CN1076784A - Magnetic corpuscular immune separating agent and preparation - Google Patents

Abstract

The present invention has prepared a kind of novel immune release agent, and this separating agent is by Fe ₃O ₄Microparticle magnetic nuclear, the high molecular polymer thin layer that is wrapped in magnetic nuclear surface and chemical crosslinking are formed at the antibody on thin layer surface.When being used as the separating agent of immunoassay, non-specific bond is low, and suspending power is good, has solved the problem of using aldehyde radical solid phase and magnetic solid-phase separating agent in the immunoassay.

Description

Magnetic corpuscular immune separating agent and preparation

The present invention relates to a kind of new immune release agent and preparation thereof.

In panimmunity is analyzed, all need to separate the bound fraction and the free fraction of antigen, antibody.Improve isolation technics and can not only make analytical approach fast and simple and easy, and, therefore be the important content that immune analysis method is learned research always improving precision of analysis and repeatability has important value.

Application solid phase isolation technics is the important improvement in the immune analysis method evolution, and development antibody is one of frontier nature research topic by the immune release agent of chemical crosslinking on magnetic corpuscular.Wherein, microcrystalline cellulose magnetic antibody is set up (Forrest, GC.etal: " Immu-noassays for Clinical Chemisty(Edited by W.M.Hunter, etal) ", Churchill Livingstone, p147-162,1983; Read, GF.etal: the same, p163-169).But preparation fibrin magnetic microparticle program is loaded down with trivial details, tediously long; In addition, need before this microparticle cross-linking antibody earlier cellulosic hydroxyl activation to be active functional group that used activating reagent is poisonous or expensive, activation products instability, the also difficult control of activation condition.

Aldehyde radical is active functional group, need not to activate can be under common condition direct crosslinked amino-compound.Once the someone used chemical polymerization (Pourfarzaneh, M.etal:Methods Biochem.Anal., 28:267,1982; Margel, S.etal:J.Cell Sci., 56:157,1982) and the irradiation polymerization method (Rembaum, A:U.S.Patent 4413070,1983; Chang, M.etal:Methods Enzymol., 112:150,1985) preparation polyacrolein magnetic corpuscular.But these method complexity, and the sign that is mainly used in cell by the separating agent that these magnetic corpusculars make with separate, and affinity chromatography separates, when as the separating agent of immunoassay, then non-specific bond is very high; Relatively poor with suspending power, influence immunoreactive completeness.These two problems never solve.

The objective of the invention is to prepare a kind of new magnetic corpuscular separating agent, this separating agent has suspending power preferably in the immune response medium, and non-specific bond is low.

To the effect that of the present invention:

1, prepared Fe with chemical precipitation method ₃O ₄Microparticle is examined as magnetic.

Precipitation reaction is carried out in aqueous medium.Fe ⁺⁺⁺With Fe ⁺⁺The mole ratio of water soluble salt is 1.5-2.0: 1; Precipitation agent and Fe ⁺⁺⁺And Fe ⁺⁺The gram equivalent of salt is than being 5-8: 1, and precipitation agent is ammonium hydroxide, NaOH or potassium hydroxide.Temperature of reaction is 5-20 ℃, and the reaction time is 1-2 hour.

2, wrap up the polyacrolein thin layer with the irradiation polymerization method on magnetic nuclear surface, as magnetic carrier.

Polyreaction is carried out in aqueous suspensions.The composition of suspension is: magnetic nuclear 0.5-1.0%(W/V); The acryl aldehyde and second monomer be 3-6%(V/V altogether), wherein second monomer accounts for 10-40%(V/V); Emulsifying agent 0.5-1%(W/V).Irradiation bomb is ⁶⁰Co, the irradiation accumulated dose is 5 * 10 ³-1 * 10 ⁴Gy, exposure time is 2-4 hour.

Above-mentioned second monomer is an alkene class carboxylic acid, as acrylic or methacrylic acid; Emulsifying agent is lauryl sodium sulfate, sodium dodecylsulphonate or neopelex.

3, the crosslinked soluble antibody of magnetic carrier becomes magnetic antibody.

Cross-linking reaction is carried out in the weak acidic buffer suspension.The composition of suspension is: magnetic carrier 20-50mg/ml, water-soluble antibody and 0.1mol/L damping fluid.Temperature of reaction is 15-25 ℃, and the reaction time is 10-20 hour.

Above-mentioned antibody can be the antiserum crossed through the ammonium sulfate precipitation method purifying or the dried frozen aquatic products of pure gamma Globulin, and addition is pressed the titre of antibody and determined.The pH5-7 of damping fluid.

4, residual aldehyde radical functional group on the closed magnetic antibody particle becomes the magnetic resolution agent.

Capping carries out in the alkaline buffer suspension.The composition of suspension is: magnetic antibody 20-50mg/ml and the 0.1mol/L damping fluid that contains sealer.Temperature of reaction is 15-25 ℃, and the reaction time is 3-5 hour.

Above-mentioned sealer is low-molecular-weight amino-compound, example hydrochloric acid azanol, monoethanolamine, glycocoll or tri methylol amino methane.The pH8-10 of damping fluid.

As above the physics of Zhi Bei magnetic nuclear, magnetic carrier and magnetic resolution agent, chemistry and immunology performance are:

They all are the particles of black.Use transmission electron microscopy observation, the outward appearance of magnetic nuclear is irregular particle, and its mean diameter and relative standard deviation thereof are respectively 10-50nm and 30-40%.The outward appearance of magnetic carrier is magnetic nuclear surface parcel one layer of polymeric; The capacity of crosslinking protein is 200-300mg/g.Half settling time of magnetic resolution agent in the alkaline buffer that contains 1% bovine serum albumin(BSA) is 30 minutes; It is the magnetic sheet surface of 0.15 tesla after 3-5 minute that 3 centimetres of high suspensions place magnetic induction density, and magnetic particle wherein is occluded in container bottom fully, inclines can not run off when supernatant; The damping fluid suspension of magnetic resolution agent was 40 ℃ of placements at least 7 days, and 4 ℃ of placements at least 6 months, its usability can not change.

With the isolation technics of existing immunoassay relatively, the advantage of this magnetic resolution agent is: the preparation of (1) separating agent is easy, save time; Raw material is easy to get, and cost is low.Be convenient to produce in batches.(2) easy to use, save time, avoided in the analytic process stirring and centrifugal.Be suitable for analyzing the robotization of batch samples and routine analyzer.(3) excellent storage stability.Transporting than under the high ambient temperature, do not influencing the usability of product.(4) be common to multiple radiommunoassay project, as triiodo thryonine (T ₃), anti-T ₃, free T ₃, thyroxine (T ₄), free T ₄, thyrotropic hormone, thyroglobulin (TG) and TG antibody.Non-specific bond is low, and good reproducibility meets the requirement of radioimmunoassay method quality control index.

Embodiment

Example 1. chemical precipitation methods prepare Fe ₃O ₄Microparticle

Dissolve 9.21 gram FeCl respectively ₃6H ₂O and 3.87 gram FeCl ₂4H ₂O is cooled to about 10 ℃ in 80ml and 40ml distilled water.25mlNH ₄OH(contains NH ₃25-28% W/V) adds 55ml distilled water, is cooled to about 10 ℃.Mix Fe ⁺⁺⁺And Fe ⁺⁺Salt solusion is in two mouthfuls of reaction bulbs, and reaction bulb places ice-water bath, and the oar formula stirs and drips NH down ₄OH solution reacted 1 hour the about 3ml/ of speed second, formed black Fe ₃O ₄The microparticle suspension.Suspension magnetic sheet precipitation separation.Precipitation is fully washed with 0.9%NaCl, washs with distilled water then.At last, the precipitation adding distil water becomes suspension.Sampling, oven dry is weighed.Suspension places 4 ℃ of preservations.

Fe ₃O ₄The yield of microparticle is 4.81 grams, and mean diameter and relative standard deviation thereof are respectively 10.4nm and ± 34%.

Example 2. irradiation polymerization legal systems are equipped with polyacrolein microparticle magnetic carrier

9.6ml methacrylic acid adds the Fe that acryl aldehyde 38.4ml and example 1 make ₃O ₄Microparticle aqueous suspensions 200ml(48.1mg/ml), adding distil water is to 914ml.Feed N ₂Flowed 20 minutes, and added 0.1g/ml lauryl sodium sulfate 48ml then.Airtight container places on the upset stirrer, makes the suspension in the container be convertible stirring.The suspension warp ⁶⁰Co gamma ray radiator irradiation 3 hours, accumulated dose are 8 * 10 ³Gy.Polymerizate is precipitation separation on magnetic sheet.Precipitation is fully washed with 0.9%NaCl, washs with distilled water then.At last, the precipitation adding distil water becomes suspension.Sampling, oven dry is weighed.Suspension places 4 ℃ of preservations.

The yield of magnetic carrier is 4.66 grams; 4 ℃ of storages at least 6 months, the performance of cross-linking antibody did not change.

Example 3. magnetic carrier cross-linking antibodies

The magnetic carrier suspension 86ml(46.6mg/ml that example 2 makes), place precipitation separation on the magnetic sheet.Precipitation adds 0.1mol/L NaH ₂PO ₄-Na ₂HPO ₄Damping fluid (to call PB in the following text) PH5.4 washes once.Precipitation adds the anti-rabbit anti-serum of 40ml donkey (through (NH ₄) ₂SO ₄Precipitation method purifying contains total protein 31.8mg/ml, and the titre of gamma Globulin G is 1: 32.To call DXR in the following text), 0.2mol/L PB pH5.4 80ml and distilled water 40ml.Airtight container places on the upset stirrer and stirred 10 hours, and room temperature is 15 ℃.Reaction product is precipitation separation on magnetic sheet.Precipitation is washed 2 times with 0.1mol/L PB pH8.0.Be precipitated as magnetic antibody.

As above program is if add simultaneously ¹²⁵I-DXR, measures in the supernatant at the different suspension precipitation separations of constantly getting of reaction as the tracer agent of DXR cross-linking reaction ¹²⁵The I radiocounting, the result shows: reacted 5 hours, and reached molecular balance substantially; Reacted 10 hours, in the supernatant ¹²⁵The I counting is 16.1% of addition.The total protein concentration of known DXR is 31.8mg/ml, so the capacity of the magnetic carrier crosslinking protein that makes of example 2 is 267mg/g.

Residual aldehyde radical on example 4. closed magnetic antibodies

The magnetic antibody that example 3 obtains adds 0.1mol/L PB pH 9.5 160ml that contain 1% oxammonium hydrochloride and becomes suspension.Suspension such as example 3 stirrings 5 hours, magnetic separation and washing.Precipitation is washed 1 time with 0.075mol/L barbitol buffer solution pH8.5 again.Precipitation adds 1% bovine serum albumin(BSA)-0.1% NaN ₃-0.075mol/L barbitol buffer solution pH8.5 becomes the 5mg/ml suspension, is magnetic resolution agent suspension, place 4 ℃ standby.

Using method and the effect of example 5. magnetic resolution agent in radiommunoassay

This magnetic resolution agent can be used for radiommunoassay, and using method is: after antigen is finished with the reaction of its first antibody, add magnetic resolution agent 0.5mg/ pipe, 37 ℃ of incubations 10 minutes; Add the washing of 0.9% NaCl 1ml/ pipe.Reaction tube placed on the magnetic sheet after 3-5 minute, and reaction tube is inverted together with magnetic sheet, the supernatant that inclines, and place on the multi-layer absorbent paper and flow to end raffinate.And then carry out after this routine analyzer.

This magnetic resolution agent is used for T ₃And T ₄Radiommunoassay, non-specific bond rate is respectively 4.3% and 1.8%, zero pipe specific bond rate is respectively 57.8% and 58.3%; The coefficient of variation: in batch＜6%, between batch＜12%; The related coefficient of typical curve is respectively-0.9994 and-0.9990; The effect that is used for other radiommunoassay project is similar.These results all meet the requirement of radiommunoassay quality control index.This magnetic resolution agent and is made separating agent with classical liquid double antibody+polyglycol, analyzes T simultaneously ₃Or T ₄The quality controling serum of three kinds of variable concentrations, the result of two kinds of separating agents coincide each other.

Claims (4)

1, a kind of polyacrolein magnetic corpuscular immune separating agent is examined, is wrapped in the high molecular polymer thin layer and the chemical crosslinking on magnetic nuclear surface and forms at the antibody on thin layer surface by magnetic, it is characterized in that can be used as the separating agent of immunoassay.

2, a kind of method for preparing the described separating agent of claim 1, the high molecular polymer thin layer of this separating agent is that magnetic is examined reaction in containing the aqueous solution of monomer and is wrapped in magnetic nuclear surface, what it is characterized in that monomer consists of the acryl aldehyde and second monomer, and the two proportioning (volume) is 9: 1-6: 4.

3, second monomer according to claim 2 is characterized in that composition is an alkene class carboxylic acid.

4, the preparation method of magnetic nuclear according to claim 2 is Fe ⁺⁺⁺With Fe ⁺⁺Salt solusion in drip alkali precipitation and get, it is characterized in that Fe ⁺⁺⁺Salt and Fe ⁺⁺The mole ratio of salt is 1.5-2.0: 1, and alkali is 5-8 with the gram equivalent ratio of molysite: 1.