CN101158690A - Immune syntony scattering spectrometry for rapid measuring copper blue protein - Google Patents
Immune syntony scattering spectrometry for rapid measuring copper blue protein Download PDFInfo
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- CN101158690A CN101158690A CNA2007100506047A CN200710050604A CN101158690A CN 101158690 A CN101158690 A CN 101158690A CN A2007100506047 A CNA2007100506047 A CN A2007100506047A CN 200710050604 A CN200710050604 A CN 200710050604A CN 101158690 A CN101158690 A CN 101158690A
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Abstract
The present invention discloses a simple and rapid immune resonance scattering spectroscopy for the determination of ceruloplasmin, which mainly makes use of the specific immune binding reaction between goat anti-human ceruloplasmin and ceruloplasmin under the condition of PEG in the 37 DEG C water bath for production of immune complex particles. The resonance scattering is existed in the complex particles. The concentration of the ceruloplasmin is linear with the strength of the resonance scattering when the concentration of ceruloplasmin is in a certain range, thereby establishing a novel method for the rapid determination of the ceruloplasmin. The present invention has the advantages that: compared with the prior art, the determination method has simple instrument, easy operation, high sensitivity and selectivity, and easy achievement of the reaction conditions. The dosage of the goat anti-human CER reagent is less and the cost is low.
Description
Technical field:
The present invention relates to the assay method of albumen, particularly a kind of immune syntony scattering spectrometry of energy fast measuring CER.
Background technology:
CER belongs to α
2-globulin (CER) is a kind of α-glycoprotein of cupric, is unique blue protein in human and the vertebrate body.The CER detection method is a lot of at present, as according to its oxidase characteristic, uses p-phenylenediamine (PPD) (PPD) method; According to its immunological characteristic, assay method has enzyme linked immunosorbent assay, radio immunoassay, immunoprecipitation colorimetric analysis, rocket immunoelectrophoresis analytic approach, immune electrodiffusion analytic approach, in addition, also have pulse laser sound analysis method, application rate nephelometric turbidity method etc., more than these methods to have plenty of sensitivity not high, has plenty of complex steps, have because matrix, reaction conditions etc. are inconsistent, and the result lacks comparability, the antiserum consumption that has is big, and the purity requirement height, not easy to operate.
Summary of the invention:
The objective of the invention is for overcoming the deficiencies in the prior art, and the immune syntony scattering spectrometry of the easy fast measuring CER of a kind of energy is provided.
Realize that purpose technical scheme of the present invention is: utilize CER and goat-anti human ceruloplasmin under the uniform temperature water bath condition, produce the specific immunity association reaction and generate the compound particulate of immunity, there is resonance scattering in this compound particles, CER in the finite concentration scope along with the increase of its concentration, its resonance scattering intensity is linear to be strengthened, adopt fluorospectrophotometer that the immune complex that generates is carried out synchronous scanning and obtain resonance scattering spectroscopy, according to the linear relationship of resonance scattering intensity and CER concentration, fast measuring goes out CER in the serum.
Use immune syntony scattering spectrometry fast measuring CER, comprise the steps:
(1) test system of the known CER concentration of preparation: pipette 0.30mL pH value successively and be 6.4 phosphate buffered solution (PBS), 0.20mL dilute 25 times goat-anti people CER solution, the CER solution of a certain amount of 10.8 μ g/mL, 0.50mL mass concentration is that 15% PEG-6000 solution is in 5mL band scale test tube, be settled to 2.0mL with redistilled water, mixing, incubation 15min in 37 ℃ of water-baths;
(2) do not add CER with the method for step (1) and prepare the blank system;
(3) get above-mentioned system respectively in quartz cell, synchronous scanning excitation wavelength lambda ex and emission wavelength lambda em on fluorospectrophotometer (λ ex=λ em) excite and launch slit to be 5.0nm, and mensuration voltage is 450V, obtain the resonance scattering spectroscopy of system, measure the scattering strength I at 490nm place
RS, reagent blank is I
0, calculate Δ I
RS=I
RS-I
0
(4) with Δ I
RSConcentration relationship workmanship to CER makes curve;
(5) method according to step (1) prepares detection architecture, and wherein the CER of Jia Ruing contains CER but the measured object of unknown concentration, obtains the Δ I of measured object
RS
(6), calculate the concentration of the CER of measured object according to the working curve of (4).
The concentration range of linearity that this method detects CER is 27～927 μ g/ml.
Advantage of the present invention is:
(1) compare with existent method, the instrument of this assay method is simple, and is easy and simple to handle, and anti-interference strong, highly sensitive, selectivity is good, and reaction conditions reaches easily.
(2) reagent only is phosphate buffered solution, goat-anti people CER solution, CER solution, PEG-6000 solution, and used goat-anti people CER solution and CER solution concentration are lower, and reagent dosage is few and all nontoxic.
Description of drawings:
Fig. 1 is the CER immune syntony scattering spectrogram of the embodiment of the invention,
Curve a to e represents a among the figure; PH6.4 PBS-goat-anti people CER-37.5mg/mL PEG6000; B:a-0.027 μ g/ml CER; C:a-0.324 μ g/ml CER; D:a-0.648 μ g/ml CER; E:a-0.972 μ g/ml CER.
Embodiment:
The following examples have been set forth the immune syntony scattering spectral analysis ratio juris that the present invention sets up CER:
Polyglycol (PEG) is the polymkeric substance of ethylene glycol, is a kind of polysaccharide that does not have the electric charge linear molecule, and PEG can cause the immune complex precipitation, and precipitation has reversibility, and precipitated activity of proteins is unaffected, can be used for the mensuration of immune complex.There are complementarity and compatibility on structure and the space between CER and the goat-anti people CER, immune response can take place, generate insolubilized immune complexes, its acting force comprises electric charge gravitation, Van der Waals force, hydrogen bonded power, acting force such as hydrophobic, reaction between the two has higher specificity, and there is resonance scattering in the immune complex of generation.Na at PH6.4
2HPO
4-NaH
2PO
4(PBS) in the damping fluid, CER along with the increase of its concentration, in the linear enhancing of the resonance scattering intensity at 490nm place, sets up the immune syntony scattering spectrographic method of CER in view of the above in the finite concentration scope.
Embodiment:
A kind of immune syntony scattering spectrometry of energy fast measuring CER comprises the steps:
(1) tested systems of the known CER concentration of preparation, pipetting 0.30mL pH value with microscale sampler respectively is that 6.4 phosphate buffered solution is in 5 5mL band scale test tube, add 0.20mL respectively in every test tube and dilute 25 times goat-anti people CER solution (Shang Haibei adds reagent company limited to be provided), pipette the CER solution 0 that concentration is 10.8 μ g/mL with microscale sampler then, 5,60,120,180 μ L inject 5 test tubes respectively, add the 0.50mL mass concentration more respectively and be 15% PEG-6000 solution, be settled to 2.0mL with redistilled water, the difference mixing, incubation 15min in 37 ℃ of water-baths;
(2), do not add CER and prepare the reagent blank system according to the method for step (1);
(3) respectively get in right amount in different quartz cells, go up synchronous scanning excitation wavelength lambda ex and emission wavelength lambda em (λ ex=λ em) at Cary Eclipse fluorospectrophotometer (U.S. Varian company), excite and launch slit and be 5.0nm, mensuration voltage is 450V, obtain the resonance scattering spectroscopy of system, measure the scattering strength I at 490nm place
RS, not add the resonance scattering intensity I that CER solution records
0Be blank, calculate Δ I
RS=I
RS-I
0Value;
(4) with Δ I
RSLinear relationship workmanship to CER concentration makes curve, i.e. Δ I
RS=0.1505c+4.8656, R
2=0.9968.
(5) according to the method for step (1), wherein add the CER of unknown concentration, according to the method for step (3), try to achieve the Δ I of the CER measured object of unknown concentration
RSBe 45.20;
(6) according to the working curve of (4), the CER concentration of obtaining measured object is 267.97ng/mL.
Claims (3)
1. the immune syntony scattering spectrometry of a fast measuring CER, it is characterized in that: assay method comprises the steps:
(1) test system of the known CER concentration of preparation: pipette 0.30mL pH value successively and be 6.4 phosphate buffered solution, 0.20mL dilute 25 times goat-anti people CER solution, the CER solution of a certain amount of 10.8 μ g/mL, 0.50mL mass concentration is that 15% PEG-6000 solution is in 5mL band scale test tube, be settled to 2.0mL with redistilled water, mixing, incubation 15min in 37 ℃ of water-baths;
(2) do not add CER with the method for step (1) and prepare the blank system;
(3) get above-mentioned system respectively in quartz cell, on fluorospectrophotometer, equal emission wavelength lambda em (λ ex=λ em) at excitation wavelength lambda ex, excite and launch slit and be 5.0nm, measure voltage and be synchronous scanning under the condition of 450V, obtain the resonance scattering spectroscopy of system, measure the scattering strength I at 490nm place
RS, reagent blank is I
0, calculate Δ I
RS=I
RS-I
0
(4) with Δ I
RSConcentration relationship workmanship to CER makes curve;
(5) method according to step (1) prepares detection architecture, and wherein the CER of Jia Ruing contains CER but the measured object of unknown concentration, obtains the Δ I of measured object
RS
(6), calculate the concentration of the CER of measured object according to the working curve of (4).
2. the immune syntony scattering spectrometry of fast measuring CER as claimed in claim 1 is characterized in that: in the scanning of the described resonance spectrum of step (2), answering synchronous scanning excitation wavelength lambda ex and emission wavelength lambda em is λ ex=λ em.
3. the immune syntony scattering spectrometry of fast measuring CER as claimed in claim 1 is characterized in that: the concentration range of linearity of measuring CER is 27～927 μ g/ml.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100506047A CN101158690A (en) | 2007-11-19 | 2007-11-19 | Immune syntony scattering spectrometry for rapid measuring copper blue protein |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100506047A CN101158690A (en) | 2007-11-19 | 2007-11-19 | Immune syntony scattering spectrometry for rapid measuring copper blue protein |
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|---|---|
| CN101158690A true CN101158690A (en) | 2008-04-09 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103954599A (en) * | 2014-05-08 | 2014-07-30 | 重庆医科大学 | Method for determining active site concentration of monoclonal antibody by means of fluorescence titration |
-
2007
- 2007-11-19 CN CNA2007100506047A patent/CN101158690A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103954599A (en) * | 2014-05-08 | 2014-07-30 | 重庆医科大学 | Method for determining active site concentration of monoclonal antibody by means of fluorescence titration |
| CN103954599B (en) * | 2014-05-08 | 2016-06-08 | 重庆医科大学 | A kind of fluorescence titration measures the method for monoclonal antibody avtive spot concentration |
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Open date: 20080409 |