CA1054050A - Gel column device and use in blood purification and immunoassay - Google Patents
Gel column device and use in blood purification and immunoassayInfo
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- CA1054050A CA1054050A CA198,533A CA198533A CA1054050A CA 1054050 A CA1054050 A CA 1054050A CA 198533 A CA198533 A CA 198533A CA 1054050 A CA1054050 A CA 1054050A
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Abstract
ABSTRACT OF THE DISCLOSURE
A method and device for immunological determinations in which use is made of a volume of a hydrophilic, insoluble, porous gel particles having binding proteins entrapped therein, and in which a fluid to be tested is introduced into the volume of gel particles whereby some of the binding sites are occupied followed by exposure of the gel particles to radio active tagged material followed by measurement of the radio activity of the unbound or bound tagged material. 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 material followed by washing to effect removal of the unbound material, and then drying.
A method and device for immunological determinations in which use is made of a volume of a hydrophilic, insoluble, porous gel particles having binding proteins entrapped therein, and in which a fluid to be tested is introduced into the volume of gel particles whereby some of the binding sites are occupied followed by exposure of the gel particles to radio active tagged material followed by measurement of the radio activity of the unbound or bound tagged material. 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 material followed by washing to effect removal of the unbound material, and then drying.
Description
This invention relates to clinical techniques for immunological determinations and more particularly to radio-immunoassay by manual and preferably by automated technique.
Description is made of a device and technique for immunological determinakions of a fluid system in which use is made of a measured volume of dried, insoluble, yet highly hydrophilic gel particles in which there are entrapped one or more binding proteins or combinations of binding proteins and radio active tag material. The dry gel particles are characterized by pores of a size that permit entry of low molecular weight compounds into the intragel volume but in-sufficient to permit entry of large molecular weight compon~
` ents which remain in the extra gel volume, thereby to achieve ;~
a desirable fractionation which excludes high molecular weight interferences otherwise present to interfere with blood or serum assays. ~ ~ -Application of liquid sample to the measured volume of dry gel particles is determined by water regain of the gel thereby to provide a pip~less method of sample application based on use of highly reproducible dry-volume aliquots of the dried or lyophilized gel particles.
The dry gel particles can be preloaded with radio -` active tagged hormone and standard hormone either during ~5 preparation of the dry gel particles, or by af~er 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 `; 3 long periods of time without deviation or loss in binding ;
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activity Thus, there i5 described a radloimmunoassay system which provides for automatic exclusion of higll 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 The application is addressed to an assay syst0m and device which is characterized by manual operation in a single use, with the device being discarded after the assay has been completed ~lowever, it is desirable and it is an object of this inv0ntion to provide a system which is also capable of automation for multiple use without loss in binding activity of the antibody gel particles thereby to afford reproducible results which makes the system for radioimmunoassay much more attractive, at least from the standpoint of cost, time, inventory and results The present invention provides 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 radio-active tag material entrapped within the gel particles and characterized by a predetermined water regain value and pores of a size which permit diffusion of small molecules into the intro gel volume, but insufficient to permit entry ` of high molecular weight component and means for transmission of fluid for determination into and out of said volume.
.. . The present invention also provides a method for radioimmu~oassay with a device as.above comprising the step of exposing the dry gel particles to a fluid system of the blood, blood plasma or serum to be determined to wet (rehydrate) the gel particles whereby molecular weight components in the fluid system small enough to penetrate the pores enter into the intra gel ~volume while components of larger molecular weight remain in the extra gel volume, radioactive tag material being pre-incorporated into the fluid system and being automatically released when the sample fluid hydrates the gel ~ ;.
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particles, incuhating thc system and then washlng the gel particles with at least one void volume of a wash solution to separate the unbound tag materlal from the bound tag material, and then measuring the radio activity of the bound or ullbollnd tag material.
Proteins havillg a binding capaclty for certain speci~ic smaller molecules and wllicll can be readily immobilized in the polymer gel are il]ustrated by various protein globulins and antibodies.
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 o~ being assayed in a system of the type described include, for example, angiotensin, insulin, growth hormone, gonadotropic hormone, parathyroid hormone, glucagon, cortisol, protoglandin, corticoster-oids, cyclic fatty acid .
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hormones and estrogens, and drugs such as digoxin, thyrosin, morphene~ digitalis, and the like. By way of example, specific corticosteroid binding globulins, immobili~ed by entrapment in the gel system, permit assay oE corticosteroid hormones in addition to the many polypeptide hormones. 7S
gamm~-globulin (150,000 m.w.) can be used to assay for angio-tension (1,000 m.w.).
The invention will hereinafter be described in greater detail by reference to specific examples.
1~ Example 1 A gel, in which dilute anti-angiotensin immune serum containing 7S gamma globulin (m.w. 150,000) is entrapped in a gel matrix having a pore size to enable diffusion of angiotensin (m.w. 1,000) for assay may be prepared as follows:
8 grams of acrylamide and 1 gram of 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 suitable 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 0.02 ml of ~,N,N',N-tetramethyl-ethylene-diamine is added, followed by 0.2 mg of sodium hydrosulfite~
The mixture, in a suitable vessel, is stoppered and agitated and then is exposed to light from an ordinary tungsten light ; bulb to induce polymerization. Polymerization is complete in 5-15 minutes. The reaction vessel is continuously chilled in an ice water bath, as needed, to prevent the heat gener-ated by the exothermic polymerization reaction from adversely affecting the binding protein.
The antibody gel is fragmented by pushing through ; 30 a #40 mesh brass screen. The gel particles are collected on :: . .. ~ . . . .
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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 7.4 phosphate buffer. The gel particles are now ready for the drying procedure.
Two fundamentally different drying procedures have been employed including (1) lyophilization and (2) dehydra-1~ tion in acetone ethanol or other low boiling alcohol, followed by air drying. ~either method was associated with loss of antibody activity. ~owever, ethanolic dehydration i9 pre-erred 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 solu-tion 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 hermetic- ;
ally sealing the lyophilized gel particles, with the entrapped antibody activity, at temperatures of 37C, 25C, 4C and -20C for over two years without detectable 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-; 25 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 3 I and guinea pig antisera for use against insulin can be ~4~ ~`
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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 elongated barrel at the other end for drawing fluid for unidirectional flow through the needle 12 into the body portion 16 of the syringe in response to the generation of volume conditions upon ~ith-drawal of the plunger.
- Ad~acent the needle end portion, the syringe is sub-divided by a porous barrier 18 into a calibrated portion 20 packed with a column of prescribed 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 permit ~0 passage of the gel particles. In the illustrated modification, 33 mg of dry gel particles are packed in the volume of 0.14 ml enclosed by the porous barrier made up of about two-thirds ; 1 :
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.
- Example 3 ~ ~;
Method of use: ~;
In manual use, the needle 10 is inserted in the sample (serum, plasma or whole b~ood drawn directly from a vein) and the plunger is pulled back to draw some of the _5_ ~5~S~
liquid sample into the syringe 10 in an amount to at least fill the volume 20 below the barrier ~8 and preferably in an amount to at least cover the net.
As the sample is drawn into the syrinye, the highly hydrophilic ge] particles 22 absorb a predictable and reproduc-ible portion of low molecular weight component in the sample by reh~dration. To prevent coagula-tion, when testing fresh whole ' ~ G~ 0 a ~ ~ 1c~ ,~ f blood or plasma, it is desirable to incorporate a ~s~gulant 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 present in the column~ measured by weight or by volume. As a result, sample size can automatic-ally be controLled by the amount and water regain ~alue 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 solu-tion, such as a 0.1 M phosphate buffer or any other physio-logical 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 to five void volumes in order to insure the re-moval of sample component otherwise remaining in the extra gel ~ volume. The low molecular weight polypeptide hormone (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 :;
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:-. i . --molecular weight interferences are removed with the wash solu-tion used to flush the column 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 Eill the void volume and preferably in an amount greater than one void volume.
The unit is then 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 activity.
Thereafter, a buffered wash solution, 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 un-bound hormones from bound hormones. The column is flushed with sufficient volume for a length of time substantially 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 measur-ing 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.
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The described technique and materials are capable of a number of modifications and ramifications.
; The formulation of the gel of which the particles are formed is not significant since use can be made of any . -- ' ' .
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gel forming material which is capable of being reduced into porous, insoluble, highly hydrophilic particles of controlled pore size. ThuS, other highly hydrophilic, insolu~le gels capable of the desired pore size and drying or lyophilization can be used but it is preferred to make use of the polyacryl-amide 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 the monomer not only to provide the desired pore size but to give the gel the density desired for rapid sedimentation in partic-ulate form and to provide sufficient rigidity to the gel particles. The ratio of one part by weight of cross linking ayent to 4 to 10 parts by weight monomer is sufficient to give a gel having a total gel polymer concentration in the range of 12-35%.
Tha 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, gonadotropic hormone, - 20 parathyroid hormone, cortisol, digoxin, digitalis or thyroxin.
By way of modification, the radioactive tag hormone can also be incorporated in calibrated amounts within gel particles. These gel particles which, upon rehydration, re-lease radioactively tagged hormone, can also be added in precise amount by weight or volume to the dry antibody gel particles, used as the binding reagent in the RIA system so : that the addition of radioactively tagged hormone into the .: , RIA system is automatic. In this arrangement, it is desir-able to make use of an amount of tagged hormone or drug which 3 will not completely satisfy the binding sites available in ,: ~
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the gel particles before the unknown sample is drawn into thecolumn.
In a similar fashion, a standard amount of drug or hormone can be incorporated into the gel recipe for release upon rehydration 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 which is automatically released upon rehydration of the gel particles.
~ Example 4 The foregoing modification contemplates the forma-tion of dry gel particles containing the tag hormone in a manner similar to that for producing the dry gel particles containing the binding protein, but separate and apart there-Erom. The gel particles containing the tag hormone and the gel particles containing the antibody or binding protein would then be mixed 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 form a pre-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 3 sites. This can be accomplished by the addition of the tag : --9-- :.
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hormone to the dry gel particles from a non-aqueous medium.
Thus incorporation of the tag hormone is carried out when the antibody gel p~rticles are dry -- which should preclude pre-mature binding of the tag hormone to antibody binding sites.
Example 5 Dry gel particles with immobilized antibody activity, prep~red in accordance with Examples 1 and 2, are conkacted 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 chroma-tographic column having clean air 10wing therethrough to effect alcohol removal. ; ,~
The resulting dry gel particles have both antibody binding protein activity and tagged hormone in a non-complex (non-bound) form. The tagged hormone will not diffuse to the binding site until the gel undergoes rehydration with the `~ test sample, as in Example 4. At such time, the tagged hor-mone is released to compete with the sample hormone for com-plexing to the binding sites.
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The water regain value of the gel particles deter-mines how much sample reaches the antibody binding protein contained in the intra gel compartment. The water regain of the gel will depend somewhat on the per cent polymer and per cent cross linking agent 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 ~ot convenient, because of changes in 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 em-ployed in which a Xnown 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 :
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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 vol~ne, then barriers such as a nylon net should be provided at opposite ends to confine the particles into the chromato-graphic column while enabling the various 1uids to pass therethrough into and out of the column of particles.
The porous barrier func~ions automatically to separate bound from unbound radio activity which occurs dur-ing the flushing o the column. This porous structure func-tions also as an anticonvection barrier, which discourages mixing of any solution sequestered outside the gel particle compartment.
Since the volume of gel particles is known and the water regain is constant, 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 ultimately exposed. ThuS many of the -~
. steps critical to prior immunoassays have been eliminated with equal or greater reproducibility and accuracy of test results~
Since the antibody is insolubilized and immobilized within - 25 the gel particlesg conventional centrifugation and washing steps9 normally employed in current procedures, can be eli:,`
inated in their entirety~in the preparation and use of a test unit of the type described.
Similarly, since the unknown and tag material taken 3 up depends on such known values as volume and water regain, .:'~ , .
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the critical steps of pipetting and the numerous errors as-sociated therewith are eliminated thereby to increase the accuracy of the anaLysis.
By immobilizing the antibody binding reagent in the yel, the centrifugation step of conventional RIA can be eliminated. Furthermore, by engaging the antibody binding protein in a gel polymer matrix, high molecular weight inter-ference 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 in~ections 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 manùally applied for single assays9 after which the device is discarded. It would, of course, be desirable to be able to ma~e multiple use of the device~ without loss of binding -s activity, and it would be still more desirable to be able to automate the operation for multiple use thereby to effect considerable savings in time and materials, as well as re-` producibility of the results.
It has been found, in accordance with the practiceof this invention, that the binding sites of the antibody gel particles in the chromatographic column of the test device 3 can be returned to their original state to enable re-use as . .
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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.
Unbound radio activity can be washed from the column, in the manner previously described, with a variety o~ 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 10 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 anti-;~body gel particles can be made the subject of analysis, as ~ ` ~
15 by gamma or liquid scintillation type detectors~ to arrive ~ ;
at the assay value for the previous sample.
Following elution of the bound radio activit~, the column of gel particles is washed with a buffer for removal of the acid or hyperthermic 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 'co enable rapid dehydration and collapse of the antibody gel particles. Upon evaporation of the organic solvent these yel particles have been returned to cheir original dry state. collapse of the gel particles, which occurs with solvent drying, is preferred because it prevents entrapment `of large amounts of air which effect reproducibility of the quantity of sample or water regain and/or is released as 3 tiny bubbles on rehydration.
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For detachment of the bound radio activity and bound hormones rom 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 pre-ferably an aqueous solution of hydrochloric acid. The acidicsolution is introduced into the gel column in an amount cor-responding to at least one gel volume and preferably more than one gel ~olume but less than three.
Removal of the acidic medium with the detached ~ -radio activity and hormones is effected by washing with a . , .
buffer solution~ such 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 particles, usually one to five volumes will be sufficient.
The organic solvent is employed primarily to Pffect -removal of aqueous medium and to permit rapid drying and ~
collapse of the gel particles in the column. For this pur- ~-pose, it is desirable to make use of an organic solvent which - does not denature the binding activity o the entrapped anti-~ body and which forms a constant boiling mixture with aqueous ;~
, medium to effect water removal and in which the organic "'~ solvent is highly volatile to facilitate rapid removal and ` permit rapid drying of the gel particles. Representative of suitable organic solvents are acetone, methyl alcohol, ethyl alcohol~ low boiling esters, and the like.
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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 . ~, ::"
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~.Q54~V , temperature which would destroy any of the antibody binding sites. The stream of drying air accelerates volatilization oE the organic solvent for rapid and complete removal.
The thus regenerated antibody gel particles in the column are returned to their original state including col-lapsed 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-chromatographic, unidirectional flow- ;
ing stream configuration of analysis and regeneration enables complete automation of the use and regeneration of the device merely by the addition of a precision syringe or proportion-ating pump operatively connected with the ingoing end of the device to force the 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 sequenc~
- ing the materials for flow through the device.
An automated system o~ 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 ap-~` proaches. Gel antibody RIA does not require dilution of plasma to reduce or eliminate high molecular weight interfer- ~ ~ ;
ences. ThUS reaction kinetics need not be slowed by reason of dilution in the system.
3 Manual methods of radioimmunoassay (RIA) have :: , :
generally called for incubation of several hours in order for the system to reach equilibrium. However, in the described automated system, it is no longer necessary to reach equili-brium 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 pro~ein.
Thus, an important further concept of this inven-tion resides in the utilization of dry gel particles of a - predetermined pore size and water regain value whereby use .
can be made thereof to remove a calibrated increment of sample for testing independent 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 regain value, in equal amounts measured by volume or weight, will reproducibly take up precise amounts of liquid containing molecules below a maxi-mum size for subsequent test, independent of the size of the sample and without the need for the conventional less accur-- ate practice of pipetting a measured amount of the sample.
.:. 25 Water regain value of the 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 ~0 samples are exposed.
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A still further important concept of this inven-tion resides in the utilization of the dry gel particles per se~ with controlled pore size, as a means an~ method to eE-fect separation of high molecular weight interfering compon-5 ents 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 com- ~ ~`
ponents or molecules but insufficient to permit entry of high 10 molecular weight components or molecules. Thereafter the gel ; particles can be flushed with a reagent to wash out the highmolecular weight components from the extra gel volume or void, leaving an aliquot portion of the low molecular weight com-ponents or molecules entrapped within the intra gel volume 15 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 ~ s20 of the desired small pore size and high water regain, finds particular value in separation of high molecular weight inter-' fering 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 25 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 molecular weight interfering components. The latter remain in the extra gel `~ volume from which they can be flushed for separation from the 3 low molecular weight components entrapped wi-thin the gel 31 particles.
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Because the gel particles are dry and hydrophilic, the watex regain can be employed as a means to measure ou-t a sample without pipetting. The intra gel compartment has a water regain that is reproducible Erom column to column, whether overEilled or underfilled and the amount of sample removed will be exactly the same. Because of the entry of components below a particular molecular weight, while holding out higher molecular weight components incapable of entry through the pores, a measured increment of sample, puri~ied from the standpoint of a par~icular molecular weight frac-tion, 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 component can be tested in situ or eluted from the gel particles for use or testing. Thus one can achieve the important combina-tion of elution and measurement of sample volume simultan-eously with separation.
Chemical assays in the clinical laboratory general-ly require the addition of a precise amount of sample to the ~A ' 20 assay system. Analysis of whole blood sample frequently re-quires separation of serum or plasma from red blood cells and ~ -` deproteinization before the serum or plasma sample is added to the assay system. ThiS type of sample preparation clas-sically has required centrifuyation. However, the invention - 25 described herein allows an automatic selection and separation of a precise low molecular weight fraction away from both cel-lular and protein elements of blood. A variety of low molecu~
weight anticoagulants, for example, EDTA, citrate, and hep~ri~
~` can be incorporated in the dry gel particles and activated 3 upon contact with whole bllood. In this way, clotting of the .. -: .
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assay sample is prevented. :
Thus it will be apparent that the concepts des-cribed find application clinically in chemistry laborator-ies as well as in radioimmunoassays.
It will be understood that various changes and modi~ications can be made in the details of construction, operation and use without departing from the spirit of the invention, especially as deEined in the following claims.
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Description is made of a device and technique for immunological determinakions of a fluid system in which use is made of a measured volume of dried, insoluble, yet highly hydrophilic gel particles in which there are entrapped one or more binding proteins or combinations of binding proteins and radio active tag material. The dry gel particles are characterized by pores of a size that permit entry of low molecular weight compounds into the intragel volume but in-sufficient to permit entry of large molecular weight compon~
` ents which remain in the extra gel volume, thereby to achieve ;~
a desirable fractionation which excludes high molecular weight interferences otherwise present to interfere with blood or serum assays. ~ ~ -Application of liquid sample to the measured volume of dry gel particles is determined by water regain of the gel thereby to provide a pip~less method of sample application based on use of highly reproducible dry-volume aliquots of the dried or lyophilized gel particles.
The dry gel particles can be preloaded with radio -` active tagged hormone and standard hormone either during ~5 preparation of the dry gel particles, or by af~er 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 `; 3 long periods of time without deviation or loss in binding ;
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activity Thus, there i5 described a radloimmunoassay system which provides for automatic exclusion of higll 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 The application is addressed to an assay syst0m and device which is characterized by manual operation in a single use, with the device being discarded after the assay has been completed ~lowever, it is desirable and it is an object of this inv0ntion to provide a system which is also capable of automation for multiple use without loss in binding activity of the antibody gel particles thereby to afford reproducible results which makes the system for radioimmunoassay much more attractive, at least from the standpoint of cost, time, inventory and results The present invention provides 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 radio-active tag material entrapped within the gel particles and characterized by a predetermined water regain value and pores of a size which permit diffusion of small molecules into the intro gel volume, but insufficient to permit entry ` of high molecular weight component and means for transmission of fluid for determination into and out of said volume.
.. . The present invention also provides a method for radioimmu~oassay with a device as.above comprising the step of exposing the dry gel particles to a fluid system of the blood, blood plasma or serum to be determined to wet (rehydrate) the gel particles whereby molecular weight components in the fluid system small enough to penetrate the pores enter into the intra gel ~volume while components of larger molecular weight remain in the extra gel volume, radioactive tag material being pre-incorporated into the fluid system and being automatically released when the sample fluid hydrates the gel ~ ;.
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particles, incuhating thc system and then washlng the gel particles with at least one void volume of a wash solution to separate the unbound tag materlal from the bound tag material, and then measuring the radio activity of the bound or ullbollnd tag material.
Proteins havillg a binding capaclty for certain speci~ic smaller molecules and wllicll can be readily immobilized in the polymer gel are il]ustrated by various protein globulins and antibodies.
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 o~ being assayed in a system of the type described include, for example, angiotensin, insulin, growth hormone, gonadotropic hormone, parathyroid hormone, glucagon, cortisol, protoglandin, corticoster-oids, cyclic fatty acid .
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hormones and estrogens, and drugs such as digoxin, thyrosin, morphene~ digitalis, and the like. By way of example, specific corticosteroid binding globulins, immobili~ed by entrapment in the gel system, permit assay oE corticosteroid hormones in addition to the many polypeptide hormones. 7S
gamm~-globulin (150,000 m.w.) can be used to assay for angio-tension (1,000 m.w.).
The invention will hereinafter be described in greater detail by reference to specific examples.
1~ Example 1 A gel, in which dilute anti-angiotensin immune serum containing 7S gamma globulin (m.w. 150,000) is entrapped in a gel matrix having a pore size to enable diffusion of angiotensin (m.w. 1,000) for assay may be prepared as follows:
8 grams of acrylamide and 1 gram of 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 suitable 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 0.02 ml of ~,N,N',N-tetramethyl-ethylene-diamine is added, followed by 0.2 mg of sodium hydrosulfite~
The mixture, in a suitable vessel, is stoppered and agitated and then is exposed to light from an ordinary tungsten light ; bulb to induce polymerization. Polymerization is complete in 5-15 minutes. The reaction vessel is continuously chilled in an ice water bath, as needed, to prevent the heat gener-ated by the exothermic polymerization reaction from adversely affecting the binding protein.
The antibody gel is fragmented by pushing through ; 30 a #40 mesh brass screen. The gel particles are collected on :: . .. ~ . . . .
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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 7.4 phosphate buffer. The gel particles are now ready for the drying procedure.
Two fundamentally different drying procedures have been employed including (1) lyophilization and (2) dehydra-1~ tion in acetone ethanol or other low boiling alcohol, followed by air drying. ~either method was associated with loss of antibody activity. ~owever, ethanolic dehydration i9 pre-erred 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 solu-tion 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 hermetic- ;
ally sealing the lyophilized gel particles, with the entrapped antibody activity, at temperatures of 37C, 25C, 4C and -20C for over two years without detectable 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-; 25 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 3 I and guinea pig antisera for use against insulin can be ~4~ ~`
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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 elongated barrel at the other end for drawing fluid for unidirectional flow through the needle 12 into the body portion 16 of the syringe in response to the generation of volume conditions upon ~ith-drawal of the plunger.
- Ad~acent the needle end portion, the syringe is sub-divided by a porous barrier 18 into a calibrated portion 20 packed with a column of prescribed 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 permit ~0 passage of the gel particles. In the illustrated modification, 33 mg of dry gel particles are packed in the volume of 0.14 ml enclosed by the porous barrier made up of about two-thirds ; 1 :
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.
- Example 3 ~ ~;
Method of use: ~;
In manual use, the needle 10 is inserted in the sample (serum, plasma or whole b~ood drawn directly from a vein) and the plunger is pulled back to draw some of the _5_ ~5~S~
liquid sample into the syringe 10 in an amount to at least fill the volume 20 below the barrier ~8 and preferably in an amount to at least cover the net.
As the sample is drawn into the syrinye, the highly hydrophilic ge] particles 22 absorb a predictable and reproduc-ible portion of low molecular weight component in the sample by reh~dration. To prevent coagula-tion, when testing fresh whole ' ~ G~ 0 a ~ ~ 1c~ ,~ f blood or plasma, it is desirable to incorporate a ~s~gulant 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 present in the column~ measured by weight or by volume. As a result, sample size can automatic-ally be controLled by the amount and water regain ~alue 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 solu-tion, such as a 0.1 M phosphate buffer or any other physio-logical 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 to five void volumes in order to insure the re-moval of sample component otherwise remaining in the extra gel ~ volume. The low molecular weight polypeptide hormone (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 :;
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:-. i . --molecular weight interferences are removed with the wash solu-tion used to flush the column 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 Eill the void volume and preferably in an amount greater than one void volume.
The unit is then 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 activity.
Thereafter, a buffered wash solution, 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 un-bound hormones from bound hormones. The column is flushed with sufficient volume for a length of time substantially 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 measur-ing 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 number of modifications and ramifications.
; The formulation of the gel of which the particles are formed is not significant since use can be made of any . -- ' ' .
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gel forming material which is capable of being reduced into porous, insoluble, highly hydrophilic particles of controlled pore size. ThuS, other highly hydrophilic, insolu~le gels capable of the desired pore size and drying or lyophilization can be used but it is preferred to make use of the polyacryl-amide 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 the monomer not only to provide the desired pore size but to give the gel the density desired for rapid sedimentation in partic-ulate form and to provide sufficient rigidity to the gel particles. The ratio of one part by weight of cross linking ayent to 4 to 10 parts by weight monomer is sufficient to give a gel having a total gel polymer concentration in the range of 12-35%.
Tha 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, gonadotropic hormone, - 20 parathyroid hormone, cortisol, digoxin, digitalis or thyroxin.
By way of modification, the radioactive tag hormone can also be incorporated in calibrated amounts within gel particles. These gel particles which, upon rehydration, re-lease radioactively tagged hormone, can also be added in precise amount by weight or volume to the dry antibody gel particles, used as the binding reagent in the RIA system so : that the addition of radioactively tagged hormone into the .: , RIA system is automatic. In this arrangement, it is desir-able to make use of an amount of tagged hormone or drug which 3 will not completely satisfy the binding sites available in ,: ~
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the gel particles before the unknown sample is drawn into thecolumn.
In a similar fashion, a standard amount of drug or hormone can be incorporated into the gel recipe for release upon rehydration 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 which is automatically released upon rehydration of the gel particles.
~ Example 4 The foregoing modification contemplates the forma-tion of dry gel particles containing the tag hormone in a manner similar to that for producing the dry gel particles containing the binding protein, but separate and apart there-Erom. The gel particles containing the tag hormone and the gel particles containing the antibody or binding protein would then be mixed 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 form a pre-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 3 sites. This can be accomplished by the addition of the tag : --9-- :.
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hormone to the dry gel particles from a non-aqueous medium.
Thus incorporation of the tag hormone is carried out when the antibody gel p~rticles are dry -- which should preclude pre-mature binding of the tag hormone to antibody binding sites.
Example 5 Dry gel particles with immobilized antibody activity, prep~red in accordance with Examples 1 and 2, are conkacted 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 chroma-tographic column having clean air 10wing therethrough to effect alcohol removal. ; ,~
The resulting dry gel particles have both antibody binding protein activity and tagged hormone in a non-complex (non-bound) form. The tagged hormone will not diffuse to the binding site until the gel undergoes rehydration with the `~ test sample, as in Example 4. At such time, the tagged hor-mone is released to compete with the sample hormone for com-plexing to the binding sites.
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The water regain value of the gel particles deter-mines how much sample reaches the antibody binding protein contained in the intra gel compartment. The water regain of the gel will depend somewhat on the per cent polymer and per cent cross linking agent 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 ~ot convenient, because of changes in 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 em-ployed in which a Xnown 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 :
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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 vol~ne, then barriers such as a nylon net should be provided at opposite ends to confine the particles into the chromato-graphic column while enabling the various 1uids to pass therethrough into and out of the column of particles.
The porous barrier func~ions automatically to separate bound from unbound radio activity which occurs dur-ing the flushing o the column. This porous structure func-tions also as an anticonvection barrier, which discourages mixing of any solution sequestered outside the gel particle compartment.
Since the volume of gel particles is known and the water regain is constant, 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 ultimately exposed. ThuS many of the -~
. steps critical to prior immunoassays have been eliminated with equal or greater reproducibility and accuracy of test results~
Since the antibody is insolubilized and immobilized within - 25 the gel particlesg conventional centrifugation and washing steps9 normally employed in current procedures, can be eli:,`
inated in their entirety~in the preparation and use of a test unit of the type described.
Similarly, since the unknown and tag material taken 3 up depends on such known values as volume and water regain, .:'~ , .
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the critical steps of pipetting and the numerous errors as-sociated therewith are eliminated thereby to increase the accuracy of the anaLysis.
By immobilizing the antibody binding reagent in the yel, the centrifugation step of conventional RIA can be eliminated. Furthermore, by engaging the antibody binding protein in a gel polymer matrix, high molecular weight inter-ference 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 in~ections 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 manùally applied for single assays9 after which the device is discarded. It would, of course, be desirable to be able to ma~e multiple use of the device~ without loss of binding -s activity, and it would be still more desirable to be able to automate the operation for multiple use thereby to effect considerable savings in time and materials, as well as re-` producibility of the results.
It has been found, in accordance with the practiceof this invention, that the binding sites of the antibody gel particles in the chromatographic column of the test device 3 can be returned to their original state to enable re-use as . .
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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.
Unbound radio activity can be washed from the column, in the manner previously described, with a variety o~ 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 10 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 anti-;~body gel particles can be made the subject of analysis, as ~ ` ~
15 by gamma or liquid scintillation type detectors~ to arrive ~ ;
at the assay value for the previous sample.
Following elution of the bound radio activit~, the column of gel particles is washed with a buffer for removal of the acid or hyperthermic 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 'co enable rapid dehydration and collapse of the antibody gel particles. Upon evaporation of the organic solvent these yel particles have been returned to cheir original dry state. collapse of the gel particles, which occurs with solvent drying, is preferred because it prevents entrapment `of large amounts of air which effect reproducibility of the quantity of sample or water regain and/or is released as 3 tiny bubbles on rehydration.
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For detachment of the bound radio activity and bound hormones rom 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 pre-ferably an aqueous solution of hydrochloric acid. The acidicsolution is introduced into the gel column in an amount cor-responding to at least one gel volume and preferably more than one gel ~olume but less than three.
Removal of the acidic medium with the detached ~ -radio activity and hormones is effected by washing with a . , .
buffer solution~ such 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 particles, usually one to five volumes will be sufficient.
The organic solvent is employed primarily to Pffect -removal of aqueous medium and to permit rapid drying and ~
collapse of the gel particles in the column. For this pur- ~-pose, it is desirable to make use of an organic solvent which - does not denature the binding activity o the entrapped anti-~ body and which forms a constant boiling mixture with aqueous ;~
, medium to effect water removal and in which the organic "'~ solvent is highly volatile to facilitate rapid removal and ` permit rapid drying of the gel particles. Representative of suitable organic solvents are acetone, methyl alcohol, ethyl alcohol~ low boiling esters, and the like.
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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 . ~, ::"
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~.Q54~V , temperature which would destroy any of the antibody binding sites. The stream of drying air accelerates volatilization oE the organic solvent for rapid and complete removal.
The thus regenerated antibody gel particles in the column are returned to their original state including col-lapsed 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-chromatographic, unidirectional flow- ;
ing stream configuration of analysis and regeneration enables complete automation of the use and regeneration of the device merely by the addition of a precision syringe or proportion-ating pump operatively connected with the ingoing end of the device to force the 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 sequenc~
- ing the materials for flow through the device.
An automated system o~ 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 ap-~` proaches. Gel antibody RIA does not require dilution of plasma to reduce or eliminate high molecular weight interfer- ~ ~ ;
ences. ThUS reaction kinetics need not be slowed by reason of dilution in the system.
3 Manual methods of radioimmunoassay (RIA) have :: , :
generally called for incubation of several hours in order for the system to reach equilibrium. However, in the described automated system, it is no longer necessary to reach equili-brium 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 pro~ein.
Thus, an important further concept of this inven-tion resides in the utilization of dry gel particles of a - predetermined pore size and water regain value whereby use .
can be made thereof to remove a calibrated increment of sample for testing independent 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 regain value, in equal amounts measured by volume or weight, will reproducibly take up precise amounts of liquid containing molecules below a maxi-mum size for subsequent test, independent of the size of the sample and without the need for the conventional less accur-- ate practice of pipetting a measured amount of the sample.
.:. 25 Water regain value of the 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 ~0 samples are exposed.
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A still further important concept of this inven-tion resides in the utilization of the dry gel particles per se~ with controlled pore size, as a means an~ method to eE-fect separation of high molecular weight interfering compon-5 ents 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 com- ~ ~`
ponents or molecules but insufficient to permit entry of high 10 molecular weight components or molecules. Thereafter the gel ; particles can be flushed with a reagent to wash out the highmolecular weight components from the extra gel volume or void, leaving an aliquot portion of the low molecular weight com-ponents or molecules entrapped within the intra gel volume 15 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 ~ s20 of the desired small pore size and high water regain, finds particular value in separation of high molecular weight inter-' fering 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 25 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 molecular weight interfering components. The latter remain in the extra gel `~ volume from which they can be flushed for separation from the 3 low molecular weight components entrapped wi-thin the gel 31 particles.
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Because the gel particles are dry and hydrophilic, the watex regain can be employed as a means to measure ou-t a sample without pipetting. The intra gel compartment has a water regain that is reproducible Erom column to column, whether overEilled or underfilled and the amount of sample removed will be exactly the same. Because of the entry of components below a particular molecular weight, while holding out higher molecular weight components incapable of entry through the pores, a measured increment of sample, puri~ied from the standpoint of a par~icular molecular weight frac-tion, 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 component can be tested in situ or eluted from the gel particles for use or testing. Thus one can achieve the important combina-tion of elution and measurement of sample volume simultan-eously with separation.
Chemical assays in the clinical laboratory general-ly require the addition of a precise amount of sample to the ~A ' 20 assay system. Analysis of whole blood sample frequently re-quires separation of serum or plasma from red blood cells and ~ -` deproteinization before the serum or plasma sample is added to the assay system. ThiS type of sample preparation clas-sically has required centrifuyation. However, the invention - 25 described herein allows an automatic selection and separation of a precise low molecular weight fraction away from both cel-lular and protein elements of blood. A variety of low molecu~
weight anticoagulants, for example, EDTA, citrate, and hep~ri~
~` can be incorporated in the dry gel particles and activated 3 upon contact with whole bllood. In this way, clotting of the .. -: .
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assay sample is prevented. :
Thus it will be apparent that the concepts des-cribed find application clinically in chemistry laborator-ies as well as in radioimmunoassays.
It will be understood that various changes and modi~ications can be made in the details of construction, operation and use without departing from the spirit of the invention, especially as deEined in the following claims.
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Claims (19)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A device for immunoassay of blood, blood plasma or serum compri-sing 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 characterized by a predetermined water regain value and pores of a size which permit diffusion of small molecules into the intra gel volume, but insufficient to permit entry of high molecular weight component and means for transmission of fluid for determination into and out of said volume.
2 A device as claimed in claim 1 in which the tag material and the binding protein are in separate dry gel particles.
3. A method for radioimmunoassay with a device claimed in claim 1 comprising the step of exposing the dry gel particles to a fluid system of the blood, blood plasma or serum to be determined to wet (rehydrate) the gel particles whereby molecular weight components in the fluid system small enough to penetrate the pores enter into the intra gel volume while components of larger molecular weight remain in the extra gel volume, radioactive tag material being pre-incorporated into the fluid system and being automatically released when the sample fluid hydrates the gel particles, incubating the system and then washing the gel particles with at least one void volume of a wash solution to separate the unbound tag material from the bound tag material, and then measuring the radio activity of the bound or unbound tag material.
4. The method as claimed in claim 3 in which the tag material and the binding protein are in separate dry gel particles.
The method as claimed in claim 3 in which the tag material and the binding protein are in the same dry gel particles.
6. A device as claimed in claim 1 in which the gel is a polyacryl-amide.
7. A method as claimed in claim 3 in which the gel particles are formed of a polyacrylamide.
8. The method as claimed in claim 3 which includes the steps of regenerating the volume of gel particles for re-use in subsequent immunolog-ical determinations comprising the steps of exposing the volume of gel particles to acidic or hyperthermic wash solution to effect detachment of material bound to the binding sites of the protein, washing the volume of gel particles to elute the unbonded material, washing the volume of gel particles with a highly volatile organic liquid which has an affinity for water, and then drying the volume of gel particles to return the gel particles to the dried state for subsequent use in an immunological determination.
9. The method as claimed in claim 8 in which the acidic material is an aqueous solution having a pH below 2.5.
10. The method as claimed in claim 8 in which the acidic medium is a solution of hydrochloric acid.
11. The method as claimed in claim 8 in which the gel particles treated to effect unbounding of the materials are washed with an aqueous buffer solution.
12. The method as claimed in claim 8 in which the buffer solution is a phosphate buffer.
13. The method as claimed in claim 8 in which the wash solution is introduced into the volume in an amount of at least one volume.
14. The method as claimed in claim 8 in which the highly volatile organic liquid with which the gel particles are washed is selected from the group consisting of low molecular weight alcohol, acetone and ester.
15. The method as claimed in claim 14 in which the organic liquid is an organic solvent introduced in an amount of at least one volume.
16. The method as claimed in claim 8 in which the gel particles are dried by passing air through the volume of gel particles to effect removal by ovaporation of the organic liquid.
17. The method as claimed in claim 8 in which the dry gel particles are arranged in the form of a column and in which the materials are passed through the column in one direction for unidirectional flow.
18. The method as claimed in claim 17 in which the materials are passed through the column from an inlet at one end through an outlet at the other.
19. The method as claimed in claim 8 in which the gel particles are subjected to sequential regeneration by unidirectional flow of materials therethrough to provide an automated system for multiple immunological determinations from the same volume of dry gel particles containing binding protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA313,491A CA1062610A (en) | 1973-05-01 | 1978-10-16 | Gel column device and use in blood purification and immunoassay |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/356,092 US4138474A (en) | 1973-05-01 | 1973-05-01 | Method and device for immunoassay |
| US356093A US3925017A (en) | 1973-05-01 | 1973-05-01 | Preparation of dry, porous gel particles having high water regain for liquid sampling |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1054050A true CA1054050A (en) | 1979-05-08 |
Family
ID=26999121
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA198,533A Expired CA1054050A (en) | 1973-05-01 | 1974-04-30 | Gel column device and use in blood purification and immunoassay |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JPS5013525A (en) |
| CA (1) | CA1054050A (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2718700A1 (en) * | 1977-04-27 | 1978-11-02 | Hans A Dipl Chem Dr Thoma | METHOD FOR TOTAL DETERMINATION OF HORMONES AND PHARMACA |
| JPS54111892A (en) * | 1978-02-17 | 1979-09-01 | Ii Mairusu Rooton | Method and device for inspecting numerous data |
| US4244694A (en) * | 1978-03-31 | 1981-01-13 | Union Carbide Corporation | Reactor/separator device for use in automated solid phase immunoassay |
| JPS5837482Y2 (en) * | 1978-04-19 | 1983-08-24 | 共立エコ−物産株式会社 | Sprayer water supply system |
| JPS5510596A (en) * | 1978-06-20 | 1980-01-25 | Damon Corp | Test kit for free species quantitative analysis and method therefor |
| JPS5883260A (en) * | 1981-11-12 | 1983-05-19 | Sekisui Chem Co Ltd | Filler for liquid chromatography |
| JPS58154655A (en) * | 1981-11-16 | 1983-09-14 | Sekisui Chem Co Ltd | Filler for liquid chromatography |
| US4477578A (en) * | 1982-03-04 | 1984-10-16 | Medical & Scientific, Inc. | Method and apparatus for performing assays |
| JP4942753B2 (en) * | 2005-09-27 | 2012-05-30 | センター フォア アプライド プロテオミクス アンド モレキュラー メディスン | How to isolate an analyte from a sample |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3711247A (en) * | 1970-03-02 | 1973-01-16 | Curtis Nuclear Corp | Method for determination of thyro-binding capacity of blood proteins |
| US3710117A (en) * | 1970-04-27 | 1973-01-09 | Yissum Res Dev Co | Vitro test system for assessing thyroid function |
-
1974
- 1974-04-30 CA CA198,533A patent/CA1054050A/en not_active Expired
- 1974-05-01 JP JP49048364A patent/JPS5013525A/ja active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5013525A (en) | 1975-02-13 |
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