CN1289926A - Process for preparing human placenta lactogen as diagnostic reagent - Google Patents
Process for preparing human placenta lactogen as diagnostic reagent Download PDFInfo
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- CN1289926A CN1289926A CN 99113260 CN99113260A CN1289926A CN 1289926 A CN1289926 A CN 1289926A CN 99113260 CN99113260 CN 99113260 CN 99113260 A CN99113260 A CN 99113260A CN 1289926 A CN1289926 A CN 1289926A
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Abstract
A process for preparing human placonta lactogen hemocyte and single immunodiffusion plate to diagnose pregnancy specificity such as hypertension syndrome, slow fetus development, etc. includes such steps as extracting HPL from human placenta, affinity chromatography twice and preparing said hemocyte and diffusion plate. Its advantages include simgle component, simple method and operation, stable performance and correct result.
Description
The present invention relates to a kind of diagnosis blood cell, unidirectional immunodiffusion plate, specifically relate to HPL's (HPL) diagnosis blood cell, the preparation method and the application of unidirectional immunodiffusion plate.
HPL (HPL) is by the emiocytosis of placenta plamoditrophoblast, measure the content of HPL in pregnant woman's blood, can reflect the variation of placenta volume and function, at present radioimmunologies that adopt more, though measured value is credible, but because radioactivity decay and need valuable Medical Devices is difficult for popularizing basic hospital.
The purpose of this invention is to provide a kind ofly under pregnant late period, mature or prolonged pregnancy situation, measure the diagnosis blood cell of HPL value, the preparation method and the application of unidirectional immunodiffusion plate.
The preparation method of HPL among the present invention (HPL) diagnosis blood cell, unidirectional immunodiffusion plate adopts following steps:
The purification of 1HPL
1.1 saturated ammonium sulfate is saltoutd
Normal term birth puerpera placenta 5-10, the homogenate of application organizes bruiser, adding 2 times of volume physiological saline stirred 20-40 minute, centrifugal 3000-6000r/min20 minute, go precipitation, supernatant is saltoutd with 50% saturated ammonium sulfate, 4000-10000r/min20 minute, abandon supernatant, the precipitation use physiological saline solution, with normal saline dialysis to there not being NH
+
1.2 sephadex G200 column chromatography
Get sephadex G20010 gram, (2.5 * 100cm) use 0.1-0.3mol/LNaCl and 0.05mol/Ltris-HCl damping fluid (PH8.0) balance to handle the dress post routinely, get the concentrating sample 10ml that saltouts, flow velocity 10-80ml/h collects eluent respectively with every pipe 2-10ml, use the inspection of HPL blood cell reagent and respectively manage concentration, collect HPL high concentration pipe and merge, use 50% saturated ammonium sulphate, dialysis is to there not being NH
+
1.3 HPL coupling sepharose4B affinity chromatography
Get the sepharose4B10-40ml that Sweden pharmacia company produces, ice bath after 0.5mol/LNaCl and cold distilled water drip washing, the cyanogen bromide 3.0g/10ml that adds new preparation, transfer pH value with 0.2mol/LNaOH, make it to maintain about 5-12, reacted 5-20 minute, and drained, add through 0.1mol/LNaHCO with Buchner funnel
3(PH7-11) stirring of the HPL antibody of dialysis is spent the night for 4 ℃, adorn post next day, be washed till 280nmOD value＜0.01 with 0.01mol/L (0.5mol/LNaCI) phosphate buffer till, collect whole eluents, measure the 280nmOD value through ultraviolet spectrophotometer, calculating its coupling rate is 78.2%.The enriched sample 5ml that merges is added (1.5 * 20cm) 0.01mol/L phosphate buffer (PH6-8) wash-outs in the HPL antibody affinity chromatography, flow velocity 10-60ml/h is washed till 280nmOD value＜0.02 o'clock, use 0.1mol/L glycocoll-HCl damping fluid (PH2.4) desorption instead, flow velocity 10-80ml/h, collect the HPL high concentration region, concentrate desalination with 50% saturated ammonium sulfate.
1.4 anti-people's whole blood antibody coupling sepharose4B affinity column preparation
Sepharose4B is identical with the HPL antibody coupling, and its coupling rate is 7.28%.To concentrate desalination HPL sample 5ml and add in anti-people's whole blood antibody affinity chromatography, (1.5 * 20cm) 0.01mol/L phosphate buffer (PH7.0) wash-outs are washed speed for 30ml/h, collect the first albumen absorption peak, merge each pipe and concentrate, and freeze-drying is the pure product of HPL.
2.HPL sero-fast preparation
Select the 2kg male rabbit, in two backs vola injection Bacille Calmette-Guerin 5-40mg that lives, after 5-20 days around lymph node multi-point injection, HPL antigen adds Fu Shi Freund's complete adjuvant 0.5-4ml, (every kg body weight 50-400 μ gHPL antigen).The one all immunity of later every interval once after 5 immunity, try blood and tire (two-way agar diffusion method) when reaching 1: 64 times arteria carotis bloodletting separation of serum.
3. the erythrocytic hydroformylation of people " O " type
Get healthy male " O " type red blood cell, after glutaraldehyde, formaldehyde, pyroracemic aldehyde are handled, be made into 10% hydroformylation blood cell suspension.
4.HPL antiserum sensitization hydroformylation blood cell
Get the centrifugal back of 10% hydroformylation blood cell and be made into the 1-10% blood cell with the 0.1mol/L sodium acetate, with HPL IgG be diluted to 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml respectively, 20 μ g/ml concentration are mixed with equivalent hydroformylation blood cell, reaction is 1-3 hour under 20-60 ℃ of constant temperature shaking bath, phosphate buffer flush away through containing the 0.1-3% rabbit anteserum is not in conjunction with IgG, be made into 1% blood cell suspension, produce HPL freeze-drying diagnosis blood cell.
5. with HPL antiserum 0.02mol/LTris-HCL, 0.15mol/LNaCl, 0.05%NaN
3Be diluted to 100,150,200 times,, add the 1-4% agar solution that equal-volume boils 40-70 ℃ of back cooling respectively, mix bed board in 56 ℃ of deactivation 10-40 minutes.Agar thickness 1-4mm, punching diameter 1-5mm, pitch-row 0.5-3cm.
The evaluation of the HPL of the present invention's preparation:
1.8% polyacrylamide gel disc electrophoresis, conventional preparation separation gel and concentrated glue, the pure product of HPL of getting 50 μ g purification carry out the polyacrylamide gel disc electrophoresis, and electric current 2mA/ pipe presents a band with the dyeing of 1% amino black.
2. the immunoelectrophoresis result shows: the HPL that purifies in this chamber only presents an immunoprecipitation arc with HPL antiserum and pregnant whole serum.
3. two-way immune cross matching result shows: the HPL of purification and anti-HCG, and anti-AFP, anti-SP, anti-human whole serum does not have the immunoprecipitation line, and with anti-HPL serum and anti-pregnant whole serum tangible precipitation line is arranged, thereby shows that the HPL that is purified is the pure product of galactagogin.
More than the method introduced by " experiment immunization learns a skill " of three kinds of methods carry out.
4. checklist is non-specific to the immunodiffusion plate: get 9 parts of non-pregnant woman woman serum, 9 parts of male sex's serum add each 30 μ l in the unidirectional immune plate agar of the HPL hole respectively, place 24 hours result of determination of 37 ℃ of constant temperature, all do not have precipitation ring and occur, illustrate that the unidirectional immune plate of HPL does not have nonspecific reaction.
5. stability is checked: the unidirectional immune plate of HPL is deposited compared in 4 ℃ of different months, every batch all adds each 30 μ l of same sample, place 37 ℃ of constant temperature observed in 24 hours, find to place 6 months with interior immune plate, its precipitation ring size and sharpness indifference, and place immune plate more than 6 months, the precipitation ring sharpness is slightly poor, because immune plate is long standing time, causes HPL antiserum inactivation.Through observing relatively, immune plate term of life should be advisable with interior at 6 months, before every batch of use, all carried out quality testing with the HPL standard.
Be described in detail the present invention below in conjunction with embodiment.
The purification of 1HPL
1.1 saturated ammonium sulfate is saltoutd
10 on normal term birth puerpera placenta, the homogenate of application organizes bruiser, adding 2 times of volume physiological saline stirred 30 minutes, centrifugal 4000r/min20 minute, go precipitation, supernatant is saltoutd with 50% saturated ammonium sulfate, 5000r/min20 minute, abandon supernatant, the precipitation use physiological saline solution, with normal saline dialysis to there not being NH
+
1.2 sephadex G200 column chromatography
Get sephadex G20010 gram, (2.5 * 100cm) use 0.2mol/LNaCl and 0.05mol/Ltris-HCl damping fluid (PH8.0) balance to handle the dress post routinely, get the concentrating sample 10ml that saltouts, flow velocity 60ml/h collects eluent respectively with every pipe 5ml, use the inspection of HPL blood cell reagent and respectively manage concentration, collect HPL high concentration pipe and merge, use 50% saturated ammonium sulphate, dialysis is to there not being NH
+
1.3HPL coupling sepharose4B affinity chromatography
Get the sepharose 4B30ml that Sweden pharmacia company produces, ice bath after 0.5mol/LNaCl and cold distilled water drip washing, the cyanogen bromide 3.0g/10ml that adds new preparation, transfer pH value with 0.2mol/LNaOH, make it to maintain about 11, reacted 10 minutes, and drained, add through 0.1mol/LNaHCO with Buchner funnel
3(PH9) stirring of the HPL antibody of dialysis is spent the night for 4 ℃, adorn post next day, be washed till 280nmOD value＜0.01 with 0.01mol/L (0.5mol/LNaCI) phosphate buffer till, collect whole eluents, measure the 280nmOD value through ultraviolet spectrophotometer, calculating its coupling rate is 78.2%.The enriched sample 5ml that merges is added (1.5 * 20cm) 0.01mol/L phosphate buffer (PH7.4) wash-outs in the HPL antibody affinity chromatography, flow velocity 30ml/h is washed till 280nmOD value＜0.02 o'clock, use 0.1mol/L glycocoll-HCl damping fluid (PH2.4) desorption instead, flow velocity 60ml/h, collect the HPL high concentration region, concentrate desalination with 50% saturated ammonium sulfate.
1.4 anti-people's whole blood antibody coupling sepharose4B affinity column preparation
Sepharose4B is identical with the HPL antibody coupling, and its coupling rate is 7.28%.To concentrate desalination HPL sample 5ml and add in anti-people's whole blood antibody affinity chromatography, (1.5 * 20cm) 0.01mol/L phosphate buffer (PH7.0) wash-outs are washed speed for 30ml/h, collect the first albumen absorption peak, merge each pipe and concentrate, and freeze-drying is the pure product of HPL.
2.HPL sero-fast preparation
Select the 2kg male rabbit, in two backs vola injection Bacille Calmette-Guerin 20mg that lives, after 10 days around lymph node multi-point injection, HPL antigen adds Fu Shi Freund's complete adjuvant 2ml, (every kg body weight 150 μ gHPL antigens).The one all immunity of later every interval once after 5 immunity, try blood and tire (two-way agar diffusion method) when reaching 1: 64 times arteria carotis bloodletting separation of serum.
3. the erythrocytic hydroformylation of people " O " type
Get healthy male " O " type red blood cell, after glutaraldehyde, formaldehyde, pyroracemic aldehyde are handled, be made into 10% hydroformylation blood cell suspension.
4.HPL antiserum sensitization hydroformylation blood cell
Get the centrifugal back of 10% hydroformylation blood cell and be made into 5% blood cell with the 0.1mol/L sodium acetate, with HPLIgG be diluted to 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml respectively, 20 μ g/ml concentration are mixed with equivalent hydroformylation blood cell, reaction is 1.5 hours under 45 ℃ of constant temperature shaking baths, phosphate buffer flush away through containing 1% rabbit anteserum is not in conjunction with IgG, be made into 1% blood cell suspension, produce HPL freeze-drying diagnosis blood cell.
5. with HPL antiserum 0.02mol/LTris-HCL, 0.15mol/LNaCl, 0.05%NaN
3Be diluted to 100,150,200 times,, add 2.4% agar solution that equal-volume boils 56 ℃ of back coolings respectively, mix bed board in 56 ℃ of deactivation 10-40 minutes.Agar thickness 3.0mm, punching diameter 4.0mm, pitch-row 1.5cm.
Application Example of the present invention:
Get 10 μ l pregnancy serums and be diluted to usefulness to be detected such as 100 times with 1ml salt solution, on V-type Microhemagglutination plate, respectively add sample dilution water 25 μ l from 5 holes, the 1st hole to the, the serum 25 μ l that get 100 times of dilutions are added to the 1st hole, take turns doing 2 times with micro sample adding appliance from the 1st hole and be diluted to the 4th hole, the 5th hole is a blank, freeze-drying HPL diagnosis blood cell dissolves with blood cell dilution water 2ml and shakes up, use 4 ℃ of placement backs of spending the night, get 25 μ l dilution blood cell and respectively add 25 μ l from 5 holes, the 1st hole to the, put 37 ℃ after shake is even, 1 hour taking-up result of determination.
Criterion: HPL can agglutinating reaction (positive) occur with HPL antibody sensitized blood cell in the pregnancy serum, otherwise, as do not contain in the serum or concentration lower, aggegation (feminine gender) does not then appear, multiply by the susceptibility (0.125 μ g/ml) of sensitization blood cell according to the serum maximum dilution multiple that hemagglutination occurs, just can determine the concentration of HPL in the serum.
Decision method:
Blood cell all sinks at the bottom of the hole negative, is divided into each stage of 1-4 according to the aggegation degree, and aggegation is positive at 2 above decidables.
Computing formula:
The susceptibility (μ g/ml) of the serum diluting multiple * diagnosis blood cell in HPL content in the serum (μ g/ml)=hemagglutination hole.
Get pregnant woman's ear-lobe blood 0.1ml with glass capillary, isolate serum, draw 15 microlitres with micro sample adding appliance and add (it is outer to note not wanting overfolw hole) in the agar hole, after application of sample finishes, placed 20-30 minute, treat to add again after serum has precipitated in the hole 15 microlitre serum, add up to 30 microlitres, build plastic plate, level is put in people's temperature box, spreads 24 hours observationss under 37 ℃ of conditions, writes down the immunoprecipitation ring diameter (mm) of institute's test sample product respectively, from typical curve, can find the HPL value, promptly get the HPL content of this sample.To calibrate the blood cell susceptibility be 0.025 μ g/ml by using the HPL standard items, determine pregnant 38 weeks above be normal value greater than 5 μ g/ml, 5 μ g/ml are dangerous values for police circles are worth less than 5 μ g/ml.Wherein the preparation method of typical curve is: the HPL titer that adds 4 variable concentrations in the agar plate hole respectively, every hole application of sample 30 μ l, put 37 ℃ of constant temperature of people and observe deposit ring diameter after 24 hours, the agar plate of being done after 150 times of dilutions of discovery selection, the immunoprecipitation ring diameter is more clear, and its diffusion ring diameter is respectively 9mm, 6.5mm, 5.2mm, 4.7mm, is horizontal ordinate with the standard content, titer immunoprecipitation ring diameter is an ordinate, the drawing standard working curve.(the HPL standard of the German Benring of reference research institute)
Claims (2)
1, a kind of HPL diagnoses the preparation method of blood cell, unidirectional immunodiffusion plate, and this method adopts following steps:
(1) purification of HPL
(1.1) saturated ammonium sulfate is saltoutd
Normal term birth puerpera placenta 5-10, the homogenate of application organizes bruiser, adding 2 times of volume physiological saline stirred 20-40 minute, centrifugal 3000-6000r/min20 minute, go precipitation, supernatant is saltoutd with 50% saturated ammonium sulfate, 4000-10000r/min20 minute, abandon supernatant, the precipitation use physiological saline solution, with normal saline dialysis to there not being NH
+
(1.2) sephadex G200 column chromatography
Get sephadex G20010 gram, (2.5 * 100cm) use 0.1-0.3mol/LNaCl and 0.05mol/Ltris-HCl damping fluid (PH8.0) balance to handle the dress post routinely, get the concentrating sample 10ml that saltouts, flow velocity 10-80ml/h collects eluent respectively with every pipe 2-10ml, use the inspection of HPL blood cell reagent and respectively manage concentration, collect HPL high concentration pipe and merge, use 50% saturated ammonium sulphate, dialysis is to there not being NH
+
(1.3) HPL coupling sepharose4B affinity chromatography
Get the sepharose4B10-40ml that Sweden pharmacia company produces, ice bath after 0.5mol/LNaCl and cold distilled water drip washing, the cyanogen bromide 3.0g/10ml that adds new preparation, transfer pH value with 0.2mol/LNaOH, make it to maintain about 5-12, reacted 5-20 minute, and drained, add through 0.1mol/LNaHCO with Buchner funnel
3(PH7-11) stirring of the HPL antibody of dialysis is spent the night for 4 ℃, adorn post next day, be washed till 280nmOD value＜0.01 with 0.01mol/L (0.5mol/LNaCI) phosphate buffer till, collect whole eluents, measure the 280nmOD value through ultraviolet spectrophotometer, calculating its coupling rate is 78.2%.The enriched sample 5ml that merges is added (1.5 * 20cm) 0.01mol/L phosphate buffer (PH6-8) wash-outs in the HPL antibody affinity chromatography, flow velocity 10-60ml/h is washed till 280nmOD value＜0.02 o'clock, use 0.1mol/L glycocoll-HCl damping fluid (PH2.4) desorption instead, flow velocity 10-80ml/h, collect the HPL high concentration region, concentrate desalination with 50% saturated ammonium sulfate.
(1.4) anti-people's whole blood antibody coupling sepharose4B affinity column preparation
Sepharose4B is identical with the HPL antibody coupling, and its coupling rate is 7.28%.To concentrate desalination HPL sample 5ml and add in anti-people's whole blood antibody affinity chromatography, (1.5 * 20cm) 0.01mol/L phosphate buffer (PH7.0) wash-outs are washed speed for 30ml/h, collect the first albumen absorption peak, merge each pipe and concentrate, and freeze-drying is the pure product of HPL.
(2) the sero-fast preparation of HPL
Select the 2kg male rabbit, in two backs vola injection Bacille Calmette-Guerin 5-40mg that lives, after 5-20 days around lymph node multi-point injection, HPL antigen adds Fu Shi Freund's complete adjuvant 0.5-4ml, (every kg body weight 50-400 μ gHPL antigen).The one all immunity of later every interval once after 5 immunity, try blood and tire (two-way agar diffusion method) when reaching 1: 64 times arteria carotis bloodletting separation of serum.
(3) the erythrocytic hydroformylation of people " O " type
Get healthy male " O " type red blood cell, after glutaraldehyde, formaldehyde, pyroracemic aldehyde are handled, be made into 10% hydroformylation blood cell suspension.
(4) HPL antiserum sensitization hydroformylation blood cell
Get the centrifugal back of 10% hydroformylation blood cell and be made into the 1-10% blood cell with the 0.1mol/L sodium acetate, with HPLIgG be diluted to 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml respectively, 20 μ g/ml concentration are mixed with equivalent hydroformylation blood cell, reaction is 1-3 hour under 20-60 ℃ of constant temperature shaking bath, phosphate buffer flush away through containing the 0.1-3% rabbit anteserum is not in conjunction with IgG, be made into 1% blood cell suspension, produce HPL freeze-drying diagnosis blood cell.
(5) with HPL antiserum 0.02mol/LTris-HCL, 0.15mol/LNaCl, 0.05%NaN
3Be diluted to 100,150,200 times,, add the 1-4% agar solution that people's equal-volume boils 40-70 ℃ of back cooling respectively, mix bed board in 56 ℃ of deactivation 10-40 minutes.Agar thickness 1-4mm, punching diameter 1-5mm, pitch-row 0.5-3cm.
2, a kind of HPL diagnoses blood cell, the application of unidirectional immunodiffusion plate aspect the judgement placental function.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99113260 CN1111283C (en) | 1999-09-24 | 1999-09-24 | Process for preparing human placenta lactogen as diagnostic reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99113260 CN1111283C (en) | 1999-09-24 | 1999-09-24 | Process for preparing human placenta lactogen as diagnostic reagent |
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| Publication Number | Publication Date |
|---|---|
| CN1289926A true CN1289926A (en) | 2001-04-04 |
| CN1111283C CN1111283C (en) | 2003-06-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99113260 Expired - Fee Related CN1111283C (en) | 1999-09-24 | 1999-09-24 | Process for preparing human placenta lactogen as diagnostic reagent |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102272604A (en) * | 2008-11-17 | 2011-12-07 | 盖茨和圣托马斯英国国民健康制度保险信托基金会 | Pregnancy testing |
-
1999
- 1999-09-24 CN CN 99113260 patent/CN1111283C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102272604A (en) * | 2008-11-17 | 2011-12-07 | 盖茨和圣托马斯英国国民健康制度保险信托基金会 | Pregnancy testing |
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| Publication number | Publication date |
|---|---|
| CN1111283C (en) | 2003-06-11 |
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Owner name: CHANGCHUN DELAIFU BIOISYSTECH CO., LTD. Free format text: FORMER OWNER: JILIN PROVINCE FAMILY PLANNING SCIENCE AND TECHNOLOGY RESEARCH INSTITUTE Effective date: 20091023 |
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Effective date of registration: 20091023 Address after: Jilin province Changchun Luyuan Economic Development Zone, 6.5 km Patentee after: Changchun de Laifu Biotechnology Co. Ltd. Address before: Changchun provincial family planning science and Technology Research Institute, No. 2 Garden Road, Nanguan District, Jilin, China Patentee before: Jilin Family Planning Sciences and Technology Inst |
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Granted publication date: 20030611 Termination date: 20130924 |