CN1210267A

CN1210267A - Reagent box for determining plasma protein A related to pregnancy by enzyme combination quantity

Info

Publication number: CN1210267A
Application number: CN 97116236
Authority: CN
Inventors: 唐棣; 都兴稼; 李晨阳; 王云龙
Original assignee: CHINA BIOLOGICAL ENGINEERING Co
Current assignee: CHINA BIOLOGICAL ENGINEERING Co
Priority date: 1997-09-04
Filing date: 1997-09-04
Publication date: 1999-03-10

Abstract

A reagent box for quantitativel enzyme-linked test of plasma protein A associated with pregnancy comprises box body, coated reaction board with antigen concentration of 1mg/ml and dual diffusion titer higher than 1 : 32, standard reagents including freeze dried plasma protein A associated with pregnancy with purity higher than 95%, quality-controlled serum with same components as standard one and enzyme-labelled antigen with protein content of 0.2-1.5 mg/ml, and other auxiliary reagents. Its advantages include reasonable contained substances, stable performance, correct test and convenient application.

Description

The kit of determining plasma protein A related to pregnancy by enzyme combination quantity

The present invention relates to the kit of a kind of kit of determining plasma protein A related to pregnancy by enzyme combination quantity, particularly a kind of determining plasma protein A related to pregnancy by enzyme combination quantity.

Abnormal fetus is fetus, newborn child's illness and dead major reason, is to influence the key factor that population quality improves, and for reducing the birth of abnormal fetus to greatest extent, should carry out pre-natal diagnosis to fetus as early as possible.At present, methods for prenatal diagnosis is more, for example: female blood AFP pH-value determination pH, B ultrasonic etc., though B ultrasonic is diagnosed safety, no wound, the kind that diagnoses the illness is limited.Immediate development a kind of diagnostic method that utilizes the pregnant albumen Prenatal Screening abnormal fetus in pregnant woman's blood, the foundation of this method is: PAPP-A (PAPP-A) content is with stablizing increase pregnant age, PAPP-A is a kind of big molecular saccharides albumen by the secretion of placenta syneytiotrophoblast, this property of protein is stable, detect the PAPP-A level and can be directly acquainted with the placenta situation, reflect the fetal in utero growth indirectly.

The kit that the purpose of this invention is to provide a kind of determining plasma protein A related to pregnancy by enzyme combination quantity (PAPP-A), reagent stable performance in this kit, assembly is reasonable, is suitable for, and detects accurately, and is easy to use.

For achieving the above object, the present invention takes following design proposal: the kit of this determining plasma protein A related to pregnancy by enzyme combination quantity comprises: the kit box body, place bag in the box body by reaction plate and the packaged reagent of difference, reagent has: standard items, quality controlled serum, enzyme labelled antibody, and their composition is respectively:

Standard items: the freeze-dried powder of pregnancy-associated plasma protein-A, its purity are 〉=95%,

Quality controlled serum: the freeze-dried powder of pregnancy-associated plasma protein-A, its purity are 〉=95%,

Enzyme labelled antibody: with the antibody of the crosslinked anti-pregnancy-associated plasma protein-A of rabbit of horseradish peroxidase, its protein content 0.2-1.5mg/ml,

Bag is by reaction plate: the antibody that is used to wrap quilt is the antibody of the anti-pregnancy-associated plasma protein-A of rabbit, antibody concentration 1mg/ml, and double diffusion tires＞and 1: 32.

Kit comprises mentioned reagent and bag at least by outside the reaction plate, for convenience, and also can pack into concentrated cleaner, developer A, developer B, stop buffer, sticker, semi-logarithmic scale paper etc.

Described concentrated cleaner is a phosphate buffer, and developer A is 1% tetramethyl benzidine (TMB), and developer B is a hydrogen peroxide, and stop buffer is a 1M sulfuric acid.

The purity of above-mentioned pregnancy-associated plasma protein-A (PAPP-A) is very high, can make the user more reliable as the typical curve of data basis of reference, additional in this kit have a quality controlled serum lyophilized products, and whether successful it can make each sign that detects, in order to avoid the conclusion that work makes mistake.

One, the preparation of standard items:

1, collect serum: collect the pregnant woman's venous blood that was higher than for 30 weeks pregnant week, 3000 rev/mins centrifugal 20 minutes, get serum in container, put in-20 ℃ of refrigerators and preserve.

2, the purifying of pregnancy-associated plasma protein-A (PAPP-A):

1. saturated ammonium sulfate is slightly carried:

Because PAPP-A dissolves in the solution of saturated ammonium sulfate and serum composition (volume ratio that is serum and saturated ammonium sulfate is 2: 1), therefore, when in serum, adding saturated ammonium sulfate to above-mentioned concentration, the PAPP-A dissolving, other albumen are removed with precipitation form, add saturated ammonium sulfate solution in the supernatant behind the precipitation separation, when supernatant: during saturated ammonium sulfate=1: 1 (volume ratio), the PAPP-A precipitate and separate.

2. Sephadex G200 chromatography:

Chromatography is got the blood serum sample of 5% bed volume, sample concentration 6mg/ml, and application of sample is collected, and eluent is the Tris-Hcl damping fluid.Measure the OD value under each collection tube 280nm, the 260nm wavelength, calculate protein concentration.Through the SDS-PACE vertical slab electrophoresis, detect its purity, will have highly active PAPP-A and merge, concentrate with hollow fiber ultrafiltration membrane.

3. DEAE-Sephadex A-50 ion-exchange chromatography

Chromatography: sample size is 10% of a bed volume, and sample concentration is 5mg/ml,

With 0.03M PB is eluent, and Nacl concentration increases gradually from 0-0.9M, and the continuous gradient wash-out is collected the 3rd peak, through concentrating.

4. immune affinity absorption:

Adsorbent is CNBr-sepharose 4B, soaks and abundant swelling with diluted acid, makes itself and the coupling of anti-normal person's whole serum antibody protein, and above-mentioned immunosorbent is drained, and adds sample, reprints absorption, and reservation filtrate is washed till 0D with damping fluid with adsorbent ₂₈₀＜0.02.Adsorbent reactivation, standby.

5. purity is reached the PAPP-A freeze-drying more than 95%, and take out a part as quality controlled serum and freeze-drying, the weight of standard items generally is controlled at 0.5-2.5 μ g in every kit, and the weight of quality controlled serum dried frozen aquatic products is 0.3-1.0 μ g, and above-mentioned weight can be made secondary PAPP-A assay.

Two, the preparation of enzyme labelled antibody:

1, immune animal:

The rabbit of selecting about 2 kilograms stimulates animal with the injection of 5-10mg Bacille Calmette-Guerin earlier, after 7-10 days as immune animal, PAPP-A is made Fu Shi Freund's complete adjuvant antigen liquid, adopt the immunity for the first time of subcutaneous multiple spot, after 2 weeks, the subcutaneous immunity second time of groin, after 2 weeks, examination blood and survey are tired.

2, antiserum separates, and identifies and preservation:

After treating that immune serum is tired up, the way of the disposable bloodletting of kill animals is adopted in sero-fast collection, and rabbit can be adopted 70ml left and right sides whole blood.Sterile cryogenic, many centrifuging and taking serum of 5000g.

Immune serum is identified:

1. mensuration-the double immunodiffusion of antibody titer;

2. antibody content is measured-contrary single-phase SRID;

3. antibody specificity-immune purity and cross reactivity

3, purifying antibody and mark

1. purifying antibody: add saturated ammonium sulfate (SAS) in serum, the volume ratio of serum and saturated ammonium sulfate is 2: 1, makes antibody heavy fixed, centrifuging, and antibody purity reaches more than 95%,

2. antibody labeling:

Adopt the glutaraldehyde two step method horseradish peroxidase (HRP) to be marked on the PAPP-A antibody packing ,-20 ℃ of freezing preservations.

4, bag is by reaction plate

The PAPP-A antibody of above-mentioned purity is made into the NaHCO that concentration is 1mg/ml ₃(PH=9.6) damping fluid, 100 μ l/well wrap by reaction plate, filter, and inferior daily confining liquid 100 μ l/well are in 37 ℃ of sealings 2 hours, freeze-drying, standby.

Kit quality standard: standard items S ₁0D450 〉=0.16; 10 parts of negative findingses detect errorless; The accuracy sample is measured CV＜15% (batch variation), 2-8 ℃ of preservation; Linear r＞0.98.

Advantage of the present invention: the assembly of this kit is reasonable, and stable performance is measured accurately, and is easy to use.

The using method of this kit:

1, the variable concentrations liquid of preparation standard items:

1. with 25 times (concentration is 0.1M during detection) of concentrated cleaning solutions dilution;

2. add cleaning fluid 700 μ l in the bottle of standard items, vibration evenly;

3. add 400 μ l cleaning fluids in other 4 clean containers, these 4 volumes are designated as S ₁, S ₂, S ₃, S ₄

4. from 2., get 200ul liquid and add S ₄In the bottle;

5. from S ₄Bottle is got 200ul to S ₃In the bottle;

6. from S ₃Bottle is got 200ul to S ₂In the bottle;

7. from S ₂Bottle is got 200ul to S ₁In the bottle.

Except that the standard items of above-mentioned variable concentrations, also need add testing sample to bag and (be become different multiples with cleaning solution dilution on the reaction plate hole according to pregnant Zhou Butong, for example: the serum in pregnant woman 9-14 week is with 1: 20 times of dilution), bag included by the hole of reaction plate-and quality controlled serum, blank test use the hole, after relevant each hole adds above-mentioned various test solution, wash each hole, add enzyme labelled antibody subsequently, add developer A, developer B mixed liquor after the washing, add the stop buffer stopped reaction again, with the blank zeroing, in 450nm wavelength place colorimetric, write down each hole OD value with microplate reader.Measure the statistical procedures of numerical value, adopt method of percentiles, wherein the 5th and the 95th percentile is represented the lower limit and the upper limit of PAPP-A normal reference range respectively, and the 50th percentile is a median.

Claims

1, a kind of kit of determining plasma protein A related to pregnancy by enzyme combination quantity, it is characterized in that it comprises: the kit box body, place bag in the box body by reaction plate and the packaged reagent of difference, reagent has: standard items, quality controlled serum, enzyme labelled antibody, and their composition is respectively:

2, the kit of determining plasma protein A related to pregnancy by enzyme combination quantity according to claim 1 is characterized in that: every box Packing Unit of described standard items is 0.5-2.5 μ g, and the every box Packing Unit of quality controlled serum is 0.3-1.0 μ g.

3, the kit of determining plasma protein A related to pregnancy by enzyme combination quantity according to claim 1 is characterized in that also including in the kit:

Concentrated cleaner: phosphate buffer;

Developer A: tetramethyl benzidine;

Developer B: hydrogen peroxide;

Stop buffer: 1M sulfuric acid;

Sticker;

Semi-logarithmic scale paper.