CN1005220B - Process for using cow and horse serms preparing radioactive immune and biochemical determination of quality-controlling - Google Patents
Process for using cow and horse serms preparing radioactive immune and biochemical determination of quality-controlling Download PDFInfo
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- CN1005220B CN1005220B CN87100515.8A CN87100515A CN1005220B CN 1005220 B CN1005220 B CN 1005220B CN 87100515 A CN87100515 A CN 87100515A CN 1005220 B CN1005220 B CN 1005220B
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Abstract
The present invention relates to a method for preparing a quality controlling substance of radioactive immune measurement and biochemical measurement by using cow serum or horse serum. The method of the present invention is characterized in that cow serum or horse serum is used as raw material; after a method of affinity chromatography or ion exchange chromatography is used for specifically eliminating certain substances to be measured or/and influencing measured hormones, biological active substances, enzymes and electrolytes in serum, the serum is used as a substrate for preparing the quality controlling substance of biochemical measurement and radioactive immune measurement of the substance to be measured; a certain quantity of known substances to be measured are added to the substrate to be prepared into a quality controlling product for measuring the substance to be measured by a radioactive immune method or a biochemical method. The present invention has the advantages of abundant raw material source, low cost, secure use, advanced preparation method and stable and reliable product quality.
Description
The invention belongs to the preparation method of biological products (diagnostic reagent).Products obtained therefrom can be used as the Quality Control thing of radio-immunity (RIA) and biological chemistry detection, is to be used for monitoring to put exempting from and the essential reagent of biochemical measurement accuracy.
At present the Quality Control thing preparation method who generally adopts in the world is to select hepatitis B virus surface antigen feminine gender (HBsAg(one)) normal person or patients serum, after simple process, allocate making.
For example:
Ministry of Health of the People's Republic of China's medicine and biological products assay institute (1981) once add 10% sugar with normal person's pooled serum and stirred 6～8 hours with acticarbon, and 4 ℃ adsorbed 24 hours, and the 4000rpm low-temperature centrifugation is got supernatant through 2kg/cm
2The nitrogen pressure filtration sterilization obtains low T
3, T
4Pure product, packing, T is made in freeze-drying
3, T
4Put and exempt from the Quality Control thing.
Shanghai City family planning research institute (1983) preparation reproductive hormone is put and is exempted from the Quality Control thing, is to collect to contain the higher or lower human serum of certain hormone, and allotment is made mutually.
In recent years appear in the serum of " rejecting " certain material, add the method for tested reference material.Adopt a small amount of label L H of adding and FSH in human serum as Edinburgh, Britain laboratory (1982), have the agar gel of anti-LH of globefish and the anti-FSHIgG of globefish to handle, after the mixing, spend the night with excessive coupling.Leaching serum, adding excessive coupling has goat-anti globefish IgG agar gel to handle, to remove anti-LH of a small amount of globefish and the FSHIgG that contains in the serum.This method still can not be removed LH and FSH fully, and the LH and the FSH that contain mark in its serum still have 10% of commercial weight.
Biochemical test Quality Control thing has report to replace the human serum person that does the raw material with animal blood serum in recent years, but does not all adopt affinity chromatography and ion exchange chromatography to handle serum.
Said method is owing to adopt human serum to make raw material mostly, and a large amount of the collection is difficult for, and the serum homogenieity is poor, and hepatitis b surface antigen positive rate height is produced in enormous quantities and the long preservation difficulty, and cost is higher.And, also impossible certain determinand of special removal of above-mentioned disposal route or chaff interference, thereby can not get real " zero " serum that does not contain certain component to be measured, so of low quality, the poor stability of Quality Control thing; And the technology of disposal route is complicated, and material therefor is many can only disposablely to be used.
The objective of the invention is: improving the quality, reducing cost, guaranteeing under the preceding topic safe in utilization, seeking new material, new method that a special quality control thing host material is produced.
Main points of the present invention are: with horse serum or cow's serum is raw material, after the filtration sterilization, handle through various specific affinity chromatographys or ion-exchange chromatography, make " zero " serum that does not contain certain determinand or chaff interference, add a certain amount of certain hormone, bioactivator, enzyme or the pure product of electrolyte again, be mixed with comprise zero, the radio-immunity or the biochemistry quality control thing of basic, normal, high this material of concentration.
Advantage of the present invention is: raw material sources are abundant, and cost is low, and is safe in utilization.Preparation method advanced person, stable and reliable product quality.
Fig. 1 is put to exempt from and biochemistry quality control thing flow sheet; Fig. 2 is the affinity column preparation technology flow process of hormone or bioactivator; Fig. 3 is the preparation of enzyme affinity column.
Embodiment:
One, produces hormone and go the technological process of bioactivator serum following (to go the example that is prepared as of growth hormone (hGH) serum, referring to process chart I, II).
1. the purification of anti-hGHIgG: will resist hGH serum 10ml to contain the dilution of 2M/LNaCl damping fluid, and be added on (1 * 10cm) separation and purification of supA-Ago-Gel (supA-Sepharose4B) chromatographic column with equal-volume Tris/HCl(0.2M.pH8.6.After do not have albumen and flow out, use 0.05MHAc/HCl(pH3.0 with the same buffer drip washing of 0.1M) buffer solution elution, the protein peak of collection eluent adds isopyknic 0.1MH immediately
3PO
4(pH9.0) be neutralized to pH8.6, this is activated specific IgG (recovery can reach 80%).
2. the preparation of anti-hGHIgG-Ago-Gel affinity column:
1. use prior art I, II, III (referring to appendix 1) activated agarose gel, (affinity column is made in 5～10mgIgG/ml) the ratio couplings in the wet glue of 10～30mgIgG/lgm with the reactant liquor that contains anti-IgG under suitable separately reaction conditions.Collect the forward and backward liquid of coupling and make ultraviolet spectrometry mensuration at the 280nm place, be calculated as follows coupling rate and coupling capacity:
IgG（gm）＝OD280nm+1.3×V（ml）
Or the direct value (gm) that on ultraviolet light examination criteria curve, checks in IgG.
IgG(gm after coupling capacity=(IgG(gm before the coupling)-coupling))/wet glue (gm)
IgG(gm after coupling rate=(IgG(gm before the coupling)-coupling))/the preceding IgG(gm of coupling) * 100%
2. after using this mucilage binding post (1 * 10cm), through 0.1MTris/HAc(pH7.7) damping fluid drip washing balance, hGH horse or cow's serum solution (1ug/ml) 10ml(that usefulness is added with the slight trace thing generally are not more than bed volume) the mistake column chromatography.The radioactivity tale of its albumen peak value of synchronous monitoring, drip washing equilibrium liquid and eluent, calculate affine capacity by following formula:
The total CPM/(protein peak+leacheate of affine capacity=eluting peak+eluting peak+eluent) total CPM * hGH total amount=can remove/chromatography of hGH amount
Coupling rate is the post of 20%(1 * 10cm), once can handle serum 200ml.
3. go the preparation of hGH serum:
In normal ox, horse serum, add 0.05%NaN
3Anticorrosion, after the seitz filter filtration sterilization, once add column chromatography on the 200ml serum, collect the serum before and after the chromatography, detect with the RIA method, be sure of chromatography after Bo/T% raise, hGH is zero, after Nsub/T% was constant substantially, it was standby to put 4 ℃ of storages.Chromatographic column with the Tris/HAC damping fluid drip washing balance of pH7.7, is put 8 ℃ of preservations (can use repeatedly) then with the hGH of the 0.05MHAc/HCl buffer solution elution occlusion of pH3.0.
Two, produce enzyme serum technological process following (referring to I process chart III):
1. the preparation of specific substrate-Ago-Gel affinity column:
1. with reference to above-mentioned 2 1. condition and methods, the substrate of enzyme-specific as part, is coupled on the good Ago-Gel of activation, measure its coupling capacity and coupling rate with method.
2. with this gel dress post, affine capacity and once accessible serum amount are measured, calculated to reference 2 method 2..
2. after ox, horse serum being handled with reference to above-mentioned 3 method, cross column chromatography, the serum specimen of getting before and after the chromatography detects the content of its certain enzyme with radioimmunoassay method or biochemical method, be sure of that behind the chromatography be zero, and it is standby to put 4 ℃ of storages.
Three, produce the technological process following (referring to appendix 2) of electrolyte serum:
1. go the preparation of negative ion serum:
With the cationite dress post (prior art IV) of AE-.DEAE-.QAE-one class, ox, horse serum are crossed column chromatography, behind the various negative ion in the specificity occlusion serum, the serum behind the collection chromatography is negative ion serum.Chromatographic column, can be recycled through balance regeneration with behind the eluent wash-out of cation.
2. the preparation of decationizing serum:
With anionoid exchangers such as CM-.phospho or sup-dress post (prior art V), with reference to above-mentioned 1 method chromatography, the serum behind the collection chromatography is decationizing serum.This chromatographic column with anion-containing eluent wash-out after, through balance regeneration, can use repeatedly.
Four, various putting exempted from and the preparation of biochemical measurement Quality Control thing (put with preparation hGH to exempt from the Quality Control thing be example):
Ox or horse serum with the above-mentioned hGH of going are matrix, and the hGH dried frozen aquatic products of dilution WHO First International normative reference is made into and contains hGH0.0,2.0,10.0 and the Quality Control thing of 30.0ng/ml, and be distributed into 0.25ml/ and prop up, freeze-drying can be for sale or use.
It is raw material that the present invention adopts horse, cow's serum, and cost descends 70～99%, and the serum homogenieity is good, and is safe in utilization.
The present invention adopts affinity chromatography or ion exchange chromatography to handle serum simultaneously, has both removed wherein contained various chaff interferences specifically, has kept other and the irrelevant active component of mensuration in the serum again.Thereby detect serum behind the chromatography with the RIA method, and every mass parameter obviously improves (table 1), and the Quality Control thing (table 2) of the serum preparation that is better than choosing, and uses the hGH-RIA Quality Control stable in properties of this matrix preparation, and the recovery is near the desirable recovery (table 3).
Through paired data T check, Nsub/T% does not have significant difference before and after the chromatography, and Bo/T% raises, and statistical significance (P＜0.05) is arranged.
* the reference targets value detects 8 gained averages of hGH Quality Control thing for my chamber oneself.
Training seminar's value is 11 groups of inferior measured averages of student of twice training seminar.
Big average is that above-mentioned Quality Control thing is distributed to 22 units of domestic 17 provinces and cities and faces half a year on probation, amounts to the average of measuring for 254 times.
Appendix:
1. prior art I, II, III are referring to LKB(Denmark) the product introduction handbook of company-affinity chromatography uses and correlation technique (Pract icalguide for use in affinitychromatography).
Prior art I: glutaraldehyde method;
Prior art II: bis-epoxy reagent and chloropropylene oxide method;
Prior art III: divinyl sulfone method;
2. prior art IV, V are referring to the principle and the method (Ion Exchange Chromatograph-Principles and Methods) of Pharmacia Fine Chemicals-ion-exchange chromatography.
Reference:
1., Fu Licheng etc., T
3, T
4The foundation and the application of radio-immunity medicine box quality controling serum.184 pages of China's Journal of Nuclear Medicine 23 phases of volume of nineteen eighty-two.
2., Liu Shifan etc., the preparation of quality controling serum and application in radiommunoassay.116 pages of China's Journal of Nuclear Medicine 32 phases of volume of nineteen eighty-three.
③、W.M.Hunter.Internal Quality Control and External Quality Assessment.In“Radioimmunoassay Design and Quality Control.”Editor Jan I.T.Rorell.Pregamon Press.1982.P69。
4., the Ministry of Public Health of Beijing army rear service portion check professional cooperation group." preparation of quality controling serum and preservation ".Chinese journal of medical examination was rolled up 235 pages of 4 phases in 1979 2.
The hGH-RIA measured value (N=10) of horse serum before and after table 1. chromatography
Behind the preceding chromatography of chromatography
Nsub/T% Bo/T% hGH ng/ml Nsub/T% Bo/T% hGH ng/ml
3.35±0.43 35.68±1.23 0.0 3.82±0.65 37.26±1.22 0.0
Before and after table 2. chromatography single normal human serum with mix the horse serum comparison
Behind the preceding chromatography of chromatography
Nsub/T% Bo/T% hSH TSH INS- Nsub/T% Bo/T% hGH TSH Ins.
（ng/ml） （/ml） （/ml） （ng/ml） （/ml） （/ml）
Horse serum 3.39 35.6---5.37 54.73 0.000＜1.6 6.27
Human serum 8.84 51.02 0.095--6.62 53.83 0.028 3.0 26.56
±10.79 ±1.88 ±0.82 ±1.37
The big average of table 3.hGH Quality Control thing, target value and desired value are relatively
QC1 ng/ml QC2 ng/ml QC3 ng/ml
X±SD CV% X±SD CV% X±SD CV%
Desired value 2.00 10.00 30.00
(compound concentration) measures number of times
Reference targets value n=8 1.85 ± 0.14 7.57 10.13 ± 0.82 8.09 31.27 ± 1.17 3.74
Training seminar value n=11 1.98 ± 0.51 25.76 10.22 ± 0.85 8.32 27.17 ± 3.77 13.88
Big average n=254 2.13 ± 0.25 11.93 10.35 ± 0.79 7.67 30.81 ± 4.04 13.11
Average n=273 2.12 ± 0.26 12.36 10.34 ± 0.97 7.71 30.68 ± 3.95 12.86
The recovery 106. ± 13% 103.4 ± 9.7% 102.3 ± 13%
Appendix:
1, prior art I, II, III are referring to LKB(Denmark) the product introduction handbook of company-affinity chromatography uses and correlation technique (Practical guide for use in affinitychromatography).
Prior art I: glutaraldehyde method;
Prior art II: bis-epoxy reagent and chloropropylene oxide method;
Prior art III: divinyl sulfone method;
2, prior art IV, V are referring to the principle and the method (Ion Exchange Chromatograph-Principles and Methods) of Pharmacia Fine Chemicals_ ion-exchange chromatography
Reference:
1., Fu Licheng etc., T
3, T
4The foundation and the application of radio-immunity medicine box quality controling serum.184 pages of China's Journal of Nuclear Medicine 23 phases of volume of nineteen eighty-two.
2., Liu Shifan etc., the preparation of quality controling serum and application in radiommunoassay.116 pages of China's Journal of Nuclear Medicine 32 phases of volume of nineteen eighty-three.
③、W.M.Hunter.Internal Quality Control and External Quality Assessment.In“Radioimmunoassay Design and Quality Control.”Editor Jan I.T.Rorell.Pregamon Press.1982.P69
4., the Ministry of Public Health of Beijing army rear service portion check professional cooperation group." preparation of quality controling serum and preservation ".Chinese journal of medical examination was rolled up 235 pages of 4 phases in 1979 2.
Claims (4)
1; the present invention relates to a kind of ox of using; horse serum prepares the method for radio-immunity and biological chemistry detection Quality Control thing; be to remove ox; contained chaff interference in the horse blood; determinand; make the various matrix serum that do not contain chaff interference and original determinand; add a certain amount of known certain hormone on demand again; bioactivator; the pure product of enzyme or electrolyte; be mixed with and comprise zero; low; in; this material of high concentration is (as hormone; enzyme; electrolyte etc.) Quality Control thing; it is characterized in that: with specific antibodies; substrate or the moon; cationite; make stationary phase; with ox or horse serum as moving phase; cross certain wherein contained hormone of column chromatography specific adsorption; bioactivator; enzyme or the moon; kation is prepared into various matrix serum.
2, according to the method for the various matrix serum of the described preparation of claim 1, when preparation is removed hormone and is removed the matrix serum of bioactivator, it is characterized in that: earlier with A albumen-Ago-Gel affinity chromatography system, the specific IgG that from the serum of anti-this hormone or bioactivator, separates purification antihormones or bioactivator, again with this specific IgG and agarose gel coupling, make specific affinity chromatography system, the affinity chromatography system that this is specific is used to handle horse or cow's serum, the method that preparation is removed hormone and removed the matrix serum of bioactivator.
3, according to the method for the various matrix serum of the described preparation of claim 1, when enzyme matrix serum is removed in preparation, it is characterized in that: elder generation is with substrate or the inhibitory enzyme and the agarose gel coupling of enzyme undetermined, make affinity chromatography system undetermined, be used to handle the method that enzyme matrix serum is removed in the preparation of horse or cow's serum.
4, according to the method for the various matrix serum of the described preparation of claim 1, when electrolytical matrix serum was removed in preparation, it is characterized in that: with the moon or cation resin exchange chromatographic column, handle horse or cow's serum earlier, preparation did not contain the matrix serum of yin, yang ion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN87100515.8A CN1005220B (en) | 1987-01-26 | 1987-01-26 | Process for using cow and horse serms preparing radioactive immune and biochemical determination of quality-controlling |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN87100515.8A CN1005220B (en) | 1987-01-26 | 1987-01-26 | Process for using cow and horse serms preparing radioactive immune and biochemical determination of quality-controlling |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN87100515A CN87100515A (en) | 1988-08-31 |
| CN1005220B true CN1005220B (en) | 1989-09-20 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN87100515.8A Expired CN1005220B (en) | 1987-01-26 | 1987-01-26 | Process for using cow and horse serms preparing radioactive immune and biochemical determination of quality-controlling |
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|---|---|
| CN (1) | CN1005220B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101046479B (en) * | 2007-02-25 | 2011-08-31 | 清华大学 | Process of preparing human serum base matter containing no target protein |
| CN102621335A (en) * | 2011-01-27 | 2012-08-01 | 首都医科大学附属北京安定医院 | Blood product and preparation method thereof |
| CN107505460A (en) * | 2017-08-07 | 2017-12-22 | 嘉兴博泰生物科技发展有限公司 | C reactive protein quality for POCT controls the preparation method of product |
| CN109443876A (en) * | 2018-12-14 | 2019-03-08 | 郑州安图生物工程股份有限公司 | Hepatic fibrosis markers quality-control product and preparation method thereof |
| CN110981963B (en) * | 2019-09-23 | 2021-09-03 | 山东绿都生物科技有限公司 | Preparation method of monoclonal antibody for replacing anti-rat-rabbit secondary antibody |
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1987
- 1987-01-26 CN CN87100515.8A patent/CN1005220B/en not_active Expired
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