CN106353507A - Kit for detecting serum amyloid protein and application thereof - Google Patents

Kit for detecting serum amyloid protein and application thereof Download PDF

Info

Publication number
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
Authority
CN
China
Prior art keywords
buffer
reagent
polystyrene latex
serum amyloid
stabilizer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201610711795.6A
Other languages
Chinese (zh)
Inventor
华权高
来祥兵
徐春雷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wuhan Life Origin Biotech Joint Stock Co Ltd
Original Assignee
Wuhan Life Origin Biotech Joint Stock Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wuhan Life Origin Biotech Joint Stock Co Ltd filed Critical Wuhan Life Origin Biotech Joint Stock Co Ltd
Priority to CN201610711795.6A priority Critical patent/CN106353507A/en
Publication of CN106353507A publication Critical patent/CN106353507A/en
Pending legal-status Critical Current

Links

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

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

A kind of test kit of detection serum amyloid protein and application thereof
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.
CN201610711795.6A 2016-08-24 2016-08-24 Kit for detecting serum amyloid protein and application thereof Pending CN106353507A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610711795.6A CN106353507A (en) 2016-08-24 2016-08-24 Kit for detecting serum amyloid protein and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610711795.6A CN106353507A (en) 2016-08-24 2016-08-24 Kit for detecting serum amyloid protein and application thereof

Publications (1)

Publication Number Publication Date
CN106353507A true CN106353507A (en) 2017-01-25

Family

ID=57844629

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610711795.6A Pending CN106353507A (en) 2016-08-24 2016-08-24 Kit for detecting serum amyloid protein and application thereof

Country Status (1)

Country Link
CN (1) CN106353507A (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107860929A (en) * 2017-11-10 2018-03-30 苏州康和顺医疗技术有限公司 The immunoturbidimetry detection reagent and method of a kind of serum amyloid A protein
CN108828225A (en) * 2018-04-25 2018-11-16 迪瑞医疗科技股份有限公司 Kit, preparation method and the detection method of serum amyloid A protein assay
CN111426846A (en) * 2020-04-08 2020-07-17 深圳市锦瑞生物科技有限公司 Kit and detection system
CN111868527A (en) * 2018-03-16 2020-10-30 富士胶片株式会社 Kit, assay kit and assay method
CN111896757A (en) * 2020-08-04 2020-11-06 武汉生之源生物科技股份有限公司 D-dimer determination kit for improving whole blood sample measurement value, preparation method and application
CN112014574A (en) * 2020-08-25 2020-12-01 上海睿康生物科技有限公司 Serum amyloid protein A detection kit coated by double-liposome nanoparticles
CN112014576A (en) * 2020-09-03 2020-12-01 北京安图生物工程有限公司 Reagent for detecting human serum amyloid A and preparation method thereof
CN112098647A (en) * 2020-02-28 2020-12-18 安徽大千生物工程有限公司 Kit for determining CK-MB based on latex enhanced immunoturbidimetry and preparation and use methods thereof
CN112326635A (en) * 2020-10-20 2021-02-05 浙江理工大学 Double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit, and preparation method and application thereof
CN112500514A (en) * 2020-11-11 2021-03-16 四川迈克生物新材料技术有限公司 Polystyrene latex microsphere and preparation method thereof
CN114137227A (en) * 2021-12-06 2022-03-04 石家庄斯巴克生物科技有限公司 Coupling method and kit for serum amyloid A antibody and magnetic beads
CN114137223A (en) * 2021-11-25 2022-03-04 安徽大千生物工程有限公司 Latex enhanced immunoturbidimetry assay kit for rapidly assaying IGF-1 and preparation and use methods thereof
CN116298317A (en) * 2023-03-14 2023-06-23 浙江夸克生物科技有限公司 Alpha fetoprotein determination kit based on latex immunoturbidimetry

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0818680A1 (en) * 1996-07-12 1998-01-14 Daiichi Pure Chemicals Co. Ltd. Agglutination immunoassay
JPH1090268A (en) * 1996-09-18 1998-04-10 Eiken Chem Co Ltd Immiunological particle agglutination method
JP2006017745A (en) * 2005-09-26 2006-01-19 Eiken Chem Co Ltd Immunological particle agglutination reaction method
CN104237522A (en) * 2012-12-03 2014-12-24 武汉生之源生物科技有限公司 Adiponectin content detection kit and preparation method thereof
CN105203771A (en) * 2015-09-14 2015-12-30 绍兴圣康生物科技有限公司 Serum amyloid protein A determination kit and use method thereof
CN105372434A (en) * 2015-08-13 2016-03-02 浙江卓运生物科技有限公司 Detection kit for human serum amyloid A protein
CN105548571A (en) * 2016-02-02 2016-05-04 潍坊三维生物工程集团有限公司 Kit and method for detecting content of serum amyloid protein A and application
CN105738617A (en) * 2016-04-01 2016-07-06 武汉生之源生物科技股份有限公司 Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0818680A1 (en) * 1996-07-12 1998-01-14 Daiichi Pure Chemicals Co. Ltd. Agglutination immunoassay
JPH1090268A (en) * 1996-09-18 1998-04-10 Eiken Chem Co Ltd Immiunological particle agglutination method
JP2006017745A (en) * 2005-09-26 2006-01-19 Eiken Chem Co Ltd Immunological particle agglutination reaction method
CN104237522A (en) * 2012-12-03 2014-12-24 武汉生之源生物科技有限公司 Adiponectin content detection kit and preparation method thereof
CN105372434A (en) * 2015-08-13 2016-03-02 浙江卓运生物科技有限公司 Detection kit for human serum amyloid A protein
CN105203771A (en) * 2015-09-14 2015-12-30 绍兴圣康生物科技有限公司 Serum amyloid protein A determination kit and use method thereof
CN105548571A (en) * 2016-02-02 2016-05-04 潍坊三维生物工程集团有限公司 Kit and method for detecting content of serum amyloid protein A and application
CN105738617A (en) * 2016-04-01 2016-07-06 武汉生之源生物科技股份有限公司 Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107860929A (en) * 2017-11-10 2018-03-30 苏州康和顺医疗技术有限公司 The immunoturbidimetry detection reagent and method of a kind of serum amyloid A protein
CN111868527A (en) * 2018-03-16 2020-10-30 富士胶片株式会社 Kit, assay kit and assay method
CN111868527B (en) * 2018-03-16 2024-05-03 富士胶片株式会社 Kit, assay kit and assay method
CN108828225A (en) * 2018-04-25 2018-11-16 迪瑞医疗科技股份有限公司 Kit, preparation method and the detection method of serum amyloid A protein assay
CN112098647A (en) * 2020-02-28 2020-12-18 安徽大千生物工程有限公司 Kit for determining CK-MB based on latex enhanced immunoturbidimetry and preparation and use methods thereof
CN111426846A (en) * 2020-04-08 2020-07-17 深圳市锦瑞生物科技有限公司 Kit and detection system
CN111896757B (en) * 2020-08-04 2023-08-29 武汉生之源生物科技股份有限公司 D-dimer determination kit for improving whole blood sample measurement value, preparation method and application
CN111896757A (en) * 2020-08-04 2020-11-06 武汉生之源生物科技股份有限公司 D-dimer determination kit for improving whole blood sample measurement value, preparation method and application
CN112014574A (en) * 2020-08-25 2020-12-01 上海睿康生物科技有限公司 Serum amyloid protein A detection kit coated by double-liposome nanoparticles
CN112014576A (en) * 2020-09-03 2020-12-01 北京安图生物工程有限公司 Reagent for detecting human serum amyloid A and preparation method thereof
CN112326635A (en) * 2020-10-20 2021-02-05 浙江理工大学 Double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit, and preparation method and application thereof
CN112500514A (en) * 2020-11-11 2021-03-16 四川迈克生物新材料技术有限公司 Polystyrene latex microsphere and preparation method thereof
CN114137223A (en) * 2021-11-25 2022-03-04 安徽大千生物工程有限公司 Latex enhanced immunoturbidimetry assay kit for rapidly assaying IGF-1 and preparation and use methods thereof
CN114137227A (en) * 2021-12-06 2022-03-04 石家庄斯巴克生物科技有限公司 Coupling method and kit for serum amyloid A antibody and magnetic beads
CN116298317A (en) * 2023-03-14 2023-06-23 浙江夸克生物科技有限公司 Alpha fetoprotein determination kit based on latex immunoturbidimetry

Similar Documents

Publication Publication Date Title
CN106353507A (en) Kit for detecting serum amyloid protein and application thereof
CN102590524B (en) Neutrophil gelatinase-associated lipocalin detection kit
CN106324239B (en) Latex microsphere preparation method and application is immunized in C reactive protein
CN101377492B (en) Bladder chalone C determining reagent kit
CN103134934B (en) Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample
CN109596843B (en) A kind of assay kit of serum amyloid A protein
CN108303544A (en) A kind of whole blood c reactive protein detection kit
CN105738617B (en) A kind of bladder chalone C latex intensified is than turbid detection kit and application thereof
CN103645323B (en) A kind of cystatin C detection kit and preparation method thereof
CN101310186B (en) Method of assaying antigen and kit to be used therein
CN103823070A (en) Cystatin C determination kit with high sensitivity
CN102944679A (en) Kit for performing retinol binding protein detection by using latex turbidimetry
CN111122874A (en) Kit for determining LN based on latex enhanced immunoturbidimetry, and preparation and use methods thereof
CN102621332A (en) Retinol binding protein assay kit based on latex particle coating
CN101893619B (en) Method for improving stability of latex suspension liquid
CN109725160B (en) Procalcitonin (PCT) detection kit
CN102636653A (en) Compounded latex particle-enveloped cystatin C detection kit
CN102662064A (en) Immunonephelometry kit for detecting lipid carrier protein related to neutrophils gelatinase and preparation method thereof
CN100396331C (en) Carrier particle latex for assay reagent and assay reagent
CN104535770A (en) Myoglobin determination kit of compound antibody
CN103308681B (en) Trypsinogen-2 detection kit and preparation method
CN109212204A (en) A kind of kit of latex immunoturbidimetry measurement d-dimer content
CN112858698B (en) D-dimer latex enhanced immunoturbidimetry kit and preparation method thereof
CN111912990B (en) Neutrophil gelatinase-associated lipocalin assay kit
CN108627652A (en) It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20170125

RJ01 Rejection of invention patent application after publication