CN1045820C - Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay - Google Patents
Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay Download PDFInfo
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- CN1045820C CN1045820C CN 93114698 CN93114698A CN1045820C CN 1045820 C CN1045820 C CN 1045820C CN 93114698 CN93114698 CN 93114698 CN 93114698 A CN93114698 A CN 93114698A CN 1045820 C CN1045820 C CN 1045820C
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Abstract
The present invention comprises: human procollagen III is extracted from periderm of induced abortion and is purified, high titer antiserum is prepared from the antigens and is labeled by <125>I, and radio immunoassay of serum precollagen III (the original PCIII) is established. The method is used for measuring the serum precollagen III contents of 852 patients and normal persons, and results indicate that liver cirrhosis and chronic active hepatitis are increased obviously, and the fibrosis degree of liver biopsy is in close direct correlation with the serum precollagen III level (r=0.984; p<0.001). The measuring method of the present invention has great diagnostic value in hepatic fibrosis and early liver cirrhosis.
Description
What the present invention relates to is medical diagnostic reagent, particularly a kind of human III type precollagen hPC III diagnostic reagent of the diagnosing liver fibrosis under measured by radioimmunoassay, its preparation method and its using method in radiommunoassay.
It is believed that liver fibrosis is the only stage which must be passed bies of various chronic active hepatitis to the cirrhosis development, after cirrhosis further causes the hepatic parenchymal cells grievous injury, could be made a definite diagnosis by the liver function experiment, but general conventional liver function test can't diagnosing liver fibrosis and early-phase hepatocirrhosis.Clinical main dependence liver biopsy is at present judged, but the method is traumatic inspection, and is subjected to the limitation of site of puncture, and is difficult to carry out dynamic observing of liver fibrosis progress.According to the formation and development process of cirrhosis and even liver cancer, world hepatopathy expert thinks and stops or the generation of the liver fibrosis of slowing down will be cured most of chronic hepatitis patients.Therefore, the early diagnosis liver fibrosis reliable markers thing of particularly setting up liver fibrosis clinically has very important significance for the formation and development that prevents cirrhosis.
In recent years, domestic and international many scholar's reports, serum III procollagen type peptide (Procollagen type III Peptide, P III P) is a diagnosing liver fibrosis serological index preferably.Assay method adopts radio immunoassay more, and used antigen extracts from the animal periderm, extracts from the ox periderm exactly as the critical material P III P in the P III P radioimmunological kit of German Beling company (Behr-ingwerke Co.) production.The report that extracts P III P from periderms such as sheep, mouse is also arranged.Its foundation as the diagnosing liver fibrosis mark is that cirrhosis is in early days with III Collagen Type VI (Collagen type III, the C III) it is main increasing, cell when synthetic collagen before form of collagen secrete the extracellular, and blood-serum P III P is an III procollagen type (Procollagen type III, the PC III) polypeptied chain that splits away off through the effect of aminoterminal inscribe peptase is so measure the metabolism status that P III P content can reflect the C III.Yet there is some difference in animals and human beings class tropocollagen molecule some antigenic determinants structurally, must bring influence to measurement result.In addition, some scholar finds that also hepatocellular inflammation and the downright bad cracking of C III so that the blood-serum P III P value that exist of all can making raise in liver, thereby blood-serum P III P value not necessarily points out collagen synthetic increase, that is is that blood-serum P III P value diagnosing liver fibrosis can be subjected to the influence of other factors and inaccurate.In addition, it is very expensive at present to put the price of the agent of being excused from an examination from external import P III P, and the ria-determination complicated operation that it is concrete needs just can obtain P III P content among the patients serum through complicated calculations.
The objective of the invention is: 1. provide a kind of and comprise that the human III type precollagen (human Procollagen type III) that extracts is the hPC III from the stillborn foetus skin of induced abortion, its antibody anti-hPC III, isotope labeling antigen
125The radioimmunological kit of I-hPC III; 2. the radioimmunoassay method of described kit is provided.
Because above-mentioned purpose, there is the content of following two aspects in the present invention.
One, a kind of precollagenous kit of measured by radioimmunoassay human III type that is used for comprises pure antigen reagent, antibody reagent, labelled antigen reagent and separating agent; It is characterized in that: pure antigen reagent behaviour III procollagen type hPC III, antibody reagent behaviour III procollagen type antibody anti-hPC III, labelled antigen reagent is labelled antigen
125I-hPC III; Wherein:
1, to reach polyacrylamide gel electrophoresis by the protein standard test of Fa Maxi (Pharmacia) company pure for described pure antigen reagent hPC III, with electrophoresis Coomassie brilliant blue R250 dyeing pinkiness, electrophoresis pattern is viewed as single district band, through the visible Liang Tiao of beta-mercaptoethanol reduction rear electrophoresis collection of illustrative plates district band, represent peptide chain Pro α respectively
l(III) and P α
l(III), the former mobility are 0.238, and protein molecular weight is 190,000, and the latter's mobility is 0.310, and protein molecular weight is 170,000;
2. described antibody reagent anti-hPC III is that hPC III immunizing rabbit obtains, and its working fluid is with 0.1% (w/v) bovine serum albumin(BSA) (BSA), 2% (v/v) normal rabbit serum (NRS) and 0.1M, and the phosphate buffered saline (PBS) of pH7.4 (PBS) is prepared;
3, described labelled antigen reagent
125I-hPC III is with 1% (w/v) BSA, and it is per 100 microlitre liquid per minute gamma-radiation count values (cpm/100 μ l) 30000 that 0.1M, the PBS of pH7.4 are mixed with concentration;
4, separating agent is 0.1M, and PBS preparation 6% (w/v) polyglycol (PEG) and the goat-anti rabbit anti-serum (second antibody) of pH7.4 obtain.
The present invention has following accompanying drawing:
Accompanying drawing 1 is the PAGE collection of illustrative plates of hPC III;
Accompanying drawing 2 is the SDS-PAGE collection of illustrative plates;
Accompanying drawing 3 is the intermediate product SDS-PAGE collection of illustrative plates in the purifying hPC III process from periderm;
Accompanying drawing 4 is liver biopsy fibrosis and blood-serum P C III correlationship statistical graph;
Accompanying drawing 6 is the micro-image of the Pathologic specimen of light level for the pathology liver fibrosis classification;
Accompanying drawing 7 is the micro-image of the Pathologic specimen of middle rank for the pathology liver fibrosis classification;
Accompanying drawing 8 pathology liver fibrosis classifications are the micro-image of the Pathologic specimen of heavy duty.
The Quality Identification method and the result of the reagent in the kit of the present invention are as follows:
1, referring to accompanying drawing 1, identify that through polyacrylamide gel electrophoresis (PAGE) and Coomassie brilliant blue R250 dyeing the hPC III of acquisition is a single district band, it is pure to illustrate that purity has reached electrophoresis, and arrow indication district band is a pink, meets the collagen feature; Referring to accompanying drawing 2,, be respectively Pro α through the visible Liang Tiao district of beta-mercaptoethanol reduction rear electrophoresis (SDS-PAGE) band
1(III) and P α
1(III), but its mobility ratio ox III procollagen type is lower slightly, and among the figure, each is district's band digitized representation different material composition vertically: 1-PC III (ox III procollagen type); 2-hPC III (human III type precollagen); 3-HMW (high molecular weight protein standard); 4-hPC I (people's I procollagen type); 5-C III (III Collagen Type VI); Wherein vertically the district is with in 2: band a-Pro α
1(III), band b-P
α 1(III).Warp and import Fa Maxi company high molecular weight protein standard (HMW) blank determination, the subunit molecular of the hPC III molecule of acquisition is respectively: Pro α
1(III) chain 190000 dalton (Dolton, D), P α
l(III) chain 170000D.And the Pro α of ox type III procollagen type
l(III) chain is 170000D, P
α 1(III) chain is 150000D.Referring to accompanying drawing 3: the intermediate product SDS-PAGE collection of illustrative plates of purifying hPC III process from periderm, see that the hPC III progressively is purified situation in the purification process, among the figure, before the 1-DEAE-32 chromatography, visible impurity component is a lot; Before the 2-DEAE-52 chromatography, visible impurity level reduces a lot; Behind the 5-DEAE-52 chromatography, obtain the pure product hPC III of clear two bands.The chromatographic step of when raw material sources are too poor, still needing and increasing: 3, before the 4-Sepha-dex G100 chromatography, as seen also be mixed with the small amount of impurities composition, behind the 5-SephadexG100 chromatography, obtain pure product hPC III.
2, the evaluation of anti-hPC III:
The antiserum of above-mentioned preparation is made serial dilution,, do not add competitive antigen, draw the antiserum dilution curve, measure it and tire by putting the method for exempting from; Immunohistochemistry: get each 10 routine liver biopsy specimen of cirrhosis and chronic active hepatitis patient, the antiserum of above-mentioned preparation is diluted to 1: 100 by tiring, this high price antiserum is used for immunohistochemistry liver biopsy specimen, collagenous fibres under optical microscope between the visible cirrhosis pseudolobuli turn out to be specific antibody.
3, with aforementioned
125I-hPC III is measured with the trichloroacetic acid method, and radiochemical purity reaches 99.6%.
Two. the radioimmunoassay method system of the reagent of indication of the present invention adopts double antibody PEG method, the imbalance method application of sample, reagent is pressed following capacity packing before the operation:
1, hPC III titer: totally 7 bottles, use S respectively
0～S
6Indicate every bottle of capacity 1ml; Its concentration is respectively S
o0 μ g/L, S
125 μ g/L, S
250 μ g/L, S
3100 μ g/L, S
4250 μ g/L, S
5500 μ g/L, S
61000 μ g/L;
2, PBS:1 bottle, 2ml;
3, anti-hPC III: 3 bottles, be total to 22ml;
4,
125I-hPC III: 2 bottles, be total to 11ml;
5, separating agent: 3 bottles, be total to 22ml;
6, quality controlled serum: 1 bottle, 1ml.
The assay method that the present invention is used for radiommunoassay is by following operation steps:
After adding 100 μ 1anti-hPC III in (1) 100 μ l titer or the patients serum's sample to be measured, placed 42～48 hours for 4 ℃;
(2) adding people's 100 μ l count values is 30000cpm's
125I-hPC III solution was placed 6 hours for 4 ℃;
(3) add separating agent 200 μ l, room temperature was placed 30 minutes;
(4) 4000 rev/mins, under 4 ℃～10 ℃ conditions centrifugal 30 minutes;
(5) measure sedimentary radiocounting value with the v calculating instrument, its value B after supernatant is abandoned in suction
xExpression (subscript x represents variable concentrations standard operation liquid or patient specimen);
(6) on semilogarithmic paper with ((B
Titer-B
NSB)/(B
o-B
NSB)) (%) (B
oRepresent maximum combined, B
TiterRepresent the titer combination, B
NSBRepresent non-specific binding) be ordinate, be horizontal ordinate drawing standard working curve with hPC III concentration of standard solution ([hPC III (μ g/L)]);
(7) with ((B
Patient specimen-B
NSB)/(B
o-B
NSB)) (%) the anti-standard working curve of looking into can obtain hPC III content among the patients serum.
It is good that the competition of the typical curve that the data of obtaining with above-mentioned operation steps are drawn suppresses relation, and drop is big, and measurement range is wide, satisfies the clinical assays requirement fully.Sensitivity is 6 μ g/L, is respectively 5.2% and 6.2% with interassay coefficient of variation in batch, and the recovery is 97.6～102.3%, and with other types collagen no cross reaction, method is stable.Above index has reached the quality standard requirement of radioimmunology.
The relation of aforementioned calculation result and liver biopsy fibrosis: 37 routine liver biopsy specimens dye through H-E, by fiberization distribution range and fibroblast proliferation degree be divided into gently, in, weigh three grades.No fibroplasia person is 0 grade.Its blood-serum P C III measurement result: 0 grade of pathology, 11 examples, blood-serum P C III are 89.5 ± 18.1 μ g/L (down together); Light level, 10 examples, 135.5 ± 20.5; Middle rank, 8 examples, 260.8 ± 39.2; Heavy duty, 8 examples, 369.8 ± 68.0.Blood-serum P C III increases the weight of with degree of hepatic fibrosis and increases progressively, and both are close positive correlation (r=0.984, P＜0.001) (referring to Fig. 4).
Fig. 5 Fig. 8 is the pathology microphoto of the liver biopsy specimen of the different degree of hepatic fibrosis of 4 routine representational performances wherein, and its corresponding blood-serum P C III content is: Fig. 5,0 grade of pathology, blood-serum P C III content are 58.9 μ g/L (down with); Fig. 6, light level, 125.3; Fig. 7, middle rank, 210.2; Fig. 8, heavy duty, 323.5.Prove measurement result of the present invention with the actual state of an illness consistent.
Reagent of the present invention and ria-determination method are through 7 large hospital clinic trial of Chongqing City tumour hospital and the whole nation, and its effect sees table: the comprehensive group number of cases means standard deviation of seven results of hospital of Chongqing City tumour hospital example number means standard deviation normal control 100 85.0 ± 17.5 265 83.4 ± 17.8 oxyhepatitises 22 131.1 ± 30.2 53 111.2 ± 30.1 chronic persistent hepatitises 18 94.9 ± 26.5 143 93.6 ± 26.2 chronic active hepatitises 13 188.2 ± 73 9 122 174.0 ± 42.6 compensatory cirrhosis 10 277.4 ± 91.0 13 210.3 ± 86.6 lose compensatory cirrhosis 20 228.2 ± 157.1 129 211.1 ± 67.9 cirrhosis merge liver cancer 46 288.7 ± 101.4 62 269.7 ± 66.6 primarys carcinoma of liver (simple form), 22 168.5 ± 39.3 cholelithiasis 12 88.0 ± 18.6 15 87.9 ± 16.6 other: hepatopathy 7 88.2 ± 28.0 23 93.4 ± 19.7
Non-hepatic disorder 11 88.6 ± 10.5 as can be seen from the above table, each group of cirrhosis and chronic active hepatitis group patients serum PC III raise the most remarkable, chronic persistent hepatitis, gall stone and other diseases all do not have remarkable rising, it is less to prove absolutely that hepatocellular inflammatory necrosis is measured influence to blood-serum P C III, and promptly the PC III can the interior liver fibrosis development degree of correct antimer.
Brief summary
1, extraction the stillborn foetus skin that you manually draw, miscarry from acrinol ethacridine, purifying human III type precollagen, confirmer's III such as subunit's mensuration procollagen type is pure product after polyacrylamide gel electrophoresis and reduction.
2, personnel selection III procollagen type immunizing rabbit obtains the high price antiserum, and confirms liver collagenous fibres pigmentable through immunohistochemical test.
3, Na
125I labelling human III procollagen type, radiochemicsl purity reaches 99.6%.
4, the serum III procollagen type of Jian Liing is put the every index of the method for exempting from and is all met the clinical assays requirement.
5, measure 281 routine serum III procollagen type results clinically: 100 routine normal controls (means standard deviation) are 85.0 ± 17.5 μ g/L (unit down slightly), and upper limits of normal is 120; Liver cirrhosis group rises the highest, and compensatory, mistake compensatory and merging liver cancer component are not 227.4 ± 91.0,266.2 ± 157.5 and 288.7 ± 101.4; It is 188.2 ± 73.9 that the chronic active hepatitis group has remarkable rising; Oxyhepatitis group and liver cancer (simple form) group slightly raises, and respectively is 131.1 ± 30.2 and 168.5 ± 39.3; Chronic persistent hepatitis, cholelithiasis and other groups all do not have obviously and increase.
6,37 routine liver biopsies are divided into by fibrosis: 0, light, in and heavy duty, its blood-serum P C III is respectively 89.5 ± 18.1,135.5 ± 20.5,260.8 ± 39.2 and 369.8 ± 68.0.Both are close positive correlation (r=0.984, P (0.001).
Good effect of the present invention is:
1, with glue before the radio immunoassay mensuration Serum III type of hPC III foundation Former index, extent of liver fibrosis in the antimer objectively is to diagnosis liver fibre The control of dimensionization and early-phase hepatocirrhosis has important value.
2, can obtain hPC III sterling with the method for the invention, make the reducing agent gained with cysteine during with chloramine-t method mark hPC III125The I-hPC III is stable The property greater than 1.5 months, obviously take the mark side of Sodium Metabisulfite as reducing agent Method is for well, and is very little to the damage of hPC III molecule, and radiochemical purity reaches 99.6%.
3, simple to operate, all the components all is mixed with using liquid, presses respectively The order adding gets final product, and needn't remake other additional work; Patients serum to be measured Needn't do any dilution and can obtain PC III content, measurement range is clinical making enough With; Sensitivity reaches 6 μ g/L, is respectively 5.2% He with interassay coefficient of variation in batch 6.2%, the rate of recovery is 97.6～102.3%, and all indexs have all reached radiation The requirement of immunization quality standard.
Claims (2)
1. one kind is used for the precollagenous kit of measured by radioimmunoassay human III type, comprises pure antigen reagent, antibody reagent, labelled antigen reagent and separating agent; It is characterized in that: pure antigen reagent behaviour III procollagen type hPC III, antibody reagent behaviour III procollagen type antibody anti-hPC III, labelled antigen reagent is labelled antigen
125I-hPC III and separating agent; Wherein:
(1) to reach polyacrylamide gel electrophoresis by the protein standard test of Fa Maxi company pure for described pure antigen reagent hPC III, with electrophoresis Coomassie brilliant blue R250 dyeing pinkiness, electrophoresis pattern is viewed as single district band, through the visible Liang Tiao of beta-mercaptoethanol reduction rear electrophoresis collection of illustrative plates district band, represent peptide chain Pro α respectively
1(III) and P α
l(III), the former mobility is 0.238, and protein molecular weight is 190,000, and the latter's mobility is 0.310, and protein molecular weight is 170,000;
(2) described antibody reagent anti-hPC III is that hPC III II immunizing rabbit obtains, and its standard operation liquid is with 0.1% (w/v) bovine serum albumin(BSA) BSA, 2% (v/v) normal rabbit serum and 0.1M, and the phosphate buffered saline (PBS) PBS of pH7.4 prepares;
(3) described labelled antigen reagent
125I-hPC III is with 1% (w/v) BSA, and it is per 100 microlitre liquid per minute gamma-radiation count values (cpm/100 μ l) 30000 that 0.1M, the PBS of pH7.4 are mixed with concentration;
(4) separating agent is 0.1M, and PBS preparation 6% (w/v) polyglycol of pH7.4 and goat-anti rabbit anti-serum are that second antibody obtains.
2. method of utilizing the described kit measurement liver fibrosis of claim 1 is characterized in that this method carries out according to the following steps:
After adding 100 μ lanti-hPC III in (1) 100 μ l titer or the patients serum's sample to be measured, placed 42～48 hours for 4 ℃;
(2) adding 100 μ l count values is 30000cpm's
125I-hPC III solution was placed 6 hours for 4 ℃;
(3) add separating agent 200 μ l, room temperature was placed 30 minutes;
(4) 4000 rev/mins, under 4 ℃ of-10 ℃ of conditions centrifugal 30 minutes;
(5) measure sedimentary radiocounting value with the γ calculating instrument, its value B after supernatant is abandoned in suction
xExpression, subscript x represents variable concentrations standard operation liquid or patient specimen;
(6) on semilogarithmic paper with ((B
Titer-B
NSB)/(B
o-B
NSB) % is ordinate, B
oRepresent maximum combined, B
TiterRepresent the titer combination, B
NSBRepresenting non-specific binding, is to make curve on the horizontal ordinate drawing standard with hpc III concentration of standard solution;
(7) with ((B
Patient specimen-B
NSB)/(B
o-B
NSB) anti-the standard working curve of looking into can obtain hPC III content among the patients serum to %.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 93114698 CN1045820C (en) | 1993-11-18 | 1993-11-18 | Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 93114698 CN1045820C (en) | 1993-11-18 | 1993-11-18 | Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1087997A CN1087997A (en) | 1994-06-15 |
| CN1045820C true CN1045820C (en) | 1999-10-20 |
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|---|---|---|---|
| CN 93114698 Expired - Fee Related CN1045820C (en) | 1993-11-18 | 1993-11-18 | Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100416273C (en) * | 2000-08-21 | 2008-09-03 | 法国公立援助医院 | Diagnosis method of fibrotic disease using biochemical markers |
| CN1306272C (en) * | 2000-11-17 | 2007-03-21 | 罗切斯特大学 | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
| ES2498369T3 (en) * | 2002-04-16 | 2014-09-24 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw. | A marker to measure liver cirrhosis |
| CN103076452B (en) * | 2012-12-30 | 2015-02-25 | 上海市内分泌代谢病研究所 | Method for testing adiponectin and application thereof |
| CN103399156B (en) * | 2013-07-30 | 2015-09-16 | 中国农业科学院北京畜牧兽医研究所 | Green fluorescent protein radioimmunoassay kits and preparation method thereof and detection method |
| CN103399158A (en) * | 2013-08-02 | 2013-11-20 | 上海市同济医院 | Liquid protein chip kit for detecting liver fibrosis degree |
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1993
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