Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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; Backqround of the Invention
This invention relates to immunoadsorbents and more
particularly to an improved immunoadsorbent for use in immuno-
assay procedures such as radioimmunoassay wherein the
- immunoadsorbent may be used for repeated assays. In a copending
divisional application, there is described and claimed a chamber
holder for the improved immunoadsorbent.
State of the Art
.~ ' .
~aaioimmunoassay is an analytical technique which
depends upon the competition (affinity) of antigen for
antigen-binding sites on antibody molecules. In practice,
standard curves are constructed from data gathered from a
plurality of samples each containing (a) the same ~nown
concentration of labelled antigen, and (b) various, but known,
concentrations of unlabelled antigen. Antigens are labelled
; with a radioactive isotope tracer. The mixture is incubated
in contact with an antibody, the free antigen is separated
¦ from the antibody and the antigen bound thereto, and then,
by use of a suitable detector, such as a gamma or beta
! 20 radiation detectorr the percent of either the bound or free
labelled antigen or both i9 determined. This procedure is
repeated for a number of samples containing various known
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1 concentrations of unlabelled antigens and the results plotted.
2 The percent of bound tracer antigens is plotted as a function
3 of the antigen concentration. Typically, as the total
4 antigen concentration increases the relative amount of the
~ tracer antigen bound to the antibody decreases. After the
6 standard graph is prepared, it is thereafter used to determine
~ the concentration of antigen in samples undergoing analysis.
~ In actual analysis, the sample in which the concen-
tration of antigen is to be determined is mixed with a known
11 amount of tracer antigen. Tracer antigen is the same antigen
12 known to be in the sample but which has been labelled with
13 a suitable radioactive isotope. The sample with tracer is
14 t~en incubated in contact with the antibody. Thereafter,
it may be counted in a suitable detector which counts the
16 free antigen remaining in the sample. The antigen bound to
17 the antibody or immunoadsorbent may also be similarly
18 counted. Then, from the standard curve, the concentration
19 of antigen in the original sample is determined. Afterwards,
the antibody or i~munoadsorbent mass is discarded.
~1 ,
22 In order to detect the percentage of antigen that
23 is bound to the antibody (bound ar.tigen) and/or the percentage
24 that remains free or unbound it is necessary to first separate
the sample into a fraction containing bound antigen and one
28 ~ontaining only free antigenO One common method for doing
27 this is to add a dextran coated charcoal to the mixture.
28 m e dextran permits the unbound antigen, of lower molecular
29 weight than the bound antigen, to pass through the dextran
and the charcoal adsorbs the free antigen. The charcoal
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1 with adsorbed free antigen is then separated from the
2 antibody ~and bound antigen) by centrifugation.
4 Another known procedure is to add to the mixture
another antibody which selectively precipitates the first
6 antibody (with the bound antigen) thus leaving in solution
q only free antigen. Classification into appropriate free
8 and bound fractions is then effected by separating the
9 precipitate from the supernatant by centrifugation or other
suitable means. Some worXers have resorted to the technique
11 of binding the antibody to tha inner walls of a plastic vessel,
12 filling the vessel with the antigen bearing sample, allowing
13 it to stand for an incubation period that typically ranges
14 from 4 to 72 hours and then separating free antigen from
bound antigen by draining and rinsing the vessel leaving
16 therein only the antibody and bound antigen. A more recently
17 deYeloped technique is to prepare the immunoadsorbent by
1~ binding the antibodies onto an insoluble cross-linked dextran.
19 ~he immunoadsorbent and antigen bearing sample are incubated
then the dextran with bound antigen is separated from the
21 solution by suitable means.
22
23 In all of the foregoing procedures, the pPrcentage
24 of labelled antigen in either or both the bound or free
fractions is determined and the standard curve used to determine
26 the antigen concentration. ~hereafter, the immunoadsorbent
27 is discarded.
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1 Although the foregoing radioimmunoassay techniques
2 have proven to be valuable tools and have gained widespread
3 acceptance, they are still not all that are to be desired
4 because the antibody ~mmunoadsorbent) is consumed with each
analysis hence must be discarded. Moreover, prior practice
6 is batc~ type and the several reagents are added to the
7 antibody in test tubes in which the separate steps, such as
1 8 incubation, rinsing and the like, are performed, thus
-¦ 9 resulting in a slow and costly operation.
11 There has been described a substantial
12 improvement in immunoassay procedures in that the same
-~ 13 immunoadsorbent may be used repeatedly for many assays by
14 releasing from the immunoadsorbent the antigen which is
~ 15 bound to the antibody mass, the latter immobilized on the
;jl 16 substrate; i.e. selectively and stoichiometrically releasing
_ 17 all of the antigen on the immunoadsorbent after the assay
18 is completed. It is to a reusable immunoadsorbent that
19 the present invention is directed.
` Description of the Prior Art
`~ 23
24 It is known from the literature that antibodies may
be isolated by use of immunologic adsorbents, the technique
26 being useful for isolation and puriication of antibodies
t 27 rather than quantitative determination thereof, see Campbell
28 et al, Proc. Nat'l._Acad. Sci. U._S~ 37 11951) 575.
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1 The use of an antibody coupled to an insoluble
2 polymer for extracting specific antigens for purposes of
3 isolating and purifying the same is described in Weetall et al,
4 Biochem. Biophys. Acta. 107 (1965) 150-152.
6 Porous glass has been described as a substrate for
7 immobilizing enzy~es, see ~7eetall, Biochem. Biophys. Acta.
8 212 ~1970) 1-7. There, glass was treated with gamma-
g aminopropyltriethoxysilane and the isothiocyanate derivative
was prepared by treatmen~ with thiophosgene. The enzyme
11 was coupled to the isothiocyanate derivative. Also described
-:: 12 in the preparation of an arylamine derivative by the reaction
13 of alkylamine glass with P-nitrobenzoyl chloride followed by
14 use of sodium dithionate to reduce the nitro grouPs. The
~j
arylamine glass was then diazotized and the enzyme coupled
16 thereto.
17
18 Weetall, in Biochem. J. (1970) 117, 257-261 also
19 describes the use of antibodies bound to porous glass through
a silane coupling agent, the immunoadsorbent being used to
21 isolate and purify specific antigens. The data given,
~2 however, shows that the reused column, in which the antigen
23 was eluted from the immobilized antibody immunoadsorbent
24 was quite erratic in performance since the recovery of
released antigen varied from 74% to 100%. See also U. S,
26 Patent 3,652,761 of March 28, 1972. While useful as an
27 isolation system, the described system has considerable
a8 objections from the standpoint of a useable tool in
29 quantitative analysis in which there must be substantially
stoichiometric release of the antigen.
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1 U. S. Patent 3,555,143 of January 12, 1971, relates
2 to radioimmunoassay procedures in which an immobilized
3 immunoadsorbent is used only once and th~n discarded. The
immunoadsorbent is a dextran (Sephadex G 25, su~erfine)
cross-linked with glycerine ether bridges and substituted
6 with p-nitrophenoxy-hydroxy-propyl ether grou~s. The nitro
groups are reduced to amine groups using sodium dithionite.
8 The Sephadex substituted with p-amino-phenoxy-hydroxy-propyl
9 groups was then treated with thiophosgene to form SePhadex
substituted with p-isothiocyanate-phenoxy-hydroxypropyl
11 groups, the antihody being bound to the latter substituted
1~ product.
13
14 A reaction widely used to insolubilize a protein
involves a covalent binding of the protein to a cyanogen
16 bromide activated cellulose matrix. The mechanism of such
17 activation is set forth in Bartling et al, Biotechnolo~y and
18 Bioenqineerinq, Vol XIV (1972) 1039-1044.
19
UO S. Patents 3,502,888 of July 13, 1971; 3,639,559
~1 of February 1, 1972, and 3,720,760 of March 13, 1972 are
22 also of interest.
23
24 Where an immobilized immunoadsorbent is to be
used only once and discarded, the long term properties of
26 the substrate are not of major consequence. Thus, materials
27 such as Sephadex (dextran) or Sepharose Sbeaded agarose
28 product) operate satisfactorily as substrates for antibodies
2~ bound thereto as described in U. S. Patent 3,555,143, supra.
Where the immunoadsorbent is to be used repeatedly,
31 certain problems arise.
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1 One of the objections is the tendency of Sephadex
2 and Sepharose type products to dehydrate, that is, the gel
3 collapses and packs to such an e~tent that flow through the
4 mass is substantially impeded and the availability of antibody
for binding antigen is altered, thus affecting the reproduc-
6 ibility and stability of the immunoadsorbent for repeated
use.
9 Glass and other solid inorganic materials offer a
10 desirable alternative because they can be formed into beads to
11 provide better flow and easier packing into a column type
12 arrangement. Such materials do not collapse and are not subject
13 to dehydration during periods of extended use. While a
~ desirable alternate, glass type products also suffer from
15 disadvantages. One of the problems is obtaining a sufficient
16 binding of the antibody to the substrate. Either an insufficient
17 initial binding takes place to provide the activity needed for
18 a quantitative analysis tool, or the activity changes over the
life of the immunoadsorbent by undesirable release of antibodies.
21 Where the glass is highly porous, as that used by the
~2 Weetall references cited, there is so much active glass surface
23 area that ample binding of the antibody takes place but non-
24 specific binding of the antigen also takes place. Thus, the
25 antigen bound to the glass is not released completely. That
~26 is, rather than having a stoichiometric release, for each
27 use thereof, as is needed for quantitative analysis, the release
28 characteristics are variable and unpredictable~ This is confirmed
~9 by the Weetall data. Since such glass is usually 96~ air or
30 void space, there is considerable active surface area of the
31 glass, not occupied by antibody which serves as an antigen
32 binding site.
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1 Anoth~r difficulty with highly porous glass
2 products is that there are multiple crevices in the pores
3 which result in trapping in the crevices and slow release
4 because of the slow diffusion in the crevices. Where a
fast response is needed, as for example in automated equipment,
6 the diffusion of the reactants is a rate limiting step and,
~ as is well known, diffusion may be a relatively slow process.
8 Thus, even if not bound to the substrate, the diffusion of
9 ~he antigen is relatively slow and thus, for the purpose of
rapid automated assay equipment, the antigen is effectively
11 bound rather than being rapidly and stoichiometrically
12 released.
13
14 Superficially porous supports are known for use
in chromatography, see for example U. S. Patent 3,505,785
16 of April 14, 1970 which describes a product commercially
17 available from E. I. du Pont de Nemours and Co. under the
18 trademarX "Zipax". These support beads for use as chromato- -
19 graphic column packing consists of a plurality of discrete
macroparticles with impervious cores and having irreversibly
21 joined thereto a coating of a series of sequentially
22 adsorbed-like monolayers of like colloidal microparticles.
23 Thus, spherical glass microbeads of about 30 microns
24 diameter include an outer porous surface crust which is
about one micron thick. Such a material, if used as a substrate
26 offers substantial surface area for the desired activity,
2q but the substrate must be properly prepared to assure the
28 proper quick response as well as stoichiometric release.
~9
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Thus, the provision of a reusable immunoadsorbent
and which stoichiometrically releases the antigen for each
assay is quite desirable. Where that immunoadsorbent is also
substantialLy free of dehydration and packs such that the
flow quality through the mass is of desirable character over
the useful life of the immunoadsorbent, a substantially improved
reusable immobilized immunoadsorbent is provided.
.. . .
Summary of the Inv`ention
The present invention provides an immobilized
immunoadsorbent for use in binding a specific antigen, the
immobilized immunoadsorbent comprising antibodies for a
specific antigen which are covalently bound to a water-
insoluble polymer coating bound to a particulate substrate
stable against dehydration and collapse.
More particularly, there is provided a reusable
immobilized immunoadsorbent for use in radioimmunoassay wherein
an antigen sample is flowed through said immunoadsorbent for
binding a specific antigen thereto and for release of said
antigen comprising:
~0 a mass of superficially porous refractory particles;
each said particle of said mass including an imper-
vious core having joined to the core sufficient
layers of microparticles to form an outer porous
coating on said core;
a water insoluble polymer material chemically bound
by silane linkages to said superficially porous
refractory particles; and
said polymer having bound thereto, by covalent bonds,
antibodies for binding a specific antigen.
In another aspect, the present invention provides
a process of forming an immobilized immunoadsorbent comprising:
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treating a particulate substrate stable against dehydration and
collapse to activate the same for binding a polymer thereto;
binding a water insoluble polymer to said activated substrate
to form a coating thereon;
activating said polymer coating to bind antibodies covalently
thereto;
and binding to said activated polymer coated substrate a mass
of antibodies having bound thereto antigen which is specific
to said antibodies.
Finally, the invention also includes a radioimmuno-
assay procedure wherein a mixture of a sample of an unlabelled
antigen of unknown concentration and a known amount of labelled
antigen is flowed into contact with an immobilized immuno-
adsorbent as described above having bound thereto antibodies
specific to said antigen, whereby a portion of the labelled
and unknown antigen is bound to the antibodies on the
immunoadsorbent thereby forming a bound fraction and an unbound
fraction; the eoneentration of the unknown antigen is
determined as a funetion of the bound or unbound fraetion, or
both; and the bound fraetion is released from the immuno-
adsorbent by rinsing the latter with an eluting
medium.
The improved immobilized immunoadsorbent is
provided for use and reuse in radioimmunoassay proeedures.
The substrate or basie matrix is stable against dehydration
and eollapse and is in the form of a mass of solid partieles
eaeh having an outer surfaee of high surface area. A typieal
sueh substrate is composed of superficially porous refraetory
partieles, each of whieh ineludes an impervious eore having
joined thereto suffieient layers of mieroparticles to form an
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outer porous coating on the core to provide a high surface
area.
To the substrate is bonded a water insoluble
polym~r material. The polymer may be bonded by treating the
substrate to form an amino alkylsilan~ derivative thereof
followed by treatment to form an isothiocyanoalkylsilane
derivative to which the polymer is bound. Typical of the
useful polymer materials is dextran.
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1 The dextran operates as a barrier to cover the
2 active sites on the substrate to ~lich antigen may be bound
3 in such a way as to interfere with subsequent assays. Since
1 4 the immunoadsorbent of this invention is used and reused,
1 5 by use of an eluting medium which separates the antigen
6 from the bound antibodies, the release of any antigen which
q may be bound to the substxate creates errors in subsequent
8 assays. The error arises because of the unpredictable and
! g unknown amount retained or released. Thus, the polymer
¦ 10 ef~ectively operates as a barrier to prevent the substrate
11 from binding antigen.
12
13 The polymer is then activated to bind antibody
14 through a covalent bond by treatment with cyanogen bromide,
the mechanism of the reaction being as described in Bartling
16 et al, supra.
lq
18 The resultant immobilized immunoadsorbant is then
19 placed in a cham~er holder of a unique structure for use
Z ~n automa~ed equ pm-~t,
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1 Brief Description of the Drawinqs
3 ~IGURE 1 is a view in perspective with a portion
4 thereof broken away and in section to illustrate diagrammat-
ically the substrate useable in accordance with this invention;
j 8 and
7 8 FIGURE 2 is a view in section of a chamber holder
- 9 in accordance with the invention described in the afore-mentioned
co-pending divisional application.
, 11
.
12 Detailed ~escription of the In~ention
13
14 The improved immobilized immunoadsorbent of the
present invention is intended for use principally in radio-
16 immunoassay procedures.
17
1~ Typical of the materials which may be quantitatively
19 determined by the system o~ the present invention are the
~ollowing: estriol, digoxin, digi~oxin, testosterone,
21 estradiol, aldosterone, progesterone, cortisol, ll-desoxy-
22 cortiosterone, ll-desoxycortisol, thyroid hormones such as
23 thyroxin (T4) triiodothyronine (T3), polypeptides such as
24 angiotensin, TSH (thyroid stimulating hormone), ACTH, GH
~growth hormone), ~P (human placento-lactogen), parathormone,
26 calcitonin, insulin, glucagen, polypeptide proteins such as
27 CEA ~carcino embrionic antigen), alphafetoprotein, interferon,
28 viruses ~uch as Australia antigen, vitamins such as D and B12
29 folic acid and drugs such as dilantin and barbiturates, to
mention only a few..
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1 The antisera for the above antigens are known, as
2 are the labelled antigens, available in the form of radio-
3 active isotope labelled materials, usually in the form of
4 the I125 isotope or H3 isotope.
_ 6 The immobilized immunoadsorbent of ~his invention
q includes a substrate with which the antibodies are relatively
_~ 8 permanently associated. In use, an unlabelled antigen sample
9 with a known concentration of labelled antigen is brought
10 into contact with the immobilized immunoadsorbent disposed
11 in a chamber holder. When brought into contact, a portion of
12 the mixture of labelled antigen and unlabelled antigen binds
13 to the specific antibody bound on the substrate. Thereafter,
14 the unbound antigen or the bound antigen or both are counted
15 and concentration of the unlabelled antigen is determined
16 from standard data.
' lq
18 Thereafter, the immobilized immunoadsorbent is
19 rinsed with an appropriate aqueous solution containing solvents
20 such as methyl alcohol, isopropyl alcohol or ethyl alcohol as
21 well as dimethyl formamide to effect a stoichiometric release
22 of the bound labelled and unlabelled antigen from the immobilized
23 immunoadsorbent. The rinsing or eluting operation effectively
24 regenerates the immunoadsorbent for reuse, and thereafter, the
25 same immunoadsorbent may be used again, repeatedly, for assays
26 of that antigen as to which the immobilized antibody is specific,
27 with washings, as described between each use.
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1 Since the antigen material is flowed into a chamber
2 supporting the immunoadsorbent which is reused, thè flow
3 characteristic of the substrate should be such that contact
4 is achieved between the supported antibodies and an~igen mixture.
5 Moreover, the substrate must be of such a type as not to
~ interfere with release of the bound antigen while retaining
7 the bound antibody. Reproducibility, stability and speed are
8 some of the advantages of the improved method, and thus the
9 substrate must be such that sufficient activity may be obtained
10 in terms of bound antibody with available antigen binding sites.
11 It is preferred, therefore,that the substrate be particulate,
12 and spherical i.e. formed of a mass of discrete particles since
13 this enhances the desirable flow-through character of not only
14 the sample mixture of labelled and unlabelled antigen, but
15 of the rinse or eluting medium as well.
16
1~ Particulate materials capable of providing the
18 needed antibody actively unknown, e.g. Sephadex, Sepharose,
19 porous glass and the like. Materials such as Sephadex and
20 Sepharose are yel type materials and over periods of extended
21 use, tend to dehydrate resulting in collapse with resulting
22 packing which impedes the flow. Materials such as porous
23 glass are so active that antigen is bound to the glass and
24 not released.
26 Thus, an important aspect of this invention is the
27 formation of a barrier coating over a particulate substrate,
28 the barrier coating operating to provide, in effect,
2~ a mask which precludes the potentialiy active sites on
30 the substrate from irreversibly binding the antigens.
31 The barrier also functions as an immobilized component of
32 the substrate to which the antibodies may be attached.
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1 Since assays are conducted in aqueous and non-aqueous solvents,
2 the barrier coating is preferably insoluble and not adversely
3 affected by the solvents and solutions used in the procedure.
4 Water insoluble polymer materials such as dextran are
preferred in accordance with this invention.
q The substrate itself is preferable a particulate
8 material resistant to dehydration and collapse. Rapid mass
9 transfer at relatively high flow rates are a function of
s~bstrate geometry, and packing character in the chamber
11 holder. A preferred substrate is a material having a controlled
12 surface porosity, superficially porous refractory particles
13 made up of discrete macroparticles with impervious non-porous
14 cores, and having joined ther to a coating of a series of
sequentially adsorbed like monolayers of like inorganic
~6 microparticles.
17
18 Referring to Fig. 1, for purposes o E illustration,
19 the superficially porous refractory particle 10 which forms
the substrate for the immobilized immunoadsorbent includes
21 a core 12 in the form of a macroparticle which is an
22 impervious non-porous core. The core 12 is shown as
23 spherical because this shape is preferred for packing
24 purposes. The core, in the form of a sphere is of a
diameter of between 5 and 500 microns in diameter and composed
26 of glass, although it may be o~E sands, ceramics, and the
27 like.
2~
29 The core.s are preferably of uniform size i.e. all
within about 50% o~E the average diameter. Affixed to the
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1 core 12 is a plurality of layers of microparticles 14 which
2 form an outer porous coating. The microparticles may range
3 in size from 5 milimicrons to 1 micron, and the number of
4 layers may be between 2 and 30. The micro-particles may
be amorphous silica, alumina, thoria and the like.
q As will be apparent, a substrate of material as
8 described has a relatively high surface area due to the
9 porous coating 15, but is relatively free of pores in the
core material. For beads of an overall diameter of 30
11 microns, and a porous crust of one micron, a surface area
12 of between 0>8 to 1.0 m2/gram is obtained, with a packed
~3 bed density of l.Sg/cc. I'he regular geometry, the stability
14 against dehydration and collapse, and the bulk renders the
above material quite exceptional as a substrate.
16
17 However, there is a tendency for such a material,
18 if used in the form described as a substrate directly for the
lg antibody material, to contain active sites which tend to
bind the antigen mixture or a component thereof in a non-
21 releasable manner. This problem may be quite objectionable
22 where the immobilized immunoadsorbent is reused, an important
23 objective in this invention. Since the accuracy and speed
24 of the assay is, in part, related to the ability of the
antibody to bind the antigen and stoichiometrically release
26 the same when rinsed, any unreleased antigen adversely
2~ affects the accuracy of subsequent assay. While a background
28 count could be taken, this is not entirely satisfactory since
2~ the retention-release phenomena tends to be non-uniform and
non-predictable,
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1 By this invention, such a tendency is eliminated
2 by the use of a barrier coating adhered to the substrate through
3 a silane coupling agent, i.e. the polymer is bound directly
4 to the outer surface portions of the substrate by silane
linkages. The polymer is then activated by treatment with
6 cyanogen bromide which covalently binds the protein (antibody)
q to the polymer activated particulate substrate~ The polymer
8 coating not only acts as a barrier, effectively to mask
9 latent active sites on the substrate proper, but offers an
active surface to which the antibody may be covalently
11 bound, a bond recognized as relatively strong.
12
13 In a typical procedure, in accordance with this
14 invention, 12 grams of particulate substrate material
~3~ micron diameter superficially porous rafractory particles,
16 as described supra) were added to a 500 ml flask to which
1~ was added 20 ml (18.84 grams) of 3-aminopropyltriethoxysilane
18 and 180 ml of toluene. The mixture was refluxed for 22
19 hours to form the aminoalkylsilane derivative of the glass
substrate. The derivative was filtered, washed with 200 ml
21 of toluene while on the filter support, and air dried,
22 followed by a second washing with 100 ml of chloroform and
23 a second air drying.
24
The isothiocyanoalkylsilane derivative was prepared
26 by trea~ing the prepared aminoalkylsilane glass derivative
27 with 16.6 ml (25 grams) of thiophosgene and 150 ml of
28 chloroform. The reaction vessel was protected from light
2~ and refluxed for 18 hours to form the descri~ed derivative,
30 which was filtered~ washed in chloroform, and air dried.
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1 The result of the steps thus far was to pxepare
2 an "activated" substrate to which the water insoluble
3 polymer may be bound.
In accordance with this invention it is preferred
6 to use dextran of a molecular weight of 70,000, although
q other materials may be used.
9 Accordingly, two hundred ml of a 1% solution of
dextran in 0.1 m sodium bicarbonate, pH 9.0, were added to
11 the "activated" substrate. The mixture was stirred for
12 three hours, filtered, washed with 300 ml of water, washed
13 with 100 ml of acetone and air dried to provide 11.6 grams
14 of polymer coated particulate substrate.
16 The remaining steps in the procedure involve
17 activation of the polymer coated substrate and, optionally,
18 the purification of the antibody, and binding the antibody to
19 the coated substrate, sometimes referred to as the conjugation
of the antibody to prepared substrate.
21
22 To activatè the dextran, twenty grams of cyanogen
23 bromide were dissolved in 200 ml of water. Cyanogen bromide
24 is quite toxic and therefore standard safety precautions are
2~ taken. The dextran coated substrate was added (1106 grams)
26 and the mixture stirred. The pH was raised from 3.6 to pH
27 10-11 using 23 ml of 6N sodium hydroxideO The pH was
28 maintained between 10 and 11 by the addition of 6N sodium
2~ hydroxide for two minutes. The activated dextran coated
substrate was then washed with 400 ml o~ water~ 400 Anl of
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1 50%, on a volume basis, of water and acetone, 400 ml of
2 25%,~o~`a volume basis, of water ancl acetone and finally
3 400 ml of acetone. The product was then air dried.
~ Treatment of the polymer coated substrate with
6 cyanogen bromide results in reaction with adjacent hydroxyl
7 groups on the polymer to form an imidocarbonate which couples
8 with the nucleophilic groups (amino) on the'antibody to
9 form the carbonic acid ester on hydrolysis. Rapid hydrolysis
10 of the imidocarbonate in aeid media results in formation of
11 a cyelic carbonate which is not as efficient in binding as
12 the imidocarbonate. Thus, care should be taken'to avoid
13 eonditions promoting cyclic carbonate foxmation.
14
i5 Simple purification of the antibody prior to
16 conjugation may optionally be conducted as follows: one
1~ ml of 18% sodium sulfate was added to 0.1 ml of the antisera.
18 The solution was vortexed and incubated for one hour to
19 precipitate gamma globulinsO The resulting mixture was then
20 eentrifuged for five minutes at 1000 xg at room temperature ''
21 followed by decanting and discarding the supernatant. The
22 pellet was suspended'in 10 ml of 18% sodium sulfate with
23 addition of 0.10 ml of water. The resulting mixture was
24 then vortexed and centrifuged again. The supernatant is
25 deeanted while the pellet was dissolved in 0.8 ml of 0.1 M
26 sodium bicarbonate solution.
27
28
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1 It is preferred in accordance with the pres~nt
2 invention to conjugate the antibody to the substrate while
3 the latter is saturated with antigen as to which it is
4 specific. The reason for this procedure is to obtain
enhanced activity by protecting the active sites on the
6 antibody during the conjugation procedure, thus, in effect
7 assuring that the antibody will assume a relation with the
8 substrate which assures availability of active sites. In a
9 sense, the binding of the active sites with antigen allows an
orientation of the antibody which reduces masking of the sites
11 by the conjugation procedure.
12
13 Thus, a 0.5 ml aliquot of a 0.1 mg/ml solution
14 of antigen, spec~fic to the antibody, was dissolved in an
ethanol-water solution (2 parts ethanol and 1 part water)
16 and dried down in a test tube with nitrogen gas. The purified
17 antisera, clissolved in 0.8 ml of 0.1 M sodium bicarbonate,
18 or 0.1 ml of antisera in 0.8 ml of 0.1 M sodium bicarbonate
1~ was added to the tube containing the dried antigen followed
by incubation for one hour in a capped culture tube.
21 Thereafter, 300 mg of the cyanogen activated polymer coated
22 substrate were added to the tube containing the antibody
23 solution and incubated for one to three days at 4C while
X4 mixing. During the incubation period the conjugation takes
place with the antibody having its antigen binding sites
26 protected by bound antigen.
27
28 After incubation, the suspension was centrifuged
2~ at 1000 xg for five minutes and the supernatant decanted
and discarded. The pellet was washed two times with 10 ml
31 of 0.5 M sodium bicarbonate solution. After each wash
32
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1 the suspension was centrifuged and the supernatant discarded.
1 2 The antibody coated substrate was then washed two times with
- 3 10 ml of 0.1 M acet~te buffer, pH 4. The antibody coated
1 4 substrate was then washed with 10 ml of 0.05 M phosphate
5 buffer, pH 7.5, containing 0.05 M sodium chloride and 0.5%
j 6 bovine serum albumin and 0.02% sodium azide as a preservative.
- 7 The resulting product was then resusp,ended in 10 ml of the
~ ~ last wash solution and stored at 4C.
;' 9
The resultant product, prior to use in an assay,
11 is rinsed with one of the solutions described to release
12 the antigen bound to the immobilized antibody. In storage,
i 13 however, it is preferred that the antigen remain bound to
14 the antibody.
16 It is possible, in accordance with this invention
17 to conjugate the antibody, in a free stat$, to the substrate.
18 This procedure involves admixing the antibody to the cyanogen
19 activated polymer coated substrate followed by incubation and
20 post treatment as already described.
21
1 22 The provision of a chamber holder for the immobilized
23 immunoadsorbent is covered by the aFore~mentioned co-pending division-
j 24 al application. Referring to Fig. 2, the chamber holder 25
~ 25 includes a supporting body 27 which is cylindrical in
1 26 shape for ease of installation. Provided within the body
27 27 is a chamber 30 which contains the immobilized immuno-
28 adsorbent matter 32, illustrated as particulate material.
j 29 The immunoadsorbent is supported in the chamber by a porous
30 plu~ 34 whose pore size is smaller than the particles making
31
32
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~ 1 up thc immunoadsorbent. An outlet 36 is provided for exit
i 2 of material flowed through the chamber through an inlet
3 38. The plug 34 is press fitted in the support body which
4 may be of a plastic material such as polypropylene or
j 5 "DELRIN". By way of example, the chamber is 1/8 inch in
j 6 diameter and 1/4 inch long while the plug is 1/8 inch by
7 1/8 inch 10 micron pore size.
- 8
I 9 Positioned in the inlet path are filter elements
41 and 43, the form 400 mesh nylon screen which bears
11 against a filter disc 43 preferably of polytetrafluoroethylene
12 and in the form of a felt, 10 micron or less in pore size~
13
14 As illustrated, the chamber 30, at each end thereof
j 15 terminates in enlarged end sections 46 and 47, in the form
16 of counter-bores each of which receives a press fitted plug
1~ 48 and 49, respectively. Each plug includes an interior
18 counter-bore, 51, 53, respectively, and a passage therethrough,
19 as illustrated. On the ends of the plugs facing the chamber
30, the plugs include a diverging conical opening 56, 57, and
21 an annular shoulder which receives an o-ring seal element,
22 as shown, the latter forming a seal in the respective
¦ 23 counter-bored ends of the support body 27.
2~
Plug 49 operates to compress filters 41 and 43
26 in place, the screen 41 operating to prevent the felt from
27 entering the conical opening 57.
i 28
! 29 * Trade Mark
31
32 - 21 -
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1 Attachment to the chamber holder is through the
2 end plugs 48, 49 and the end bores 51, 53 thereof. Thus,
3 the chamber holder is removed as a unit and replaced with
4 a chamber holder for an immunoadsorbent of an antibody
specific to the antigen being run in that particular assay.
6 When not used, the appropriately identified chamber holder may be
q stored at 4C.
9 Chamber holders as described for supporting
immobilized immunoadsorbents have been used in assays of
11 ~he various antigens identified. The immunoadsorbents of
1~ this invention have been used for over S00 assays for each
13 immunoadsorbent and still continue to function with results
14 favorable to those obtained with the classical methods, i.e.
standard deviation of between 5% and 6%.
16
17 The chamber holder includes a diverging cone
18 57 in the inlet end to the chamber for the purpose of
19 dispersing the flow over the bed of the particulate
immunoadsorbent in the chamber 30. Diverging opening 56
21 operates as a collector for the material exiting, while
22 plug 46 supports the porous plug 34 within the chamber.
23
24 While dextran has been described as a useable
polymer, the invention is not limited to that specific
26 material. Other water insoluble polymer materials with
27 available hydroxy groups for cyanogen bromide activation
28 may be used, e.g. cellulose and the like. Dextran is
2~ preferred, however, because of the considerable use of this
material in radioimmunoassay in the prior art, e.g. dextran
31 coated charcoal, and its behaviox i~ the environment is not
32 detrimental to the procedure.
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1 It will be apparent to those skilled in the art
2 that various modifications may be made with respect to the
3 subject matter herein disclosed. For example, the barrier
coating may be used to mask other substrates where activity
5 of the substrate is a potential problem. Other modifications,
6 changes and alterations will be apparent from the foregoing
7 description of illustrative forms of the invention without
8 departing from the scope of the invention as set forth
9 in the appended claims.
11
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