CN106290902A - A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof - Google Patents
A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof Download PDFInfo
- Publication number
- CN106290902A CN106290902A CN201610621021.4A CN201610621021A CN106290902A CN 106290902 A CN106290902 A CN 106290902A CN 201610621021 A CN201610621021 A CN 201610621021A CN 106290902 A CN106290902 A CN 106290902A
- Authority
- CN
- China
- Prior art keywords
- protein
- serum amyloid
- gold
- antibody
- colloidal
- 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
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/067—Pancreatitis or colitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/12—Pulmonary diseases
- G01N2800/122—Chronic or obstructive airway disorders, e.g. asthma COPD
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/34—Genitourinary disorders
- G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
Abstract
A kind of colloidal-gold detecting-card of serum amyloid A protein 1, belong to micro-biopsy survey technology field, it is characterized in that: gold-marking binding pad is coated with the SAAl antibody of purification and the coupling label of gold colloidal, parallel on nitrocellulose filter be provided with a SAA1 detection line being coated with SAA1 monoclonal antibody and a nature controlling line being coated with sheep anti-mouse igg polyclonal antibody.Its method is: the preparation of the coupling label of step one, serum amyloid A protein 1 antibody and gold colloidal, step 2, the assembling of test strips, uses gold spraying instrument to be sprayed on glass fibre element film by the coupling label of serum amyloid A protein 1 antibody Yu gold colloidal.Provide the benefit that: colloidal gold colloidal gold detection test paper strip the plan of preparation detection serum amyloid A protein 1 are applied to clinic, have simple to operate quickly, testing result understand be prone to judgements, high specificity, sensitivity height, without advantages such as instrument and equipments, therefore, it is highly suitable for the limited place of the experiment condition such as scene, outpatient service and carries out clinical sample detection.The invention provides the preparation method of above-mentioned test strips, it is adaptable to commercial production.
Description
Technical field
The invention belongs to micro-biopsy survey technology field.
Background technology
Serum amyloid A protein (serum amyloid A protein, SAA) is a kind of Acute reaction protein,
Belonging to the heterogeneous proteinoid in apolipoprotein family, relative molecular mass is about 12 000, is present in vertebrates, main
To be synthesized by hepatocyte.SAA is the general name of the albumen of a polymorphism, by being correlated with but the gene (SAA1-of respective independent protein
SAA4) composition, is vertebrate main acute reactive protein.Serum amyloid A protein 1(SAA1) by 104 aminoacid groups
Becoming, SAA1 and SAA2 has 7 amino acid whose difference, and they are acute phase protein, and the most importantly SAA1, in inflammatory reaction
In the effect played similar.SAA3 gene, owing to having 1 base to insert at the 3rd exon (at the 41st codon), produces
Coding displacement, causes producing translation termination signal at the 43rd codon, belongs to pseudogene.SAA4 is in acute phase response not
Greatly, for a dynamic balance state.
Research shows, SAA1 content in acute and chronic inflammation patients serum all changes.SAA1 is not only chronic resistance
The biomarker of plug property pneumonopathy Acute Exacerbation Period, is also that tool is estimated in acute myocardial infarction, the condition-inference of patients with coronary heart disease and prognosis
There is important reference value.Substantial amounts of research shows, at pancreatitis, hepatitis, diabetes, rheumatoid arthritis and chronic kidney
In disease case, the rising of SAA1 content has very close relationship with disease activity and disease.Clinical research table at present
Bright, in all acute phase reactive proteins, SAA1 is most sensitive to acute inflammatory reaction, and appreciation amplitude is maximum.Research shows, liver
The kinds of tumors SAA1 in the patient such as cancer, pulmonary carcinoma, breast carcinoma, carcinoma of prostate, carcinoma of endometrium all have and raise in various degree, and its
Level and the active stage of tumor, grade malignancy and Invasion and Metastasis have obvious dependency, the curative effect of reflection tumor patient and prognosis
。
In acute-phase response, through interleukin (interleukin, IL)-1, IL-6 and tumor necrosis factor
(tumor necrosis factor, TNF) stimulates, and SAA is closed by the macrophage being activated and fibroblast in liver
Become, 100～1000 times of initial concentration can be increased to.It is similar to c reactive protein (C reactive protein, CRP),
SAA is the sensitive indicator of reflection infectious disease Earlier period of inflammation, contribute to diagnosing inflammation, assess its activity, monitor its movable and
Treatment.Research shows that SAA level is significantly raised in viral infection acute stage, and CRP raises and inconspicuous, therefore SAA
It is to judge that virus infects sensitive and reliable index.SAA and CRP joint-detection contributes to patient antibacterial and infects and virus infection
Differential Diagnosis.Additionally, SAA (particularly carries out immunosuppressant therapy judging viral infection, kidney transplantation exclusion reaction patient
Patient) and more more sensitive than CRP by the cystic fibrosis patient aspect of adrenocortical hormones in treating.
Summary of the invention
It is an object of the invention to: colloidal-gold detecting-card of a kind of serum amyloid A protein 1 and preparation method thereof is provided, it
Can quantitatively, quickly, low cost detect the content of SAA1 in blood.
The technical scheme is that and include PVC base plate, glue sample pad, the gold mark combination being posted on PVC base plate successively
Pad, with detection line and the nitrocellulose filter of nature controlling line, absorption pad.
Gold-marking binding pad is coated with the SAAl antibody of purification and the coupling label of gold colloidal, flat on nitrocellulose filter
Row is provided with a SAA1 detection line being coated with SAA1 monoclonal antibody and the matter being coated with sheep anti-mouse igg polyclonal antibody
Control line.
The preparation method is that:
The preparation of the coupling label of step one, serum amyloid A protein 1 antibody and gold colloidal: stepwise dilution method determines human blood
Clear amyloid A 1 antibody is 4.4ug～4.8ug:1ml with the usage ratio of colloidal gold solution.
It is added dropwise over serum amyloid A protein 1 antibody of purification according to this usage ratio, adds final concentration of 1%
Bovine serum albumin, obtains the coupling label of serum amyloid A protein 1 antibody and gold colloidal after stirring, use low temperature hypervelocity
This coupling label is purified by centrifuging；
Step 2, the assembling of test strips: use gold spraying instrument by the coupling label of serum amyloid A protein 1 antibody Yu gold colloidal
It is sprayed on glass fibre element film, natural drying under the conditions of room temperature 37 DEG C, seals, it is thus achieved that gold-marking binding pad；Use and draw film instrument by pure
SAA1 monoclonal antibody, the sheep anti-mouse igg changed are drawn on film detection line on nitrocellulose filter and nature controlling line respectively；At Jiang
The sample pad managed, gold-marking binding pad, nitrocellulose filter are viscous successively to be posted on PVC base plate, cutting, assembles, seals, it is thus achieved that
Serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip, room temperature preservation.
Further, described colloidal gold solution uses trisodium citrate reduction method to prepare: takes 1% chlorauric acid solution 1ml, adds
The ultra-pure water entering 99ml is configured to the chlorauric acid solution of final concentration of 0.01%, after being heated to boiling, adds 1% citric acid three
Sodium 1ml also continues heating, and solution is transferred to the black-and-blue claret that eventually becomes by faint yellow, continues heating 10 points after colour stable
Clock, room temperature cools down, it is thus achieved that colloidal gold solution, 4 DEG C save backup, a diameter of 40nm of colloid gold particle.
Further, in step one, state stepwise dilution method and determine serum amyloid A protein 1 antibody and colloidal gold solution
The detailed process of usage ratio is as follows: the PH value regulating colloidal gold solution with the carbonate solution of 0.1mol/L is 8.5, to 11
Prop up in centrifuge tube and be separately added into 1ml colloidal gold solution；I.e. content after serum amyloid A protein 1 antibody stepwise dilution is respectively
1ug, 2ug, 4ug, 8ug, 10ug, 12ug, 14ug, 16ug, 18ug, 20ug, join No. 2 pipes in the order described above to No. 11
Guan Zhong, and mix with the colloidal gold solution in centrifuge tube, in No. 2 pipes to No. 11 pipes, it is separately added into the chlorine of 10% after 10 minutes
Changing sodium solution 1ml mixing, room temperature stands more than 2 hours observed results, and No. 1 pipe is blank；
No. 2, No. 3, in No. 9 pipes to No. 11 pipes, coagulation phenomenon from red to blue occurs, in No. 4 pipes to No. 8 pipes, keep gold colloidal
Redness constant；Therefore, centrifuge tube that is No. 4 pipe that gold colloidal is red constant and serum amyloid A protein 1 antibody addition is minimum
In serum amyloid A protein 1 antibody content, be the minimum steady needed for stable 1ml colloidal gold solution quantitative, minimum at this
Add 10%～20% on the basis of stable quantity to be serum amyloid A protein needed for stable 1ml colloidal gold solution 1 and resist
The actually used amount of body 1, i.e. serum amyloid A protein 1 antibody with the usage ratio of colloidal gold solution be: 4.4ug～4.8ug:
1ml。
Further, in step one, use the detailed process that this coupling label is purified by low temperature supercentrifugation
As follows: first serum amyloid A protein 1 antibody that volume is V to be centrifuged 30 minutes with 3000rpm/min at 4 DEG C, inhale
Take supernatant, abandon precipitation；Again supernatant is centrifuged 30 minutes with 10000rpm/min at 4 DEG C, supernatant discarded, uses
The PBS buffer solution of 0.01mol/L, pH7.4 is precipitated to the original volume i.e. volume V of serum amyloid A protein 1 antibody, surely
After Ding overnight: at 4 DEG C, then it is centrifuged 40 minutes with 10000rpm/min, abandons supernatant, delay with the PBS of 0.01mol/L pH7.4
Rush liquid dissolution precipitation to the 1/10 of the original volume i.e. volume V of serum amyloid A protein 1 antibody, it is thus achieved that the serum amyloid sample of purification
Protein A 1 antibody, 4 DEG C save backup；Containing 1% BSA and 0.02% sodium azide in described PBS buffer.
The invention has the beneficial effects as follows: enzyme linked immunological principle be combined with colloidal gold chromatographic technology, preparation detection serum forms sediment
The colloidal gold colloidal gold detection test paper strip of powder sample protein A 1 also is intended being applied to clinic, have simple to operate quickly, testing result understands and is prone to
Judgement, high specificity, sensitivity is high, without advantages such as instrument and equipments, therefore, be highly suitable for the experiment condition such as scene, outpatient service
Limited place carries out clinical sample detection.The invention provides the preparation method of above-mentioned test strips, it is adaptable to commercial production.
Accompanying drawing explanation
Fig. 1 is the structural representation of serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip of the present invention.
Fig. 2 is that the serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip positive findings of the present invention judges schematic diagram.
Fig. 3 is that the serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip negative findings of the present invention judges schematic diagram.
Fig. 4 is that the serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip null result of the present invention judges schematic diagram.
Detailed description of the invention
In figure: 1 is PVC base plate, and 2 is sample pad, and 3 is gold-marking binding pad, and 4 is nitrocellulose filter, 5 is detection line, 6
Being nature controlling line, 7 is absorption pad.
Serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip of the present invention, including PVC base plate 1, sample pad 2, gold mark knot
Close pad 3, with detection line 5 and the nitrocellulose filter 4 of nature controlling line 6 and absorption pad 7, sample pad 2, gold-marking binding pad 3, with inspection
The nitrocellulose filter 4 of survey line 5 and nature controlling line 6, absorption pad 7 are viscous successively to be posted on PVC base plate 1, and gold-marking binding pad 3 is coated with pure
The coupling label of serum amyloid A protein 1 gold colloidal, the detection line 5 on nitrocellulose filter 4 is coated serum amyloid protein
The monoclonal antibody of A1, the nature controlling line 6 on nitrocellulose filter 4 is coated with sheep anti-mouse igg.
The using method of serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip of the present invention is as follows:
1, with capillary glass blood sampling tube 1～2, the glass capillary that fractures after serum separates out naturally make serum (or blood) oneself
So flow out on clean glass plate, take 5ul sample for detecting with pipettor.
2, the 5ul sample drawn adds the sample well front end of test strips, and two diluents of dropping are in sample well therewith
In, result within 3～10 minutes, can be shown, when 10 minutes, read result.
Result judges as shown in Fig. 2:
1, positive: at detection line 5 and nature controlling line 6, a red stripes respectively to occur, it is determined that for the positive；5, line of detection is colored
The depth in pool changes according to the height detecting serum amyloid A protein 1 titer in sample, and the highest colour band of titer is the deepest, otherwise more
Shallow.
2, negative: to occur that red stripes do not occur in a red stripes, detection line 5 at nature controlling line 6, illustrate to detect sample
Middle serum amyloid A protein 1 exists.
3, invalid: only to have band at detection line 5 or all occur without obvious band at detection line 5 and nature controlling line 6, being considered as
ELISA test strip is invalid.
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further details.
Embodiment 1
One, the preparation of colloidal gold solution
Prepared by employing trisodium citrate reduction method:: taking 1% chlorauric acid solution 1ml, the ultra-pure water adding 99ml is configured to the denseest
Degree is the chlorauric acid solution of 0.01%, after being heated to boiling, adds 1% trisodium citrate 1ml and continues heating, and solution is by light
Yellow transfers the black-and-blue claret that eventually becomes to, continues heating 10 minutes after colour stable, and room temperature cools down latter 4 DEG C and saves backup,
Obtain colloidal gold solution.Drawing a small amount of colloid gold particle transmission electron microscope observing, colloid gold particle size is basically identical, and distribution is all
Even, diameter about 40nm side is qualified.
Two, (gold mark serum amyloid A protein 1 is anti-for serum amyloid A protein 1 antibody and the coupling label of gold colloidal
Body) preparation
(1) stepwise dilution method determines the usage ratio of serum amyloid A protein 1 antibody and colloidal gold solution: use 0.1mol/L
Carbonate solution regulation colloidal gold solution PH value be 8.5, take 11 clean centrifuge tubes, numbered No. 1 pipe is to No. 11
Pipe, often pipe adds colloidal gold solution 1ml；By after serum amyloid A protein 1 antibody stepwise dilution (be diluted to 20ug by 1ug,
I.e. serum amyloid A protein 1 antibody content be respectively 1ug, 2ug, 4ug, 8ug, 10ug, 12ug, 14ug, 16ug, 18ug,
20ug), join No. 2 pipes in No. 11 pipes according to stepwise dilution order, and mix with the colloidal gold solution in centrifuge tube, 5 points
Being separately added into the sodium chloride solution 1ml mixing of 10% after clock in No. 2 pipes to No. 11 pipes, room temperature stands observes knot in more than 2 hours
Really, No. 1 pipe is blank pipe.
Serum amyloid A protein 1 antibody addition be not enough to stable colloid gold centrifuge tube (No. 2, No. 3, No. 9 pipes are to 11
Number pipe), coagulation phenomenon from red to blue i.e. occurs, and serum amyloid A protein 1 antibody addition meets or exceeds minimum stable
The centrifuge tube (No. 4 pipes are to No. 8 pipes) of amount, then the redness keeping gold colloidal is constant.Therefore, the red constant and serum amyloid of gold colloidal
Serum amyloid A protein 1 antibody content (4ug) in the centrifuge tube (No. 3 pipes) that sample protein A 1 antibody addition is minimum, is
Stablize the minimum steady needed for 1ml colloidal gold solution quantitative, on the basis of this minimum steady is quantitative, adds 10%～20% is
The actually used amount of serum amyloid A protein 1 antibody needed for stablizing 1ml colloidal gold solution, i.e. serum amyloid A protein 1 resist
Body with the usage ratio of colloidal gold solution is: 4.4ug～4.8ug:1ml.
(2) the PH value with 0.1mol/L solution of potassium carbonate regulation colloidal gold solution is 8.5, under magnetic stirring, according to
The usage ratio (4.4ug～4.8ug:1ml) of above-mentioned serum amyloid A protein 1 antibody determined and colloidal gold solution is to colloid
Gold solution is added dropwise over serum amyloid A protein 1 antibody, continues stirring 20 minutes, add final concentration of 1% Sanguis Bovis seu Bubali pure
Albumen (BSA), is stirred for 15 minutes, it is thus achieved that serum amyloid A protein 1 antibody is golden with the coupling label of gold colloidal marks blood
Clear amyloid A 1 antibody, 4 DEG C save backup.
(3) purification of gold mark serum amyloid A protein 1 antibody
Use low temperature supercentrifugation purification gold mark serum amyloid A protein 1 antibody, to remove unlabelled serum shallow lake in solution
The gold colloidal of powder sample protein A 1 antibody and the most fully mark serum amyloid A protein 1 antibody note and being likely to be formed in the markers
Various polymer.First gold is marked serum amyloid A protein 1 antibody (volume is V) at 4 DEG C, with 3000rpm/min low speed
Centrifugal 30 minutes, careful Aspirate supernatant, discard precipitation；Again by supernatant at 4 DEG C, it is centrifuged 30 points with 10000rpm/min
Clock, supernatant discarded, it is heavy to dissolve with the PBS buffer (including 1% BSA and 0.02% sodium azide) of 0.01mol/L, pH7.4
Form sediment to original volume (refer to gold mark serum amyloid A protein 1 antibody volume V), substantially stabilized after overnight: at 4 DEG C, then with
10000rpm/min is centrifuged 40 minutes, supernatant discarded, (includes 1% BSA with the PBS buffer of 0.01mol/L, pH7.4
With 0.02% sodium azide) dissolution precipitation to original volume (refer to gold mark serum amyloid A protein 1 antibody volume V) 1/
10, it is thus achieved that gold mark serum amyloid A protein 1 antibody of purification, 4 DEG C save backup.
Three, the assembling of test strips
(1) use gold spraying instrument that the coupling label of serum amyloid A protein 1 antibody Yu gold colloidal is sprayed on glass fibre element film
On, serum amyloid A protein 1 antibody is 4.4ug～4.8ug:1ml with the coupling label of gold colloidal, under the conditions of room temperature 37 DEG C
Natural drying, seals, it is thus achieved that gold-marking binding pad 3,4 DEG C save backup；
(2) use a stroke film instrument that the monoclonal antibody of serum amyloid A protein 1, sheep anti-mouse igg are drawn film respectively in cellulose nitrate
Element film 4 on detection line 5 and nature controlling line 6 on, the monoclonal antibody of serum amyloid A protein 1, the labelled amount of sheep anti-mouse igg are divided
Other 1.2 μ g/cm, 2 μ g/cm, natural drying under room temperature condition, seal, it is thus achieved that (be coated serum amyloid protein with detection line 5
The monoclonal antibody of A1) and the nitrocellulose filter 4,4 DEG C of nature controlling line 6 (being coated with sheep anti-mouse igg) save backup；
(3) sample pad 2 handled well above-mentioned, gold-marking binding pad 3, with detection line 5 and the celluloid of nature controlling line 6
The materials such as film 4, absorption pad 7 are viscous successively to be posted on PVC base plate 1, cutting, assembles, seals, it is thus achieved that the serum amyloid sample of the present invention
Protein A 1 colloidal gold colloidal gold detection test paper strip, room temperature preservation is standby.Test strips after assembling is as shown in Figure 1.
Four, replica test
(1) repeatability detection in group:
Positive with ELISA test strip serum amyloid A protein 1 negative serum, the serum amyloid A protein 1 with a batch of present invention
Property each 30 parts of samples of serum (repeat for three times test).Result shows, the feminine gender of the ELISA test strip of the present invention, positive findings divide
Not being 30 examples, this shows that the test strips of the present invention has good repeatability.
(2) repeatability detection between group:
With ELISA test strip serum amyloid A protein 1 negative serum of the present invention of 3 different batches, serum amyloid protein
The each 30 parts of samples of A1 positive serum (repeat test for three times).Result shows, the feminine gender of each batch ELISA test strip, positive knot
Fruit is respectively 30 examples, again shows that the test strips of the present invention has good repeatability.
Claims (7)
1. a colloidal-gold detecting-card for serum amyloid A protein 1, is characterized in that: gold-marking binding pad is coated with the SAAl of purification
The coupling label of antibody and gold colloidal, parallel on nitrocellulose filter is provided with a SAA1 being coated with SAA1 monoclonal antibody
Detection line and a nature controlling line being coated with sheep anti-mouse igg polyclonal antibody.
2. a preparation method for the colloidal-gold detecting-card of serum amyloid A protein 1, its method is:
The preparation of the coupling label of step one, serum amyloid A protein 1 antibody and gold colloidal, stepwise dilution method determines human blood
Clear amyloid A 1 antibody is 4.4ug～4.8ug:1ml with the usage ratio of colloidal gold solution；
It is added dropwise over serum amyloid A protein 1 antibody of purification according to this usage ratio, adds the Sanguis Bovis seu Bubali of final concentration of 1%
Pure albumen, obtains the coupling label of serum amyloid A protein 1 antibody and gold colloidal after stirring, use low temperature ultracentrifugation
This coupling label is purified by method；
Step 2, the assembling of test strips, use gold spraying instrument by the coupling label of serum amyloid A protein 1 antibody Yu gold colloidal
It is sprayed on glass fibre element film, natural drying under the conditions of room temperature 37 DEG C, seals, it is thus achieved that gold-marking binding pad；Use and draw film instrument by pure
SAA1 monoclonal antibody, the sheep anti-mouse igg changed are drawn on film detection line on nitrocellulose filter and nature controlling line respectively；At Jiang
The sample pad managed, gold-marking binding pad, nitrocellulose filter are viscous successively to be posted on PVC base plate, cutting, assembles, seals, it is thus achieved that
Serum amyloid A protein 1 colloidal gold colloidal gold detection test paper strip, room temperature preservation；
Further, described colloidal gold solution uses trisodium citrate reduction method to prepare, and takes 1% chlorauric acid solution 1ml, adds
The ultra-pure water of 99ml is configured to the chlorauric acid solution of final concentration of 0.01%, after being heated to boiling, adds 1% trisodium citrate
1ml also continues heating, and solution is transferred to the black-and-blue claret that eventually becomes by faint yellow, continues heating 10 minutes after colour stable,
Room temperature cools down, it is thus achieved that colloidal gold solution, 4 DEG C save backup, a diameter of 40nm of colloid gold particle；
Further, in step one, state stepwise dilution method and determine the consumption of serum amyloid A protein 1 antibody and colloidal gold solution
The detailed process of ratio is as follows, and the PH value regulating colloidal gold solution with the carbonate solution of 0.1mol/L is 8.5, to 11
Centrifuge tube is separately added into 1ml colloidal gold solution；I.e. content after serum amyloid A protein 1 antibody stepwise dilution is respectively
1ug, 2ug, 4ug, 8ug, 10ug, 12ug, 14ug, 16ug, 18ug, 20ug, join No. 2 pipes in the order described above to No. 11
Guan Zhong, and mix with the colloidal gold solution in centrifuge tube, in No. 2 pipes to No. 11 pipes, it is separately added into the chlorine of 10% after 10 minutes
Changing sodium solution 1ml mixing, room temperature stands more than 2 hours observed results, and No. 1 pipe is blank；
No. 2, No. 3, in No. 9 pipes to No. 11 pipes, coagulation phenomenon from red to blue occurs, in No. 4 pipes to No. 8 pipes, keep gold colloidal
Redness constant；Therefore, centrifuge tube that is No. 4 pipe that gold colloidal is red constant and serum amyloid A protein 1 antibody addition is minimum
In serum amyloid A protein 1 antibody content, be the minimum steady needed for stable 1ml colloidal gold solution quantitative, minimum at this
Add 10%～20% on the basis of stable quantity to be serum amyloid A protein needed for stable 1ml colloidal gold solution 1 and resist
The actually used amount of body 1, i.e. serum amyloid A protein 1 antibody with the usage ratio of colloidal gold solution be: 4.4ug～4.8ug:
1ml；
Further, in step one, the detailed process using low temperature supercentrifugation to be purified this coupling label is as follows,
First serum amyloid A protein 1 antibody that volume is V is centrifuged 30 minutes with 3000rpm/min at 4 DEG C, draws supernatant
Liquid, abandons precipitation；Again supernatant is centrifuged 30 minutes with 10000rpm/min at 4 DEG C, supernatant discarded, with 0.01mol/L,
The PBS buffer solution of pH7.4 is precipitated to the original volume i.e. volume V of serum amyloid A protein 1 antibody, overnight to 4 after stablizing
At DEG C, then it is centrifuged 40 minutes with 10000rpm/min, abandons supernatant, sink with the PBS buffer solution of 0.01mol/L pH7.4
Form sediment to the 1/10 of the original volume i.e. volume V of serum amyloid A protein 1 antibody, it is thus achieved that the serum amyloid A protein 1 of purification resists
Body, 4 DEG C save backup；Containing 1% BSA and 0.02% sodium azide in described PBS buffer.
The preparation method of the colloidal-gold detecting-card of a kind of serum amyloid A protein 1 the most as claimed in claim 2, its method is:
Described colloidal gold solution uses trisodium citrate reduction method to prepare, and takes 1% chlorauric acid solution 1ml, and the ultra-pure water adding 99ml is joined
It is set to the chlorauric acid solution of final concentration of 0.01%, after being heated to boiling, adds 1% trisodium citrate 1ml and continue heating,
Solution is transferred to the black-and-blue claret that eventually becomes by faint yellow, continues heating 10 minutes after colour stable, and room temperature cools down, it is thus achieved that glue
Body gold solution, 4 DEG C save backup, a diameter of 40nm of colloid gold particle.
The preparation method of the colloidal-gold detecting-card of a kind of serum amyloid A protein 1 the most as claimed in claim 2, its method is:
In step one, described stepwise dilution method determines the concrete mistake of serum amyloid A protein 1 antibody and the usage ratio of colloidal gold solution
Journey is as follows: the PH value regulating colloidal gold solution with the carbonate solution of 0.1mol/L is 8.5, in 11 centrifuge tubes respectively
Add 1ml colloidal gold solution；By after serum amyloid A protein 1 antibody stepwise dilution i.e. content be respectively 1ug, 2ug, 4ug,
8ug, 10ug, 12ug, 14ug, 16ug, 18ug, 20ug, join No. 2 pipes in the order described above in No. 11 pipes, and with centrifugal
In pipe colloidal gold solution mixing, be separately added in No. 2 pipes to No. 11 pipes after 5 minutes 10% sodium chloride solution 1ml mix
Even, room temperature stands more than 2 hours observed results, and No. 1 pipe is blank；
No. 2, No. 3, in No. 9 pipes to No. 11 pipes, coagulation phenomenon from red to blue occurs, in No. 4 pipes to No. 8 pipes, keep gold colloidal
Redness constant；Therefore, centrifuge tube that is No. 4 pipe that gold colloidal is red constant and serum amyloid A protein 1 antibody addition is minimum
In serum amyloid A protein 1 antibody content, be the minimum steady needed for stable 1ml colloidal gold solution quantitative, minimum at this
Add 10%～20% on the basis of stable quantity and be the human serum amyloid A 1 needed for stable 1ml colloidal gold solution
The actually used amount of antibody 1, i.e. serum amyloid A protein 1 antibody with the usage ratio of colloidal gold solution be: 4.4ug～
4.8ug:1ml。
The preparation method of the colloidal-gold detecting-card of a kind of serum amyloid A protein 1 the most as claimed in claim 2, its method is:
In step one, use low temperature supercentrifugation detailed process that this coupling label is purified as follows: to be first V by volume
Serum amyloid A protein 1 antibody be centrifuged 30 minutes with 3000rpm/min at 4 DEG C, Aspirate supernatant, abandon precipitation；Again will
Supernatant is centrifuged 30 minutes with 10000rpm/min at 4 DEG C, supernatant discarded, buffers with the PBS of 0.01mol/L, pH7.4
Liquid dissolution precipitation is to the original volume i.e. volume V of serum amyloid A protein 1 antibody, after stablizing overnight: at 4 DEG C, then with
10000rpm/min is centrifuged 40 minutes, abandons supernatant, is precipitated to substance with the PBS buffer solution of 0.01mol/L pH7.4
The 1/10 of the volume V of long-pending i.e. serum amyloid A protein 1 antibody, it is thus achieved that serum amyloid A protein 1 antibody of purification, 4 DEG C of preservations
Standby；Containing 1% BSA and 0.02% sodium azide in described PBS buffer.
The preparation method of the colloidal-gold detecting-card of a kind of serum amyloid A protein 1 the most as claimed in claim 2, its method is:
In step 2, serum amyloid A protein 1 antibody is 4.4ug～4.8ug:1ml with the coupling label of gold colloidal.
The preparation method of the colloidal-gold detecting-card of a kind of serum amyloid A protein 1 the most as claimed in claim 2, its method is:
In step 2, serum amyloid A protein 1 antibody, labelled amount 1.2 μ g/cm, the 2 μ g/cm respectively of sheep anti-mouse igg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610621021.4A CN106290902A (en) | 2016-08-02 | 2016-08-02 | A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610621021.4A CN106290902A (en) | 2016-08-02 | 2016-08-02 | A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106290902A true CN106290902A (en) | 2017-01-04 |
Family
ID=57663970
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610621021.4A Pending CN106290902A (en) | 2016-08-02 | 2016-08-02 | A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106290902A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982876A (en) * | 2018-08-27 | 2018-12-11 | 浙江省人民医院 | SAA1 detection agent is preparing the application in the kit for diagnosing Henoch Schonlein purpura nephritis |
| CN110927388A (en) * | 2019-11-25 | 2020-03-27 | 益善生物技术股份有限公司 | Preparation method of CRP and SAA combined detection test strip, and prepared test strip, reagent card and kit |
| CN112782408A (en) * | 2020-12-24 | 2021-05-11 | 深圳市科曼医疗设备有限公司 | Serum amyloid A colloidal gold immunoturbidimetry detection kit |
| CN117603335B (en) * | 2024-01-19 | 2024-04-30 | 北京春雷杰创生物科技有限公司 | Human serum amyloid A mutant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879562A (en) * | 2012-09-27 | 2013-01-16 | 上海奥普生物医药有限公司 | Colloidal gold immunofiltration quantitive detection method and reagent kit |
| CN105628932A (en) * | 2016-02-01 | 2016-06-01 | 苏州东尼生物技术有限公司 | SAA (serum amyloid A) immunochromatographic test strip and preparation method and test method thereof |
-
2016
- 2016-08-02 CN CN201610621021.4A patent/CN106290902A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879562A (en) * | 2012-09-27 | 2013-01-16 | 上海奥普生物医药有限公司 | Colloidal gold immunofiltration quantitive detection method and reagent kit |
| CN105628932A (en) * | 2016-02-01 | 2016-06-01 | 苏州东尼生物技术有限公司 | SAA (serum amyloid A) immunochromatographic test strip and preparation method and test method thereof |
Non-Patent Citations (1)
| Title |
|---|
| CLAUDIA PARET 等: "Inflammatory Protein Serum Amyloid A1 Marks a Subset of Conventional Renal Cell Carcinomas with Fatal Outcome", 《EUROPEAN UROLOGY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108982876A (en) * | 2018-08-27 | 2018-12-11 | 浙江省人民医院 | SAA1 detection agent is preparing the application in the kit for diagnosing Henoch Schonlein purpura nephritis |
| CN108982876B (en) * | 2018-08-27 | 2021-09-17 | 浙江省人民医院 | Application of SAA1 detection agent in preparation of kit for diagnosing Henoch-Schonlein purpura nephritis |
| CN110927388A (en) * | 2019-11-25 | 2020-03-27 | 益善生物技术股份有限公司 | Preparation method of CRP and SAA combined detection test strip, and prepared test strip, reagent card and kit |
| CN112782408A (en) * | 2020-12-24 | 2021-05-11 | 深圳市科曼医疗设备有限公司 | Serum amyloid A colloidal gold immunoturbidimetry detection kit |
| CN117603335B (en) * | 2024-01-19 | 2024-04-30 | 北京春雷杰创生物科技有限公司 | Human serum amyloid A mutant |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2017107974A1 (en) | Detection test kit for serum psmd4 proteins and detection method and application thereof | |
| EP3242133B1 (en) | Composition and system for separating and detecting alpha-fetoprotein variant and use thereof | |
| CN103472229B (en) | A kind of carcinomebryonic antigen magnetic microparticle chemiluminescence immune assay detection kit | |
| CN101699287B (en) | Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof | |
| CN104237522A (en) | Adiponectin content detection kit and preparation method thereof | |
| CN103792353B (en) | Lipoprotein-related phospholipase A2 content detection kit and preparation method thereof | |
| CN109596843B (en) | A kind of assay kit of serum amyloid A protein | |
| CN111337691B (en) | Sensitive and stable serum procalcitonin determination kit and preparation method and application thereof | |
| CN105403693A (en) | Preparation method of magnetic particle chemiluminescence reagent | |
| CN103364558A (en) | Human tumor marker carcinoembryonic antigen (CEA) magnetic particle chemiluminiscence immunoassay kit and detection method | |
| CN102662064A (en) | Immunonephelometry kit for detecting lipid carrier protein related to neutrophils gelatinase and preparation method thereof | |
| CN106290902A (en) | A kind of colloidal-gold detecting-card of serum amyloid A protein 1 and preparation method thereof | |
| Burger | [59] Assays for agglutination with lectins | |
| CN110988364A (en) | Method for detecting GVHD (GVHD-associated cytokine) after transplantation by using flow cytometry and detection kit | |
| CN102243241A (en) | Homogeneous phase aerosol particle-type neutrophile granulocyte gelatinase-related lipid carrier protein determination kit and preparation method thereof | |
| CN106226518A (en) | Canine distemper virus colloidal gold immunochromatographydetection detection test paper bar and preparation method thereof | |
| CN105548547A (en) | Flow type array immunoassay kit for detecting lung cancer markers based on flow cytometry | |
| CN110456076A (en) | The method of multiple antibody immune magnetic beads enrichment detection CTCs | |
| CN103777026A (en) | Kit for diagnosing hepatocellular carcinoma | |
| CN206038696U (en) | Ration calprotectin detects immunochromatographic test strip | |
| CN104101715A (en) | Kit for detecting DOCK8 (dedicator of cytokinesis 8) protein and non-diagnostic DOCK8 protein detection method | |
| CN107271692B (en) | Fluorescent microsphere for marking specific high-affinity recombinant antibody and application thereof | |
| CN109738623A (en) | A kind of Serum A 1- acidoglycoprotein assay kit | |
| WO2024001044A1 (en) | Biomarker combination related to lung cancer, kit containing same, and use thereof | |
| EP2757376A9 (en) | Molecular marker for early indentification of pleural mesothelioma patients, and expression analysis method for same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170104 |