CN101581722A - Method for detecting microalbumin - Google Patents
Method for detecting microalbumin Download PDFInfo
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- CN101581722A CN101581722A CNA2009100403798A CN200910040379A CN101581722A CN 101581722 A CN101581722 A CN 101581722A CN A2009100403798 A CNA2009100403798 A CN A2009100403798A CN 200910040379 A CN200910040379 A CN 200910040379A CN 101581722 A CN101581722 A CN 101581722A
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Abstract
The invention provides a method for detecting albumin with nanogram content, which comprises the following steps: diluting an albumin standard product into concentration gradient which is properly distributed in nanogram level, adding excessive albumin antibody, and performing first incubation; adding excessive albumin labeled with radioactive isotope relative to residual albumin antibody into liquid after the incubation, and performing secondary incubation to combine partial albumin labeled with the radioactive isotope and the albumin antibody into a compound; separating the compound and determining radiocounting of the compound; determining a relational expression between the radiocounting and concentration according to the radiocounting and the concentration of the albumin in a corresponding standard product; and repeating the steps by replacing the albumin standard product by a sample to be tested, and determining the concentration of the albumin in the sample to be tested according to the measured radiocounting of the sample to be tested and the relational expression. The method can detect the albumin with the nanogram content, and has high accuracy and good stability.
Description
Technical field
The present invention relates to a kind of method that detects microalbumin, being specifically related to detection level is the albuminous method of nanogram level.
Background technology
The albumin radio immunoassay is a kind of radioimmunoassay technique that is used for measuring people's urine or body fluid albumin content, and its principle is: the albumin in testing sample, the standard items and
125The I-albumin combines with limited amount antibody competition,
125An I-albumin part is a compound with antibodies, and another part is free; The centrifugal compound part that makes is separated with free fraction, and taking precipitate is also measured its radiocounting; The albumin content in testing sample and the standard items and the radioactive intensity of compound are negative correlation, do standard with the albumin of a series of variable concentrations, can obtain typical curve, can find albumin content the sample from typical curve.The albumin radiommunoassay medicine box of Chu Shouing is mainly used in and measures albumin content in people's urine in the market, and an example of the using method of this medicine box mensuration albumin content is as follows:
(1) is 1 μ g/ml, 2 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml and 50 μ g/ml with distilled water with the dilution of albumin standard items, respectively gets 100 μ l, be designated as standard pipe B
1, B
2, B
3, B
4, B
5And B
6
(2) establish non-special pipe and zero standard pipe, be designated as NSB and B
0, add incubation liquid 300 μ l and 100 μ l respectively;
(3) get each 100 μ l of detected sample, be designated as sample hose S
1～S
N(N=1);
(4) at B
0, B
1～B
6And S
1～S
NRespectively add albumin antibody 200 μ l in the pipe;
(5) get a blank pipe, be designated as the T pipe;
(6) at T, NSB, B
0, B
1～B
6And S
1～S
NRespectively add in the pipe
125I-albumin 100 μ l fully shake up, 37 ℃ of incubations 0.5 hour;
(7) at NSB, B
0, B
1～B
6And S
1～S
NRespectively add in the pipe and separate reagent 500 μ l, fully shake up the back room temperature and left standstill 15 minutes, 3500 rev/mins centrifugal 20 minutes;
(8) supernatant discarded, on the r-calculating instrument, measure each pipe counting 60 seconds, B/B0%={[A-NSB pipe counting by formula at last]/[zero standard pipe counting-NSB manages counting] } * 100% calculate: with the calculating of the A in the count results substitution formula of standard pipe, draw the B/B of standard pipe
0The % value; Calculate with the A in the count results substitution formula of sample hose, draw the B/B of sample hose
0The % value;
(9) with the B/B of standard pipe
0The % value is an ordinate, is horizontal ordinate with the albumin concentration of standard pipe, drawing standard curve on semilogarithmic paper, and the albuminous concentration of sample is the B/B of pipe per sample
0The % value checks in from typical curve.
The sensing range of said method is 1 μ g/ml～50 μ g/ml, if the albumin content of sample exceeds 50 μ g/ml, can be with repeating step (1)～(9) again behind the diluted sample, and the result that will check in from typical curve multiply by extension rate and gets final product then.This method has the high advantage of good stability, accuracy and specificity, but its sensitivity is relatively poor, only can detect other albumin of Gamma Magnitude, for the lower albumin of content, for example synthetic other the micro-human albumin of nanogram level of in vitro culture liver cell then can't detect.And the inventor finds, though with the concentration of standard solution turn down to the nanogram rank with the production standard curve, also can't realize other the albuminous accurate detection of nanogram level.
Summary of the invention
The technical problem to be solved in the present invention is the sensitivity that improves the albumin radio immunoassay, makes it can detect content at other albumin of nanogram level.
For addressing this problem, the invention provides a kind of method that detects microalbumin, this method may further comprise the steps:
(a) the albumin standard items are diluted in the nanogram rank concentration gradient that suitably distributes, add excessive albumin antibody, and carry out the first time incubation so that they fully react;
(b) adding in the liquid behind incubation with respect to the residue albumin antibody is the albumin of excessive labelled with radioisotope, and carry out for the second time incubation so that the albumin of a part of labelled with radioisotope and albumin antibody are combined into compound, and the albumin of another part labelled with radioisotope is a free state;
(c) separate the albumin of the labelled with radioisotope of described compound and free state, and measure the radiocounting of compound;
(d) according to the definite relational expression between the two of albuminous concentration in radiocounting and the corresponding standard product;
(e) replace albumin standard items repeating steps (a)-(c) with testing sample, according to testing sample measured radiocounting and described relational expression are determined albuminous concentration in the testing sample.
In preferred implementation of the present invention, the described concentration gradient of albumin standard items is in the step (a): 2ng/m1,10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml and 200ng/ml.
In preferred implementation of the present invention, the incubation first time described in the step (a) continues 4～6 hours under 37 ℃～38 ℃ temperature.
In preferred implementation of the present invention, the radioactive isotope described in the step (b) is
3H,
125I or
131I.
In preferred implementation of the present invention, the incubation second time described in the step (b) continues 12～14 hours under 37 ℃～38 ℃ temperature.
In an embodiment of the invention, the testing sample described in the step (b) is from the hepatocellular supernatant of in vitro culture.
In the method for the invention, albumin and the albumin in the standard items in the testing sample at first fully combine with albumin antibody, (for example add radioactive isotope again
125I) albumin of mark makes itself and remaining antibodies, and the albumin of a part of labelled with radioisotope and antibodies are compound, and another part is free; Add separation agent and centrifugal, make the compound part separate with free fraction, taking precipitate is also measured its radiocounting; The albumin content in testing sample and the standard items and the radioactive intensity of compound are negative correlation, do standard with the albumin of a series of variable concentrations, can obtain typical curve, can find albumin content the sample from typical curve.
The inventive method has realized nanogram rank albumin content mensuration, has satisfied the needs of measuring the synthetic micro-human albumin of in vitro culture liver cell; Method of the present invention is diluted reagent, has reduced the especially isotope-labeled albuminous consumption of reagent, thereby has reduced the pollution of radioactive material confrontation environment.
Description of drawings
Fig. 1 is the canonical plotting of drawing according to an embodiment of the invention.
Embodiment
For methods of this invention will be better understood, will further set forth the present invention and advantage thereof by embodiment and experimental evidence below.Before describing following examples and experiment in detail, at first need definition and clarify some terms.
" radio immunoassay " as herein described is meant the technology that antigen that use is marked with radioactive isotope or antibody are measured antibody or antigen amount.For example, unmarked antigen (checking matter) with unknown quantity, when the reaction that is added on the labelled antigen of known quantity and antibody is, relies on and measure unmarked antigen how the competitiveness of labelled antigen synantibody combination suppressed to reach degree, thereby can measure the quantity of checking matter.Because sensitivity is very sharp, so trace (as the microgram rank) material that is difficult to measure with conventional method can be measured with this method.
" albumin standard items " as herein described be meant under rational storage requirement, have stability, content accurately, highly purified albumin.It is the quantitative benchmark of radio immunoassay, owing to represent the amount of measured matter with the amount of standard items, so standard items and measured matter should the chemical constitution unanimities and had identical immunocompetence.By requirement of experiment, available damping fluid is diluted to the solution of variable concentrations with standard items, is used for the production standard curve.In the present invention, use distilled water to dilute the albumin standard items.The albumin standard items that the present invention uses can be bought the albumin radiommunoassay medicine box from Beijing Atom High Tech Co., Ltd. and obtain.
" nanogram rank " as herein described is meant the scope of several nanograms to the hundreds of nanogram, as 2 nanograms, 20 nanograms, 200 nanograms or 500 nanograms, but is no more than 1000 nanograms.For " concentration gradient that suitably distributes in the nanogram rank " as herein described, those skilled in the art can determine at an easy rate that according to existing knowledge and actual needs suitable concentration gradient distributes.
" incubation " as herein described can carry out in incubation case, electric-heated thermostatic water bath or other can keep the instrument of constant temperature for a long time.
" radioactive isotope " as herein described can be
3H,
125I,
131I etc.
3H can replace the hydrogen in the organic compound and not influence original chemical property, and long half time, energy are low, is convenient to protection;
125I and
131The chemical property of I atom is more active, and labeling method is easy, can be used for the haptenic mark of polypeptide, protein and micromolecule.Some can not directly use the haptens of iodine labeling, by connecting also available iodine mark of a tyrosine.Preferably use in this article
125I carries out mark to albumin.Use among the present invention
125The I-albumin can be bought the albumin radiommunoassay medicine box from Beijing Atom High Tech Co., Ltd. and obtain.
" separation agent " as herein described can be bought the albumin radiommunoassay medicine box from Beijing Atom High Tech Co., Ltd. and obtain, and its principal ingredient is anti-sheep blood serum of donkey and PEG.
An embodiment implementing the inventive method below is provided, can detects other albumin of nanogram level by this method, this method may further comprise the steps:
(1) is 2ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml and 200ng/ml with distilled water with the dilution of albumin standard items, respectively gets 200 μ l, be designated as standard pipe B respectively
1, B
2, B
3, B
4, B
5And B
6
(2) establish non-special pipe and zero standard pipe, be designated as NSB and B
0, add incubation liquid (composition is the PBS damping fluid) 300 μ l and 100 μ l respectively;
(3) get each 200 μ l of detected sample, be designated as sample hose S
1～S
N, N=1 wherein;
(4) with the distilled water diluting albumin antibody of 3 times of volumes, at B
0, B
1～B
6And S
1～S
NRespectively add 100 μ l in the pipe, fully shake up, 37 ℃ or 38 ℃ of incubations 4～6 hours;
(5) get a blank pipe, be designated as T; At T, NSB, B
0, B
1～B
6And S
1～S
NEach adding equal-volume distilled water diluting in the pipe
125I-albumin 100 μ l fully shake up, 37 ℃ of incubations 12 hours;
(6) at NSB, B
0, B
1～B
6And S
1～S
NRespectively add in the pipe and separate reagent 500 μ l, fully shake up the back room temperature and left standstill 15 minutes, 3500 rev/mins centrifugal 20 minutes;
(7) supernatant discarded is measured each pipe counting 60 seconds on the r-calculating instrument, at last B/B by formula
0The %={[A-NSB counting]/[B
0Counting-NSB counting] } * 100% calculate: with B
1, B
2, B
3, B
4, B
5Or B
6Count results substitution formula in A calculate, draw the B/B of standard pipe
0The % value; With S
1～S
NCount results substitution formula in A calculate, draw the B/B of sample hose
0The % value;
(8) with the B/B of standard pipe
0The % value is an ordinate, and the albumin concentration of standard pipe is a horizontal ordinate, draws albumin concentration and B/B on semilogarithmic paper
0The typical curve of % value relation, the B/B of pipe per sample then
0The % value is found this B/B from typical curve
0The albumin concentration that the % value is corresponding is albuminous concentration in the sample hose.
The sensing range of said method is 2ng/ml～200ng/ml, if the albumin content of sample exceeds 200ng/ml, can be with repeating step (1)～(8) again behind the diluted sample, the result that will check in from typical curve multiply by extension rate and can obtain albuminous actual concentrations in the sample then.
Those skilled in the art can easily recognize, for the purpose of fast detecting, when implementing method of the present invention, can operate albumin standard items (being used for the drawing standard curve) and testing sample simultaneously.
Embodiment
One, reagent is prepared
Buy albumin radiommunoassay medicine box from Beijing Atom High Tech Co., Ltd., therefrom get 3 bottles of albumin standard items (dried frozen aquatic products) adding distil water and fully dissolve, being made into concentration is the albumin standard items of 1 μ g/ml, 2 μ g/ml and 5 μ g/ml; With the albumin standard items in testing sample and the albumin radiommunoassay medicine box,
125I-albumin, incubation liquid, albumin antibody, separation agent fully shake up, and balance is to room temperature.
Two, the detection of the drafting of typical curve and sample
1, the dilution of albumin standard items: get 7 clean tube, be labeled as A, B, C, D, E, F and G respectively, the albumin standard items of accurately measuring 100 μ l, 1 μ g/ml add in the A pipe, measuring 900 μ l distilled water again adds in the A pipe, fully shake up, be mixed with the albumin standard items of 100ng/ml; The albumin standard items of accurately measuring 100 μ l 100ng/ml from the A pipe add in the B pipe, measure 900 μ l distilled water again and add in the B pipe, fully shake up, and are mixed with the albumin standard items of 10ng/ml; The albumin standard items of accurately measuring 100 μ l, 2 μ g/ml add in the C pipe, measure 900 μ l distilled water again and add in the C pipe, fully shake up, and are mixed with the albumin standard items of 200ng/ml; The albumin standard items of accurately measuring 100 μ l 200ng/ml from the C pipe add in the D pipe, measure 900 μ l distilled water again and add in the D pipe, fully shake up, and are mixed with the albumin standard items of 20ng/ml; The albumin standard items of accurately measuring 100 μ l 20ng/ml from the D pipe add in the E pipe, measure 900 μ l distilled water again and add in the E pipe, fully shake up, and are mixed with the albumin standard items of 2ng/ml; The albumin standard items of accurately measuring 100 μ l, 5 μ g/ml add in the F pipe, measuring 900 μ l distilled water again adds in the F pipe, fully shake up, be mixed with the albumin standard items of 500ng/ml, the albumin standard items of accurately measuring 100 μ l 500ng/ml from the F pipe add in the G pipe, measure 900 μ l distilled water again and add in the G pipe, fully shake up, be mixed with the albumin standard items of 50ng/ml.
Get 6 clean tube, be labeled as standard pipe B respectively
1, B
2, B
3, B
4, B
5And B
6, from E, B, D, G, A, C pipe, respectively get 200 μ l albumin standard items respectively and add B
1～B
6In.
2, establish non-special pipe and zero standard pipe, be designated as NSB and B
0, add incubation liquid 300 μ l and 100 μ l respectively;
3, get the CL-1 cell line (tissue-derived: people's normal liver tissue) ordinary flat is cultivated the 1st, 3,5 and 7 day cell conditioned medium liquid, 2000 rev/mins centrifugal 2 minutes, respectively get 200 μ l and join respectively in the test tube of 4 cleanings, be designated as sample hose S
1～S
4
4, get 1 clean tube, add the 100 μ l albumin antibodies of accurately measuring, add 300 μ l distilled water again, fully shake up; At B
0, B
1～B
6And S
1～S
4In respectively add the albumin antibody 100 μ l of dilution in 1: 4, fully shake up 37 ℃ of incubations 4 hours.
The 500 μ l of 5, accurately measuring
125The I-albumin adds 500 μ l distilled water again, fully shakes up; Get 1 clean tube, be designated as the T pipe; At T, NSB, B
0, B
1～B
6And S
1～S
4In respectively add 1: 2 the dilution
125I-albumin 100 μ l fully shake up, 37 ℃ of incubations 12 hours.
6, at T, NSB, B
0, B
1～B
6And S
1～S
4In respectively add and separate reagent 500 μ l, fully shake up the back room temperature and left standstill 15 minutes, 3500 rev/mins centrifugal 20 minutes.
7, supernatant discarded is measured each pipe counting 60 seconds on the r-calculating instrument, at last B/B by formula
0%={[A-NSB manages counting]/[zero standard pipe counting-NSB manages counting] } * 100% (1) calculate: calculate with the A in the count results substitution formula of standard pipe, draw the B/B of standard pipe
0The % value; Calculate with the A in the count results substitution formula of sample hose, draw the B/B of sample hose
0The % value;
8, with the B/B of standard pipe
0The % value is an ordinate, and the albumin concentration of standard pipe is a horizontal ordinate, and (logarithmic coordinate are longitudinal axis) draws albumin concentration and B/B on semilogarithmic paper
0The relation curve of % value, the B/B of pipe per sample then
0The % value checks in the albuminous concentration of sample from the relation curve of gained.
In other embodiments, the incubation time in above-mentioned the 4th can extend to 6 hours, and the incubation time in above-mentioned the 5th can extend to 14 hours, and testing result does not change.
Three, testing result
1, with standard pipe B
1～B
6Count results substitution formula (1) in calculate B/B
0% result is as shown in table 1; B/B with standard pipe
0The % value is an ordinate, and the albumin concentration of standard pipe is a horizontal ordinate, and (logarithmic coordinate are longitudinal axis) draws albumin concentration and B/B on semilogarithmic paper
0The relation curve of % value, as shown in Figure 1.
Table 1
| The pipe number | B 0 | B 1 | B 2 | B 3 | B 4 | B 5 | B 6 |
| Albumin concentration concentration (ng/ml) | 0 | 2 | 10 | 20 | 50 | 100 | 200 |
| Radiocounting | 1999 | 1783 | 1605 | 1232 | 836 | 749 | 600 |
2, with sample hose S
1～S
4Count results substitution formula (1) in calculate B/B
0% result is respectively 1649,953,1536 and 1655, at albumin concentration and B/B
0The relation curve of % value is searched corresponding B/B
0The albumin concentration that the % value is corresponding can get S
1～S
4In albuminous concentration be respectively 9.92ng/ml, 49.75ng/ml, 13.3ng/ml and 9.75ng/ml.
Four, stability experiment
1, gets sample S in the said determination method
1～S
4Replication 4 times, the result is as shown in table 2, and variation within batch coefficient＜5% meets in the technical indicator＜10% requirement.
Table 2
| The pipe number | S 1 | S 2 | S 3 | S 4 |
| The counting of measuring for the 1st time | 1653 | 956 | 1543 | 1653 |
| The counting of measuring for the 2nd time | 1656 | 966 | 1536 | 1658 |
| The counting of measuring for the 3rd time | 1646 | 988 | 1540 | 1660 |
| The counting that the 4th is measured | 1650 | 977 | 1535 | 1656 |
2, get the sample (S that concentration is 49.75ng/ml
2) 1 all replications at interval: the radiocounting result is 960, can try to achieve interassay coefficient of variation＜5% thus, meets in the technical indicator＜10% requirement.
The albumin antibody that method of the present invention is used reaches
125Lacking of the albuminous amount ratio prior art of I-, saved resource, reduced cost and reduced pollution environment.For realizing purpose of the present invention, method of the present invention is incubation at twice: added behind the albumin antibody incubation 4～6 hours, and added
125Incubation 12～14 hours again behind the I-albumin.The present invention need reach the albumin antibody that provides in the existing medicine box
125The I-albumin dilutes, because the reaction of each material of dilution back all is in minor levels, has avoided a large amount of albumin antibody with a large amount of
125The I-albumin reaction is covered the reaction of albumin and antibody in the standard items of trace or the sample; Simultaneously, method of the present invention need add standard items (or sample) and antibody response earlier and added in 4～6 hours again
125The I-albumin has guaranteed fully combining of albumin and antibody in microstandard product or the sample.
Though the present invention is described with reference to concrete embodiment, those skilled in the art can make conspicuous modification and modification to the present invention by after reading foregoing description, and without prejudice to the intent of the present invention and essence.The present invention comprises these modifications and modification within the scope of the claims intentionally.
Claims (7)
1. method that detects microalbumin, this method may further comprise the steps:
(a) the albumin standard items are diluted in the nanogram rank concentration gradient that suitably distributes, add excessive albumin antibody, and carry out the first time incubation so that they fully react;
(b) adding in the liquid behind incubation with respect to the residue albumin antibody is the albumin of excessive labelled with radioisotope, and carry out for the second time incubation so that the albumin of a part of labelled with radioisotope and albumin antibody are combined into compound, and the albumin of another part labelled with radioisotope is a free state;
(c) separate the albumin of the labelled with radioisotope of described compound and free state, and measure the radiocounting of compound;
(d) according to the definite relational expression between the two of albuminous concentration in radiocounting and the corresponding standard product;
(e) replace albumin standard items repeating steps (a)-(c) with testing sample, according to testing sample measured radiocounting and described relational expression are determined albuminous concentration in the testing sample.
2. method according to claim 1 is characterized in that, the described concentration gradient of albumin standard items is in the step (a): 2ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml and 200ng/ml.
3. method according to claim 1 is characterized in that, the incubation first time described in the step (a) continues 4～6 hours under 37 ℃～38 ℃ temperature.
4. method according to claim 1 is characterized in that, the radioactive isotope described in the step (b) is
3H,
125I or
131I.
5. method according to claim 4 is characterized in that the radioactive isotope described in the step (b) is
125I.
6. method according to claim 1 is characterized in that, the incubation second time described in the step (b) continues 12～14 hours under 37 ℃～38 ℃ temperature.
7. method according to claim 1 is characterized in that, the testing sample described in the step (b) is from the hepatocellular supernatant of in vitro culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100403798A CN101581722A (en) | 2009-06-19 | 2009-06-19 | Method for detecting microalbumin |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2009100403798A CN101581722A (en) | 2009-06-19 | 2009-06-19 | Method for detecting microalbumin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101581722A true CN101581722A (en) | 2009-11-18 |
Family
ID=41363962
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|---|---|---|---|
| CNA2009100403798A Pending CN101581722A (en) | 2009-06-19 | 2009-06-19 | Method for detecting microalbumin |
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| Country | Link |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792315A (en) * | 2014-01-23 | 2014-05-14 | 中国计量科学研究院 | Quantifying method for human albumin inorganic mass spectrum coupling technique |
| CN104020182A (en) * | 2014-05-27 | 2014-09-03 | 中山大学附属第三医院 | Method for determination of stereo shape and distribution of protein and polypeptide drug loaded microsphere |
-
2009
- 2009-06-19 CN CNA2009100403798A patent/CN101581722A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792315A (en) * | 2014-01-23 | 2014-05-14 | 中国计量科学研究院 | Quantifying method for human albumin inorganic mass spectrum coupling technique |
| CN103792315B (en) * | 2014-01-23 | 2015-07-08 | 中国计量科学研究院 | Quantifying method for human albumin inorganic mass spectrum coupling technique |
| CN104020182A (en) * | 2014-05-27 | 2014-09-03 | 中山大学附属第三医院 | Method for determination of stereo shape and distribution of protein and polypeptide drug loaded microsphere |
| CN104020182B (en) * | 2014-05-27 | 2016-04-13 | 中山大学附属第三医院 | The assay method of load albumen, polypeptide drug microballoon solid shape and distribution |
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Application publication date: 20091118 |