CA1062610A - Gel column device and use in blood purification and immunoassay - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Hydrophilic, insoluble, porous gel particles having binding proteins entrapped therein, and in which a fluid to be tested may be intro-duced into the volume of gel particles whereby some of the binding sites are occupied. Exposure of the gel particles to radio active tagged material followed by measurement of the radio activity of the unbound or bound tagged material permits an immunological determination of the fluid. The binding proteins in the gel particles are returned to their original state for re-use by treatment with acidic medium to effect detachment of the bound mat-erial followed by washing to effect removal of the unbound material, and then drying.

Description

1(~626;1C~

This application is a dlvision from applicant's copending Canadian application Serial NoO 198,533 which claimed a device for immunoassay of blood, blood plasma or serum comprising a container housing a volume of dry, highly hydrophilic, insoluble gel particles having a binding protein and a radioactive tag material entrapped within the gel particles and characteri~ed by a predetermined water regain value and pores of a size which permit dif-fusion of small molecules into the intra gel volume, but insufficient to permit entry of high molecular weight componènt and means for transmission of fluid for determination into and out of said volume.
The present invention provides in the preparation of dry, porous gel particles of the type suitable for use in liquid sample assay and having high water regain for use in reproducible increments of liquid sample upon rehydration with the sample, mixing the gel in particle form in a non-aqueous medium, removing the gel particles from the non-aqueous medium, and then allowing the gel particles to dry before useO
The present inventi.on.also provides a process for the preparation of dry, hydrophilic, insoluble, porous gel particles having a binder protein entrapped within the gel particles and having high water regain for use in reproducible increments of liquid sample upon rehydration with the sample, said process comprising dissolving at least one monomer in a buffer solution containing binder protein and polymerising, fragmenting the gel thus produced to form gel particles, mixing the gel particles in a non-aqueous medium, removing the gel particles from the non-aqueous medium, and then allowing the gel particles to dry before useO
Application of liquid sample to the measured volume of dry gel particles is determined by water regain of the gel thereby to provide a pipetless method of sample application based on use of highly reproducible dry-volume aliquots of the dried or lyophilized gel particlesO
The dry gel particles can be preloaded with radio active tagged hormone and standard hormone either during preparation of the dry gel lO~Z6~ ~

particles, or by after treatment of the dry gel particles previously provided with the entrapped antibody.
The dry antibody-gel reagent is stable under widely diversified atmospheric conditions to enable storage over long periods of time without deviation or loss in binding activity. Thus, there is described a radio-immunoassay system which provides for automatic exclusion of high molecular weight interferences; elimination of the need to pipet or centrifuge the sample to be tested; long term stability of binding reagents stored under ambient conditions; and a reliable and accurate method of assay which has been successfully applied to the assay of angiotensin I and insulin.
According to the present invention, there is provided a process for the preparation of dryJ hydrophilic, insoluble, porous gel particles having a binder protein entrapped within the gel particles and having high water regain for use in reproducible increments of liquid sample upon rehydration with the sample, said process comprising dissolving at least one monomer in a buffer solution containing binder protein and polymerising, fragmenting the gel thus produced to form gel particles, mixing the gel particles in a non-aqueous medium, removing the gel particles from the non-aqueous medium, and then allowing the gel particles to dry before useO

2~ Proteins having a binding capacity for certain specific smaller molecules and which can be readily immobilized in the polymer gel are illus-trated by various protein globulins and antibodiesO
Gel systems having such globulins or antibodies entrapped therein can be used effectively for analyzing for a variety of antigens or haptenes, such as drugs, polypeptide hormones and steroid hormones. Antigens and haptenes susceptible of being assayed in a system of the type described include, for example, angiotensin, insulin, growth hormone, gonadotropic hormone, parathyroid hormone, glucagon, cortisol, protoglandin, cortico-steroids, cyclic fatty acid hormones and estrogens, and drugs such as dig-oxin, thyrosin, morphene, digitalis, and the like. By way of example, lO~Z610 specific corticosteriod binding globulins, immobilizcd by entrapment in the gel system, permit assay of corticosteriod hormones in addition to the many polypeptide hormones. 7S gamma-globulin (1S0JOOO m.wO) can be used to assay for angiotension (1,000 m.wO)O
The present invention taken in conjunction with the invention disclosed in copending Canadian application Serial NoO 198,533, filed April 30, 1974J will hereinafter be described in greater detail by reference to specific examples.
Example 1 A gel, in which dilute anti-angiotensin immune serum containing 7S gamma globulin ~m.w. 150,0003 is entrapped in a gel matrix having a pore size to enable diffusion of angiotensin (m.wO 1JOOO) for assay may be pre-pared as follows:
8 grams of acrylamide and 1 gram of N,N'-methylene-bis-acrylamide is dissolved to a final volume of 36 ml in sodium phosphate buffer solution (0.1 M, pH 7.4) containing a sui~able dilution of antiserum. To the resulting solution 0.1 ml of a suspension of 100 mg of riboflavin in 20 ml of distilled water and OoQ2 ml of N,N,N'JN-tetramethyl-ethylene-diamine is addedJ follow-ed by 0.2 mg of sodium hydrosulfite. The mixtureJ in a suitable vesselJ is stoppered and agitated and then is exposed to light from an ordinary tung-sten light bulb to induce polymerization. Polymerization is complete in 5-15 minutesO The reaction vessel is continuously chilled in an ice water bath, as needed, to prevent the heat generated by the exothermic polymer-ization reaction from adversely affecting the binding protein.
The antibody gel is fragmented by pushing through a #40 mesh brass screen. The gel particles are collected on a #60 mesh screen and washed with 5 liters of distilled-deionized water. The gel particles sediment rapidly and are stiff enough to tolerate moderate flow rates (0.5 to 2~0 ml/min) in a chromatographic column without plugging. After sieving, the gel particles are equiliberated with 0.1 M pH 704 phosphate buffer. The gel particles are lO~Z610 now ready for the drying procedure~
Two fundamentally different drying procedures have been employed including (1) lyophilization and (2) dehydration in acetone ethanol or other low boiling alcohol, followed by air drying. Neither method was associated with loss of antibody activity. However, ethanolic dehydration is preferred because of its simplicity and rapidity of drying. For this purpose~ the gel particles were suspended in 5 volumes of 95% ethanol and allowed to settle.
The supernatant solution was discarded and the ethanol treatment repeated one or more times. The ethanol dehydrated gel particles were then allowed to dry in room air by spreading on a nylon net.
Storage stability has been demonstrated by hermetically sealing the lyophilized gel particles~ ~rith the entrapped antibody activity~ at temperatures of 37C, 25 C, 4 C and -20 C for over two years without detect-able loss of antibody activity when the entrapped protein is in the form of anti-insulin introduced by the way of antibody sera for the anti-angiotensin in the described example. Dried gel particles containing anti-angiotensin antibody prepared in accordance with this example has also demonstrated storage stability, without loss of antibody activity for over six months.
As indicated above, rabbit antisera for use against angiotensin I and guinea pig antisera for use against insulin can be substituted for the antisera in producing the antibody gel particles of this example.

Example 2 Evaluation device:
In its simplest form~ an evaluation device for radio-immunoassay is illustrated in the form of a syringe 10, such as a 1 ml tuberculin syringe, having a syringe needle 12 at one end and a plunger 14 operative in an elonga~ed~ barrel at the other end for drawing fluid for unidirect-ional flow through the needle 12 into the body portion 16 of the syringe in ~6Z~
response to the generation of volume conditions upon withdr~wal o~ the plunger.
Adjacent the needle end portion, the syringe is subaivided by a porous barrier 18 into a calibrated portion 20 packed with a column of pre-scribed volume of the dry gel particles of Example 1, while the plunger or other withdrawal means communicates with the portion beyond the barrier 18.
In the described device, the barrier is in the form of a nylon fabric or net having openings insufficient to pcrmit passage of the gel particles. In the illustrated modification, 33 mg of dry gel particles are packed in the volumç of 0.14 ml enclosed by the porous barrier made up of about two-thirds intra gel volume and about one-third extra gel volume, with the dry gel particles having a water regain of 100 mg water per 33 mg gel particles.

xample 3 Method of use:
In manual use, the needle 10 is inserted in the sample (serum, plasma or whole blood drawn directly from a vein) and the plunger is pulled back to draw some of the liquid sample into the syringe 10 in an amount to at least fill the volume 20 below the barrier 18 and preferably in an amount to at least cover the net.
As a sample is drawn into the syringe, the highly hydrophilic gel particles 22 abs~rb a predictable and reproducible portion of low mole-cular weight component in the s~mple by rehydration. To prevent coagulation~
when tes~ing fresh whole blood or plasma, it is desirable to incorporate a anti-coagulant into the gel recipe from which the dry gel particles are formed.
The size of the sample that is taken up by the particles depends upon the water regain value of the gel as well as the amount of particles : . -: : . . , . .

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present in the column, measured by weight or by volume. As a result, sample size can automatically be controlled by the amount and water regain value of the dried gel particles thereby to make the test reproducible and accurate, independent of the amount of liquid sample that is drawn into the syringe.
After the sample has been drawn into the syringe, the void volume (extra gel volume of the miniature-syringe chromatographic column) is flushed out, as with a buffer solution, such as a 0.1 M phosphate buffer or any other physiological solution. The flushing liquid should be employed in an amount corresponding to at least one void or extra gel volume but it is preferred to make use of more than one and preferably two *o five void volumes in order to insure the removal of sample component otherwise re-maining in the extra gel volume. The low molecular weight polypeptide hor-mone (or other antigen or haptene) remains substantially entrapped within the intra gel space during this rapid column wash step for removing high molecular weight interference. Thus high molecular weight interferences are removed with the wash solution used to flush the col D of gel particles.
After the flushing step, a competing radio active "tag" hormone of known concentration is drawn into the syringe in an amount to at least fill the void volume and preferably in an amount greater than one void vol-ume.
The unit is ~hen incubated for a specific period of time, which may range from 30 seconds to 30 hours, at room temperature. It is not necessary for the radio competitive binding assay to go completely to equilibrium before final separation of the bound from the free radio act-ivity.
Thereafter, a buffered washsolution, which may be the same as that used to flush the column in the previous step, is passed through the column. This operates to separate unbound hormones from bound hormones.
The column is flushed with sufficient volume for a length of time substant-ially to complete the separation step. The total amount of tagged radio active hormone used will equal the bound plus the unbound. The radio activity bound to the binding sites in the column can be measured directly or it can be determined indirectly by measuring the radio activity of the wash solution. use can be made of standard values sufficient to construct a standard working curve having the per cent bound as the abscissa and the log scale of the standard as the base, whereby the unknown in the sample can be read directly from the curve~ in accordance with the standard procedures of current radioimmunoassay.
The described technique and materials are capable of a n~mber of modifications and ramifications.
The formulation of the gel of which ~he particles are formed is not significant since use can be made of any gel forming material which is capable of being reduced into porous~ insoluble, highly hydrophilic parti-cles of controlled pore size. Thus, other highly hydrophilic, insoluble gels capable of the desired pore size and drying or lyophilization can be used but it is preferred to make use of the polyacrylamide gels of the type described wherein an acrylamide monomer and cross linking agent are combined with sufficient cross linking agent to stabilize the pore size and support ~0 the monomer not only to provide the desired pore size but to give the gel the density desired for rapid sedimentation in particulate form and to pro-vide sufficient rigidity to the gel particles. The ra~tio of one part by weight of cross linking agent to 4 to 10 parts by weight monomer is suffic-ient to give a gel having a total gel polymer concentration in the range of 12-35~.
The binding globulin~ antibody or protein entrapped within the dry gel particles can be varied, depending upon the hormone, drug or other material to be assayed, such as angiotensin, insulin, growth hormone~ gona-dotropic hormone, parathyroid hormone, cortisolj digoxin, digitalis or , . . . .
: ~ . . . . . - ;

lO~Z610 thyroxin.
By way bf modification, the radioactive tag hormone can also bc incorporated in calibrated amounts within gel particles. These gel particl-es which~ upon rehydration~ release radioactively tagged hormone, can also be added in precise amount by weight or ~olume to the dry antibody gel particles, used as the binding reagent in the RIA system so that the addi-tion of radioactively tagged hormone into the RIA system is automatic.
In this arrangement, it is desirable to make us of an amount of tagged hormone or drug which will not completely satis~y the binding sites avail-able in the gel particles before the unkown sample is drawn into the column.
In a similar fashion, a standard amount of drug or hormone can be incorporated into the gel recipe for release upon ~ehydration of the gel particles in the test system. Thus~ for calibration purposes, the user can be provided with the convenience of pre-addition of standard to the RIA
system whichfis aut~matically released upon rehydration of the gel particles.

xample 4 The foregoing modification comtemplates the formation of dry gel particles containing the tag hormone in a manner similar to that for pr~
ducing the dry gel particles containing the binding protein, but separate and apart therefrom. The gel particles containing the tag hormone and the gel particles containing the antibody or binding protein would then be mix-ed in the amounts described and the mixture would then be employed to form -the volume in the test unit. Upon rehydration~ the tag hormone would become free to reach the antibody binding sites.
It has also been found possible to combine the antibody binding protein and the tag hormone in the same gel particles, without premature binding of the tag hormone to antibody binding sites, thereby to eliminate the need for separate preparation of the gel particles and to ~orm a pre--8- `
, ~

10~2~0 mix thereof.
For this purpose, it is important to effect the incorporation of the tag hormone in the absence of hydration which would enable the tag hormone to reach the binding sites. This can be accomplished by the add-ition of the tag hormone to the dry gel particles from a non-aqueous medium.
Thus incorporation of the tag hormone is carried out when the antibody gel particles are dry -- which should preclude premature binding of the tag hormone ~o antibody binding sites.

Example 5 Dry gel particles with immobili~ed antibody activity~ prepared in accordance with Examples 1 and 2~ are contacted with a solution of the tagged hormone in 95% ethanol or other organic alcohol or solvent.
The particles are dried as in air or in a chromatographic column having clean air flowing therethrough to effect alcohol removal.
The resulting dry gel particles have both antibody binding protein activity and tagged hormone in al~non-complex (non-bound) form.
The tagged hormone will not diffuse to the binding si~e until the gel under-goes rehydration with the test sample, as in Example 4. At such time5 the tagged hormone is released to compete with the sample hormone for complexing to the binding sites.
The water regain value of the gel particles determines how much sample reaches the antibody binding protein contained in the intra gel com-partment. The water regain of the gel will depend somewhat on the per cent polymer and per cent cross linking agen~ and more particularly on the thoroughness of the drying process. The gel reagents of the type described can be dried so that water regain will not change under the conditions of temperature and humidity used in storage. Other degrees of dryness and ~ `
water regain can be used, but are not convenient, because of changes in _9_ lO~iZt~10 hydration that occur with changes in storage temperature and humidity.
The particle size as well as the form and shape of the dry gel particles can be varied depending upon the size and shape of the test device.
Instead of a syringe, other containers can be employed in which a known volume or weight of dry gel particles can be retained and through which the liquid sample, flush solution and tag solution can be-transported for engagement with the gel particles in the amounts previously described.
In the event that a device is used in which the gel particles are retained as a column in an intermediate section of known volume, then barriers such as a nylon net should be provided at opposite ends to confine the particles into the chromatog~aphic column while enabling the various fluids to pass therethrough into and out of the column of particles.
The porous barrier functions automatically to separate bound from unbound radio activity which occurs during the flushing of the column.
This porous structure functions also as an anticonvection barrier, which discour~ges mixing of any solution sequestered outside the gel particle compartment.
Since the volume of gel particles is known and the water regain is constant7 the amount of sample taken up by the gel particles will be the same independent of the size of the sample or the amount introduced into the test unit. The same applies with respect to the fluid solution used to flush the particles for removal of material in the extra gel volume and the amount of radio active tag material to which the gel particles are ultimate-ly exposed. Thus many of the steps critical to-prior immunoassays have been eliminated with equal or greater reproducibility and accuracy of test re-sults. Since the antibody is insolybilized and immobilized within the gel particles, conventional centrifugation and washing steps, normally employed in current procedures, can be eliminated in their entirety in the prepara- .
tion and use of a test unit of the type described.

10~26~) Similarly~ since the unknown and tag material taken up depends on such known values as volume and water regain, the c~itical steps of pipetting and the numerous errors associated therewith are eliminated there-by to increase the accuracy of the analysis.
By immobilizing the antibody binding reagent in the gel, the centrifugation step of conventional RIA can be eliminated. Furthermore, by engaging the antibody binding protein in a gel polymer matrix~ high molecu-lar weight interference of RIA can be excluded. Such high molecular weight interferences which are found in some but not all biological samples, are of at least two types~ namely, (1) proteolytic enzymes, which can degrade polypeptide hormone, and (2) endogenous antibody, which can bind polypeptide hormone. Endogenous antibody that binds insulin is frequently found in patients treated with injections of bovine or porcine insulin. These high weight molecular interferences are too large to penetrate into the polymer matrix of the gel particles and are sequestered in the extra gel space, where they can be removed by washing the gel column.
The device which has heretofore been described has been manually applied for single assays, after which the device is discarded. It would, of course, be desirable to be able to make multiple use of the device, without loss of binding activity, and it would be still more desirable to be able to automate the operation for multiple use thereby to effect consider-able savings in time and materials, as well as reproducibility of the re-sults.
It has been found, in accordance with the practice of this invention, that the binding sites of the antibody gel particles in the .
chromatographic column of the test device can be returned to their original state to enable re-use as a test device, without loss of binding site, and that this can be achieved again and again in a simple and efficient manner for either manual or automated radioimmunoassay systems.

2~

Unbound radio activity can be washed from the column, in the manner previously described~ with a variety of physiological solutions at neutral pH for separation of unbound radio activity and unbound hormones from the system. Bound radio activity can be detached from the binding sites to enable elution from the antibody gel particles in the column by an acid or hyperthermic wash solution.
The unbound radio activity and hormone washed from the column or the bound radio activity eluted from the antibody gel particles can be made the subject of analysis, as by gamma or liquid scintillation type detectors, to arrive at the assay value for the previous sample.
Following elution of the bound radio activity, the column of gel particles is washed with a buffer for removal of the acid or hyper-thermic reagents. This is followed by washing with an organic solvent which is at least partially miscible with water and which is highly volatile and which forms a constant boiling mixture with water to effect removal of the buffer and to enable rapid dehydration and collapse of the antibody gel particles. Upon evap~ration of the organic solvent these gel particles have been returned to their original dry state. Collapse of the gel particles, which occurs with solvent drying, is preferred because it prevents entrap-........ .............................................................................. . , ment of large amounts of air which effect reproducibility of the quantity of sample or water ~egain and/or is released as tiny bubbles on rehydration. ~ -For detachment of the bourd radio activity and bound hormones from the binding sites of the antibody gel particles in the column~ it is ;
preferred to make use of an aqueous acidic medium having a pH of less than 2.5 and preferably an aqueous solution of hydrochloric acid. The acidic solution is introduced into the gel column in an amount corresponding to at least one gel volume and preferably more than one gel volume but less than three.
Removal of the acidic medium with the detached radio activity ,~, .. ... . .. . . . . . . ..

1~)62610 and hormones is effected by washing with a buffer solution, ~uch as an 0.1 M phosphate buffer having a pH of about 7.4 but other suitable buffers can be used as previously described. The amount of buffer passed through the column should be sufficient to rinse the acid from the column of gel part-icles, usually one to five volumes will be sufficient.
The org2nic solvent is employed primarily to effect removal of aqueous medium and to permit rapid drying and collapse of the gel particles in the column. For this purpose~ it is desirable to make use of an organic solvent which does not denature the binding activity of the entrapped anti-body and which forms a constant boiling mixture with aqueous medium to ef-fect water removal and in which the organic solvent is highly volatile to facilitate rapid removal and permit rapid drying of the gel partic]es.
Representative of suitab~e organic solvents are acetone, methyl alcohol, ethyl alcohol~ low boiling esters~ and the like.
Solvent removal and accelerated drying is effected by passing air through the column at ambient temperature or at a temperature slightly above ambient but below the temperature which would destroy any of the an~i-body binding sites. The stream of drying air accelerates volatilization of the organic solvent for ~apid and complete removal. ,~
The thus regenerated antibody gel particles in the column are returned to their original state including collapsed structure for re-use of the test device without the need for any recalibration.
It will be apparent from the foregoing description that, in the regeneration as well as in the use of the device, the materials are caused to flow through the device in one direction. Such column-chromato-; graphic, unidirectional flowing stream configuration of analysis and re-generation enables complete automation of the use and regeneration of the device merely by the addition of a precision syringe or proportionating pump operatively connected with the ingoing end of the device to force the lO~Z~10 passage of the various materials in the desired volumes therethrough; a detection means communicating with the outgoing end of the device to test for unbound radio activity or bound radio activity in the materials that are eluted from the column; and a processor-controller for sequencing the materials for flow through the device.
An automated system of the type described provides for highly reproducible applications of samples, reagents, wash solutions, solvents and air to the column of antibody gel reagent is advantageous for both manual and automated approaches. Gel ant~body RIA does not require dilu-tion of plasma to reduce or eliminate high molecular weight interferences.
Thus reaction kinetics need not be slowed by reason of dilution in the sys-tem.
Manual methods of radioimmunoassay (RIA) have generally called for incubation of several hours in order for the system to reach equili-brium. However~ in the described automated system~ it is no longer nece-ssary to reach equilibrium before carrying out the separation of bound from ~ `
unbound hormone. Response times of 5 to 10 minutes are achievable with automation with a test device of the type described.
Concepts employed in the practice of the described invention ;
have important utilization other than in radio-immunoassays. Under such circumstances~ utilization can be made of the dry gel particles without ~ -entrapped binding protein.
Thus, an important further concept of this invention resides in the utilization of dry gel particles of a predetermined pore size and water regain value whereby use can be made thereof to move a calibràted increment of sample for testing independen~ of the size of the sample. The dry gel particles take up only so much sample no matter how much reagent is passed through. For example, dry gel particles having a predetermined water re-gain value, in equal amounts measured by volume or weight, will reproducibly 1C~6Z~l~

take up precise amounts of liquid containing molecules below a maximum size for subsequent test, indepelldent of the size of the sample and without the need for the conventional less accurate practice of pipetting a measured amount of the sample.
Water regain value of ~he dry gel particles remains sufficiently constant to enable the removal of aliquot sample portions of equal amount merely by use of the more accurate and rapid techniques of measurement, as by weight or by volume, of the lyophilized gel particles to which the liquid samples are exposedO
A still further important concept of this invention resides in the utilization of the dry gel particles per se, with controlled pore size, as 2 means and method to effect separation of high molecular weight interfer-ing components or molecules from low molecular weight components or molecules, merely by contacting the liquid sample with the dry gel particles having high water regain value and a pore size sufficient to permit entry of low molecular weight components or molecules but insufficient to permit entry of high molecular weight components or moleculesO Thereafter the gel particles can be flushed with a reagent to wash out the high molecular weight components from the extra gel volume or void, leaving an aliquot portion of the low molecular weight components or molecules entrapped within the intra gel volume for testing or use. Instead, the entrapped low molecular weight components or molecules can be eluted or otherwise recovered from within the intra gel volume for testing or use separate and apart from the gel particles.
Such fractionation technique using dry gel particles of the desired small pore size and high water regain, finds particular value in separation of high molecular weight interfering compounds from low molecular weight compounds in blood plasma, whole blood, serum and the like. For such purpose, the blood or plasma is contacted with the dry gel particles having a high water regain value and a pore size sufficient to permit entry only of the low molecular weight components but insufficient to permit entry of the high :

~06~6~
molecular weight interfering components. The latter remain in the extra gel volume from which they can be flushed for separation from the low molecular weight components entrapped within the gel particles.
secause the gel particles are dry and hydrophilic, the water regain can be employed as a means to measure out a sample without pipettingO
The intra gel compartment has a water regain that is reproducible from column to column, whether cverfilled or underfilled and the amount of sample removed will be exactly the same. Because of the entry of components below a par- -ticular molecular weight, while holding out higher molecular weight components incapable of entry through the pores, a measured increment of sample, purified from the standpoint of a particular molecular weight fraction, can be removed for testing without the presence of interfering molecules and without the ;;
need for centrifuge, pipetting or the like. The low molecular weight com-ponent can be tested in situ or eluted from the gel particles for use or ~
testing. Thus one can achieve the important combination of elution and `
measurement of sample volume simultaneously with separation.
Chemical assays in the clinical laboratory generally require the addition of a precise amount of sample to the assay systemO Analysis of whole blood sample frequently requires separation of serum or plasma from red blood cells and deprotinization before the serum or plasma sample is added to the assay system. This type of sample preparation classically has required cen-trifugation. However, ~he invention described herein allows an automatic selection and separation of a precise low molecular weight fraction away from both cellular and protein elements of bloodO A variety of low molecular weight anticoagulants, for example, EDTA, citrate, and heparin, can be incor-porated in the dry gel particles and activated upon contact with whole blood.
In this way, clotting of the assay sample is prevented.
Thus it will be apparent that the concepts described find application clinically in chemistry laboratories as well as in radioimmuno-assays.

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It will be understood that various changes and modifications c~n be made in the details of construction, operation and use without departing from the spirit of the invention, especially as defined in the following claims.

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Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. In the preparation of dry, porous gel particles of the type suitable for use in liquid sample assay and having high water regain for use in reproducible increments of liquid sample upon rehydration with the sample, mixing the gel in particle form in a non-aqueous medium, removing the gel particles from the non-aqueous medium, and then allowing the gel particles to dry before use.

2. The preparation as claimed in claim 1 in which the non-aqueous medium comprises an alcohol or an alcoholic solution containing at least 50%
alcohol.

3. The preparation as claimed in claim 1 in which the non-aqueous medium is ethanol.

4. The preparation as claimed in claim 1 in which the non-aqueous medium is removed by spreading the gel particles on a drying surface and allowing the gel particles to dry.

5. A process for the preparation of dry, hydrophilic, insoluble, porous gel particles having a binder protein entrapped within the gel particles and having high water regain for use in reproducible increments of liquid sample upon rehydration with the sample, said process comprising dissolving at least one monomer in a buffer solution containing binder protein and poly-merising, fragmenting the gel thus produced to form gel particles, mixing the gel particles in a non-aqueous medium, removing the gel particles from the non-aqueous medium, and then allowing the gel particles to dry before use.

6. The process as claimed in claim 5 in which the non-aqueous medium comprises an alcohol or an alcoholic solution containing at least 50% alcohol.

7. The process as claimed in claim 5 in which the non-aqueous medium is ethanol.

8. The process as claimed in claim s in which the non-aqueous medium is removed by spreading the gel particles on a drying surface and allowing the gel particles to dry.

9. A process as claimed in claim 5 in which said monomer is an acrylamide and said gel is a polacrylamide.

10. A gel reagent comprising dry, hydrophilic, insoluble gel particles having a binder protein entrapped within the gel particles and characterised by a predetermined water reagent value and a pore size which permits dif-fusion of small molecules into the intra gel volume, but insufficient pore size to permit entry of high molecular weight molecules.

11. A method for separation of high molecular weight interfering components from low molecular weight components in blood or blood plasma comprising the steps of contacting the blood or blood plasma with dry, highly hydrophilic gel particles claimed in claim 6 and then flushing the gel particles with a reagent to wash out the high molecular weight components from the voids (extra gel void volume).