interference elimination albumen;Described reagent r2 includes buffering
Liquid, inorganic salt, surfactant, preservative, stabilizer and polystyrene latex particles mixture;Described polystyrene latex
Grain mixture is crosslinked with antiserum amyloid saa antibody;
Described polystyrene latex particles mixture is major diameter polystyrene latex particles and minor diameter polystyrene latex
The mixture of grain, described major diameter polystyrene latex particles mean diameter is 180nm~500nm, described minor diameter polyphenyl second
Alkene latex mean diameter is 32nm~82nm, major diameter polystyrene latex particles: minor diameter polystyrene latex particles=1:
(2-4).
2. according to claim 1 detection serum amyloid protein test kit it is characterised in that: described major diameter polyphenyl
Ethylene latex mean particle size is 180nm, and described minor diameter polystyrene latex mean diameter is 82nm, major diameter polyphenyl second
Alkene latex particle: minor diameter polystyrene latex particles=1:2.
3. according to claim 1 detection serum amyloid protein test kit it is characterised in that: described polystyrene colloidal
The method adopting during newborn granulate mixture crosslinking antiserum amyloid saa antibody comprises the steps:
(1) carboxylated major diameter polystyrene latex particles and minor diameter polystyrene latex particles are cleaned in buffer,
Obtain present latex particulate with described buffer is resuspended;
(2) add edc and sulfo-nhs in described present latex particulate, mix and make dissolving, room temperature reaction, anti-with buffer solution for cleaning
Described microgranule after answering, resuspended with described buffer;
(3) adopt buffer solution antiserum amyloid saa antibody;
(4) mixed by the described microgranule obtaining through step (2) with through the described antibody that step (3) obtains, room temperature reaction;
(5) use described microgranule after step (4) process for the buffer solution for cleaning, and be suspended in the coupling buffering containing hydrophilic quenching molecules
Liquid is to close excessive reaction site;
(6) the described microgranule after cleaning is processed through step (5), resuspended in buffer, obtain the latex particle being coupled antibody.
4. according to claim 3 detection serum amyloid protein test kit it is characterised in that: in described step (1)
In, add dispersant when described resuspended, described dispersant is anion surfactant.
5. according to claim 3 detection serum amyloid protein test kit it is characterised in that: in described step (2),
The concentration of described edc and sulfo-nhs is respectively 20mg/ml and 15mg/ml-18mg/ml.
6. according to claim 3 detection serum amyloid protein test kit it is characterised in that: in described step (4),
Described hydrophilic quenching molecules are ethanolamine or tris, and concentration is 100mm.
7. according to claim 1 detection serum amyloid protein test kit it is characterised in that: described buffer is selected from
3- morpholine -2-hydroxy-propanesulfonic acid, 2- (n- morpholine) ethyl sulfonic acid, 4- hydroxyethyl piperazine ethanesulfonic acid and 3- [double (the 2- hydroxyl second of n-n-
Base) amino] -2- hydroxy-propanesulfonic acid;Described surfactant is selected from TritonX x-100, lauric acid polyoxyethylene ester, Oleic acid polyoxy
Vinyl acetate and lauryl amine polyoxyethylene ether;Described stabilizer is selected from sucrose, metal chelating agent and antioxidant;Described interference eliminates
Albumen is the hbr blocker of scantibodies company;Described inorganic salt is selected from sodium chloride, potassium chloride and magnesium sulfate;Described anti-
Rotten agent is selected from sodium azide, thimerosal and proclin 300.
8. according to claim 1 or 7 detection serum amyloid protein test kit it is characterised in that: every 1 liter of reagent r1
In the consumption of each component be: buffer 7-15g, surfactant 1-5ml, preservative 0.1-0.4ml, stabilizer 8-12g, interference
Eliminate albumen 1-5ml, inorganic salt 5-15g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 0.5-5g of saa antibody, buffer 5-20g, surface
Activating agent 1-4ml, preservative 0.1-0.4ml, stabilizer 5-20g, inorganic salt 8-12g.
9. according to claim 8 detection serum amyloid protein test kit it is characterised in that: in every 1 liter of reagent r1
The consumption of each component is: buffer 9.76g, surfactant 3.6ml, preservative 0.2ml, stabilizer 10g, and interference eliminates albumen
4ml, inorganic salt 9g, balance of water;
In every 1 liter of reagent r2, the consumption of each component is: is coated the latex particle 2.2g of saa antibody, buffer 9.76g, live in surface
Property agent 2ml, preservative 0.2ml, stabilizer 10g, inorganic salt 9g, balance of water.
10. as described in any one of claim 1-9 detection serum amyloid protein test kit purposes it is characterised in that:
Described test kit is used for detecting serum amyloid protein.
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