JPS5916671B2 - Immobilized immunoadsorbent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunoadsorbents, and more particularly to improved immunoadsorbents for use in immunoassays, such as radioimmunoassays, where the immunoadsorbents can be used in repeated analyses. It relates to adsorbents.

Radiation immunosweat analysis is an analytical technique that relies on competition (affinity) of antigen for antigen-binding sites on antibody molecules.

In practice, data collected from multiple samples, each containing (a) the same and known concentration of labeled antigen, and (6) varying and known concentrations of unlabeled antigen, are used to establish a standard. Create a curve. The antigen is labeled with a radioisotope tracer. The mixture is incubated in contact with the antibody, the free antigen is separated from the antibody and the antigen bound thereto, and the bound or bound antigen is then separated using a suitable detector, such as a gamma or beta detector. The ratio of free labeled antigen or both is determined. This procedure is repeated for multiple samples containing various known concentrations of unlabeled antigen;
Then plot the results. The proportion of tracer antigen bound is plotted as a function of antigen concentration.
Typically, as the total antigen concentration increases, the relative amount of tracer antigen bound to the antibody decreases. After a standard graph is created, it is later used to determine the antigen concentration in the sample being analyzed. In the actual analysis, the sample whose concentration of antigen is to be determined is mixed with a known amount of tracer antigen.

The tracer antigen is the same antigen known to be present in the sample, but labeled with the appropriate radioactive isotope. The tracer-bearing sample is then contacted with the antibody and incubated. This can then be counted with a suitable detector, which counts the free antigen remaining in the sample. Antigens bound to antibodies or immunoadsorbents can also be counted in a similar manner.
The concentration of antigen in the original sample is then determined from the standard curve. The antibody or immunoadsorbent material is then discarded. In order to detect the proportion of antigen that is bound to antibodies (bound antigen) and/or the proportion that is free or unbound, the sample is first divided into a fraction containing bound antigen and a fraction containing only free antigen. It is necessary to separate it into fractions.

One common way to do this is to add dextran coated charcoal to the mixture. The dextran allows unbound antigen, which has a lower molecular weight than the bound antigen, to pass through the dextran, and the charcoal adsorbs the free antigen. The charcoal with adsorbed free antigen is then separated from the antibody (and bound antigen) by centrifugation. Another known method is to first prepare antibodies (with bound antigen).
Add other antibodies to the mixture that selectively precipitate
Thus only free antigen remains in solution.

Sorting into the appropriate free and bound fractions is then carried out by separating the precipitate from the supernatant by centrifugation or other suitable means. Some researchers bind antibodies to the inner wall of a plastic container, fill the container with an antigen-containing sample, let it sit for an incubation period typically ranging from 4 to 72 hours, and then drain and drain the container. A technique was used in which free antigen was separated from bound antigen by washing, leaving only the antibody and bound antigen in the container. A recently developed technique is to prepare immunoadsorbents by coupling antibodies onto insoluble cross-linked dextran. The immunoadsorbent and antigen-containing sample are incubated and the dextran with bound antigen is then separated from the solution by appropriate means. In all of the above methods, the proportion of labeled antigen in either the bound or free fractions, or both, is measured and a standard curve is used to determine the antigen concentration.

The immunoadsorbent is then discarded. Although the radioimmunoassay techniques described above have proven to be valuable tools and have received widespread acceptance, they are still far from being as perfect as they should be.

This is because the antibodies (immunoadsorbents) are consumed during each analysis and therefore have to be discarded. Furthermore,
Traditional practice is batch-based, and several reagents are added to the antibody in the test tube, e.g.
Separate steps such as washing etc. are performed, making the operation slow and expensive. U.S. Patent Application No. 342,513
The issue describes substantial improvements in immunoassay methods,
There, the antibody substance (which is immobilized on the substrate)
By releasing the bound antigen from the immunoadsorbent, the same immunoadsorbent can be used repeatedly for multiple analyses.

That is, all antigens on the immunoadsorbent are selectively and stoichiometrically released after the analysis is complete. The present invention is directed to reusable immunoadsorbents. It is known from the literature that antibodies can be isolated by the use of immunological adsorbents, and this technique is useful for isolating and purifying antibodies rather than for their quantitative analysis.

(Campbelletal, PrOc. Nat3e.
Acad. Sci. U. S. 37 (1951) 575). The use of antibodies conjugated to insoluble polymers to extract specific antigens for purposes of isolation and purification has been described by Weetallet et al.
al) (BiOcnem.Bl
Ophys. Acta. lO7 (1965) 150-1
52).

Porous glass has been described as a substrate for immobilizing enzymes (Weetal, BiOchem.BIO
phys. Acta. 2l2 (1970) 1-7)
.

There, glass is treated with gamma-aminopropyltriethoxylane and isothiocyanate derivatives are made by treatment with hand-ophosgene. The enzyme is coupled to an isothiocyanate derivative. It is also described that in the production of arylamine derivatives, an alkylamine glass and p-nitrobenzoyl chloride are reacted, followed by reduction of the nitro group using sodium dithionate. The arylamine glass is then diazotized and the enzyme is attached to it. Wheat Earl (BiOchem.J (1970) 117
, 257-261) also describe the use of antibodies bound to porous glass via silane binders, where immunoadsorbents are used to isolate and purify specific antigens. has been done.

However, the data presented indicate that reused columns, such as those from which antigen is eluted from immobilized antibody immunoadsorbents, have highly variable performance (U.S. Pat.
No. 52761, March 28, 1972). That is,
The recovery rate of the released antigen varies from 7401) to 100(:f
l). While the systems described above are useful as isolation systems, there is considerable controversy as a tool for use in quantitative analysis where the release of substantially quantitative amounts of antigen is required. U.S. Patent No. 3,555,143 (19
(January 12, 1971) concerns a radioimmunoassay method, in which the immobilized immunoadsorbent is used only once and then discarded.

The immunoadsorbent contains dextran [Sep
hadix) G25, superfine] is reduced to an amino group by sodium dithionite. The Cephadex substituted with p-aminophenoxyhydroxypropyl groups is then treated with thiophosgene to produce Cephadex substituted with p-isothiocyanatephenoxyhydroxypropyl groups; to a product substituted with . A widely used reaction to insolubilize proteins involves the covalent attachment of proteins to cellulose matrices activated with cyanogen bromide.

The mechanism of such activation is described in the literature (Bartlin
getal, BiOtechnOlOggandBlO
engineering, OlX (1972) 1039
~1044). U.S. Patent No. 350288
No. 8 (July 13, 1971), No. 3639559 (
February 1, 1972) and No. 3720760 (February 1, 1972)
March 13, 1972) is also of interest.

If the immobilized immunoadsorbent is used only once and then discarded, the long-term nature of the substrate is not very important.

For example, materials such as Sephadex (dextran) or Sepharose (a beaded agarose product) work well as substrates for binding antibodies, as described in US Pat. No. 3,555,143. For repeated use of immunoadsorbents, U.S. Application No. 34251
Certain problems arise, as described in No. 3.

One objection is that Cephadex and Cepharose type products tend to dehydrate.

That is, the gel collapses and becomes clogged to the extent that flow through the object is substantially impeded, altering the ability of the antibody to bind antigen and thus reducing the reproducibility of the immunoadsorbent for repeated use. and stability will be affected. Glasses and other solid inorganic materials provide desirable selectivity.

This is because they can be formed into beads to more easily pack into column-type arrangements and obtain better flow. Materials of this type do not disintegrate and do not suffer from dehydration even during long-term use. Although the glass type is a desirable choice, it also has drawbacks. One of the problems is getting the antibody to bind sufficiently to the substrate. Insufficient initial binding may result in deficient activity as a quantitative analytical tool, or activity may fluctuate over the life of the immunoadsorbent due to undesired antibody release. If the glass is highly porous, as used in the Wheatearl reference, there is a very large active glass surface area that will result in sufficient binding of antibodies, but also non-specific binding of antigen. Put it away.

Therefore, the antigen bound to the glass will not be completely released. That is, the stoichiometric release required for quantitative analysis does not occur with each use, and the release characteristics are variable and unpredictable. This is confirmed by Wheatear's De Ichiba. This kind of glass is usually 96%
of air or board space, there is considerable active glass surface area that is not occupied by the antibodies used as antigen binding sites. Another difficulty with highly porous glass products is the presence of numerous cracks in the pores, which leads to entrapment within the cracks and slow release due to slow diffusion within the glass array. That's true.

When a rapid response is required, such as in automated equipment, diffusion of the reactants becomes the rate-limiting step, and as is well known, diffusion is a relatively slow process.
Therefore, even when unbound to a substrate, antigen diffusion is relatively slow and, therefore, for the purposes of rapid automated analyzers, the antigen is not released rapidly and quantitatively but is rather strongly bound. There is. Superficially porous supports are known for use in chromatography (see, eg, US Pat. No. 3,505,785, April 14, 1970).

This is the first, second, and third year of my life.
It is a product marketed under the trademark "Zipax" by the Company. The support beads used as packing for chromatographic columns consist of a plurality of individual macroparticles with an impermeable core and a series of coatings irreversibly bonded thereto. The coating consists of a plurality of monolayers of colloidal microparticles that are adsorbed in sequence. For example, spherical microscopic glass beads about 30 microns in diameter include an outer porous surface skin about 1 micron thick. Such materials, when used as substrates, provide significant surface area for the desired activity, but the substrates must be properly manufactured to ensure adequate rapid response and quantitative release. No. Thus, it would be highly desirable to provide an immunoadsorbent that releases antigen stoichiometrically and can be reused after each analysis.

Substantially improved reusability is achieved if the immunoadsorbent is packed in a manner that substantially eliminates dehydration and maintains the flow through the material with desirable properties over the useful life of the immunoadsorbent. An immobilized immunoadsorbent that absorbs water is provided. According to the present invention, an improved immobilized immunoadsorbent for repeated use in radioimmunoassay is provided.

The substrate, or basic matrix, is stable to dehydration and decay and is in the form of a population of solid particles, each having an external surface of high surface area. Typically such a matrix is made of superficially porous refractory particles, each containing an impermeable core that has a layer of substantial particulates bound thereto. , forming an external porous covering over the core, giving it a high surface area. A water-insoluble polymeric material is bound to the substrate.

The polymer can be attached by treating the substrate to form an aminoalkylsilane derivative and then treating the substrate to form an isothiocyanoalkylsilane derivative to which the polymer is attached. An example of a typical useful polymeric material is dextran. The dextran acts as a barrier to cover the active sites of the substrate to which antigen can be bound, preventing subsequent analysis.

Since the immunoadsorbent of the present invention is used repeatedly by using an elution medium that separates the antigen from bound antibodies, the release of any antigen that may be bound to the substrate will introduce errors in subsequent analyses. This error occurs due to unpredictable and unknown amounts retained or released. Thus, the polymer effectively acts as a barrier to prevent the substrate from binding the antigen. The polymer is then activated by treatment with cyanogen bromide to mount the antibody through covalent bonds.

The mechanism of this reaction was explained by Bartling et al.
lingetal). The resulting immobilized immunoadsorbent was then prepared as described in US Pat. N. Third
For use in automated equipment such as that described in No. 42,513, it is placed in a chamber holder of unique construction.

The improved immobilized immunoadsorbent of the present invention is primarily intended for use in radioimmunoassays.

Representative substances that can be quantitatively measured by the system of the present invention are as follows: estriol, digoxin, digitoxin, testosterone, estradiol, aldosterone, progesterone, cortisol,
11-desoxycortiosterone, 11-desoxycortisol, thyroid hormones such as thyroxine (T4), triiodothyronine (T3), polypeptides such as angiotensin, TSH (thyroid-stimulating hormone) , ACTH. ．． GH (growth hormone), HP (human placenta-lactogen), parathormone, calcitonin, insulin, glucagen, polypeptide proteins such as CEA (carcino-embryo-antigen), alpha-fetoprotein, interferon, e.g. Viruses such as Australian Antigen, vitamins such as vitamins D and B, difolic acid, drugs such as barbiturates and cilantro. These are just a few examples.
Antisera against the above antigens are known, for example in the form of a substance labeled with a radioactive isotope, usually 1125
There are labeled antigens available in isotope or H3 isotope form.

The immobilized immunoadsorbents of the present invention include a substrate to which antibodies are relatively permanently bound.

In use, an unlabeled antigen sample containing a known concentration of labeled antigen is contacted with an immobilized immunoadsorbent placed in a chamber holder. Upon contact, a portion of the mixture of labeled and unlabeled antigen binds to the specific antibody bound on the substrate. after that,
Unbound or bound antigen, or both, are counted and the concentration of unlabeled antigen determined from standard data. The immobilized immunoadsorbent is then washed with a suitable aqueous solution containing a solvent such as methyl, isopropyl or ethyl alcohol, and dimethylformamide to remove bound labeled and unlabeled antigens from the immobilized immunoadsorbent. Stoichiometric release from the adsorbent.

The washing or elution procedure effectively regenerates the immunoadsorbent for reuse, and this same immunoadsorbent can then be used for analysis of antigens for which the immobilized antibodies will specifically act. It can be used repeatedly, with cleaning performed between each use as described above. As antigenic material flows into the chamber supporting the reused immunoadsorbent,
The flow characteristics of the substrate must be such that contact is achieved between the supporting antibody and the antigen mixture. Furthermore, the substrate must be of a type that does not prevent the release of bound antigen and retains bound antibody. Reproducibility, stability, and speed are some of the advantages of this improved method, and for example, the substrate must be such that sufficient activity can be achieved by binding antibodies with available antigen binding sites. Must be. Therefore, it is preferred that the substrate is particulate and spherical, ie made up of a population of individual particles. This is because this increases the desirable flux characteristics of the sample mixture of labeled and unlabeled antigen as well as the washing or elution medium. Particulate materials that could contain the necessary antibodies of unknown activity were, for example, Sephadex, Sepharose, porous glass, and the like.

For example, materials such as Cephadex and Cepharose are gel-type materials that tend to dehydrate with prolonged use, resulting in disintegration and the formation of blockages that impede flow. Materials such as porous glass, for example, are highly active and antigens bind to the glass and are no longer released. Therefore, an important aspect of the present invention is the formation of a barrier coating on a particulate substrate;
This barrier coating effectively acts to provide a mask to protect potentially active sites from irreversible binding of antigen.

This barrier also acts as an immobilized component of the substrate to which antibodies can be attached. Since the analysis is performed in aqueous and non-aqueous solvents, it is preferred that the barrier coating be insoluble in and not adversely affected by the solvents and solutions used in the method. Water-insoluble polymeric materials such as dextran are suitable in the present invention. The matrix itself is preferably a particulate material that is resistant to dehydration and disintegration.

Rapid mass transfer at relatively high flow rates is a function of substrate geometry and packing characteristics in the chamber/holder. The preferred substrate is a material with controlled surface porosity, which is a superficially porous refractory particle made of individual macroparticles with an impermeable, non-porous core and attached thereto a series of coatings, ie, a series of coatings consisting of a single layer of inorganic fine particles that are sequentially adsorbed. Referring to FIG. 1, shown for illustrative purposes, superficially porous refractory particles 1 forming a matrix for an immobilized immunoadsorbent.
0 contains a core 12 in the form of a macroparticle, which is an impermeable and non-porous core.

Nucleus 12 is shown as spherical. This is because this shape is suitable for filling purposes. The spherical shaped core is 5 to 500 microns in diameter and is made of glass, but may also be sand, ceramic, etc. Preferably, the nuclei are of uniform size, ie all within about 50(f) of the average diameter.

Adjunct to the core 12 are multiple layers of microparticles 14, which form an outer porous covering. The particle size can range from 5 millimicrons to 1 micron, and the number of layers can range from 2 to 30. The particulates may be amorphous silica, alumina, thoria, or the like. As can be seen, the substrate material described above has a relatively large surface area due to the porous coating 15, but there are almost no pores in the core material.

For beads with a total diameter of 30 microns and a porous skin of 1 micron, a surface area of 0.8 to 1.0 I/9 is obtained, with a packed bed density of 1.59/CC.
The regular geometry, stability against dehydration and disintegration, and bulk make the above-mentioned materials quite special as substrates. However, such substances tend to contain active sites which, if used in the form described above as substrates for antibody substances, would non-releasably bind the antigen mixture or its components. .

This problem becomes quite controversial in the case of reuse of immobilized immunoadsorbents, which is an important objective of the present invention. The accuracy and speed of the assay is, in part, related to the ability of the antibody to bind antigen and release it quantitatively upon washing, so that unreleased antigen adversely affects the accuracy of subsequent assays. . It would be possible to take a background count, but this is not entirely satisfactory. This is because the retention-release phenomenon is non-uniform and unpredictable. According to the present invention, such gradients are eliminated by using a barrier coating attached to the substrate via a silane binder.

That is, the polymer is bonded directly to the external surface portion of the substrate via silane bonds. The polymer is then activated by treatment with cyanogen bromide, thus covalently binding the protein (antibody) to the polymer-activated particulate matrix. The polymer coating not only acts as a barrier, effectively masking potentially active sites on the substrate alone, but also provides an active surface to which antibodies can be covalently attached, where the attachment is relatively strong. Can be seen. In a typical procedure, according to the invention, particulate matrix material (
Put 129 (surface porous refractory particles with a diameter of 30 microns) into a 500 ml flask, and add 3
-Aminopropyltriethoxysilane 20ml (18.
849) and 180 ml of toluene were added.

The mixture was refluxed for 22 hours to form a glass-based aminoalkylsilane derivative. Filter this derivative,
The filter was washed with 200 ml of toluene and air dried while still on the support, followed by a second wash with 100 ml of chloroform and a second air dry. The aminoalkylsilane glass derivative prepared above was treated with 16.6 ml (259) of thiophosgene and 150 ml of chloroform to prepare an isothiocyanoalkylsilane derivative.

The reaction vessel was protected from light and refluxed for 18 hours to produce the above derivative, which was filtered, washed with chloroform and air dried. As a result of such measures, an "activated" substrate is prepared to which a water-insoluble polymer can be attached.

According to the present invention, it is preferred to use Textran with a molecular weight of 70,000, but other materials can also be used.

For example, 200 ml of a 1% solution of dextran in 0.1 molar sodium bicarbonate (PH 9.1) was added to the above "activated" substrate.

The mixture was stirred for 3 hours, filtered, washed with 300 ml of water, washed with 100 ml of acetone and air-dried to yield a polymer-coated particulate substrate 11.69. The remaining steps in the procedure are activation of the polymer-coated substrate and, if necessary, purification of the antibody and binding of the antibody to the coated substrate, often referred to as conjugation of the antibody to the prepared substrate. Re+.

To activate dextran, cyanogen bromide 2
09 was dissolved in 200 ml of water.

Cyanogen bromide is extremely toxic and therefore safety regulations must be taken. Gextran coated substrate (11.69) was added and the mixture was stirred. P.H.
was increased from 3.6 to 10-11 with 23 ml of 6N sodium hydroxide. The pH was kept at 10-11 for 2 minutes by addition of 6N sodium hydroxide.

The activated dextran-coated substrate was then soaked in 400 ml of water.
11 volume basis: 50% of water (:f) and 400% of acetone
25 (f) of water and 400 of acetone on a m11 volume basis
ml and finally with 400 ml of acetone. The product was then air dried. When a polymer-coated substrate is treated with cyanogen bromide, a reaction occurs with the adjacent hydroxyl groups of the polymer to form imidocarbonate, which binds to the nucleophilic group (amino group) of the antibody and generates carbonate upon hydrolysis. Produces esters. Rapid hydrolysis of imidocarbonate in acidic media produces cyclic carbonates, which are less effective at bonding than imidocarbonate. Therefore, care should be taken to avoid conditions that promote cyclic carbonate formation. Simple purification of antibodies prior to conjugation can be performed as follows, if desired.

1 ml of 18% sodium sulfate solution was added to 0.1 ml of antiserum.

This solution was vortexed and incubated for 1 hour to precipitate γ-globulin. The resulting mixture was then heated at room temperature for 10
Centrifuged at 00Xg for 5 minutes, then decanted and discarded the supernatant. The pellets were suspended in 10 wL1 of 18(f) sodium sulfate solution by adding 0.10 ml of water. The resulting mixture was then vortexed again and centrifuged. The supernatant was decanted and the pellet was dissolved in 0.8 ml of 0.1 molar sodium bicarbonate solution. In the present invention, it is preferred to conjugate the antibody to the substrate while the substrate is saturated with antigen specific for it. The reason for this procedure is to obtain enhanced activity by protecting the active sites on the antibody during the conjugation process, i.e. to obtain a substrate that ensures the availability of the active sites. This is to effectively ensure that antibodies maintain a relationship with Binding of the active site to the antigen, in a sense, results in orientation of the antibody, which reduces masking of the active site by the conjugation process. Therefore, 1 part of 0.5 ml of a 0.1-/ml solution of the antigen specific for the antibody is dissolved in an ethanol-aqueous solution (2 parts of ethanol and 1 part of water) and evaporated using nitrogen gas. and allowed to dry in the test.

The purified antiserum was added to 0.8 mol of sodium bicarbonate.
ml, i.e. 0.1 mol sodium bicarbonate.
0.1 d of antiserum in 8 was added to the dry antigen-containing test tube, and then cultured for 1 hour in a tightly capped culture tube. after that,
Cyanogen-activated polymer-coated substrate 300Tr19 was added to the test tube containing the antibody solution and mixed with
The cells were cultured at ℃ for 1 to 3 days. During the culture period, conjugation occurred while antibodies having antigen-binding sites were protected by the bound antigen. After incubation, the suspension was
Centrifuged for 5 minutes at X9 and decanted and discarded the supernatant.

Pellets in 1.0ml of 0.5M sodium bicarbonate solution
Washed twice. After each wash, centrifuge the suspension and
Then, the supernatant liquid was discarded. The antibody-coated substrate was then washed twice with 10 ml of 0.1 molar acetate buffer (PH4). This antibody-coated substrate was then buffered in a 0.05 molar phosphate buffer containing 0.05 molar sodium chloride and 0.5 molar calf serum albumin and 0.0201 molar sodium azide as a preservative. (PH7.5) 1
I washed it with 0ml. The resulting product was then resuspended in 10 ml of the final wash solution and stored at 4°C. The resulting product is washed with one of the solutions described above to release the antigen bound to the immobilized antibody before use for analysis.

However, it is preferred that the antigen remain bound to the antibody during storage. According to the present invention, it is possible to bind antibodies in a free state to a substrate. Is this process a cyanogen-activated polymer? This involves incorporation of antibodies against the overlying substrate, followed by incubation and post-treatment as previously described. One aspect of the invention is to provide a chamber holder for immobilized immunoadsorbents.

Referring to FIG. 2, the chamber holder 25 includes a support 27 which is cylindrical in shape to facilitate the device. A chamber 30 is provided within the support 27 and contains an immobilized immunoadsorbent 32, shown as particulate material. The immunoadsorbent is supported within the chamber bar by a porous plug 34 whose pore size is smaller than the particles comprising the immunoadsorbent. Entrance 3
An outlet 36 is provided as an outlet for substances flowing from 8 through the chamber. The plug 34 is press-fit 6 to the support, which may be, for example, a plastic material such as polypropylene or DELRIN. As an example, the chamber is 1/8 inch in diameter and 1/4 inch long, while the plug is 1/8 inch by 1/8 inch and has a pore size of 10 microns. Located within the inlet passageway are filter elements 41 and 43, filter element 41 being in the form of a 400 mesh nylon screen, preferably made of polytetrafluoroethylene and having a pore size of 10 microns or less. It is supported by a filter disk 43 having a shape.

As shown, chamber 30 has enlarged end portions 46 and 47 at each end thereof, each having a counterbore configuration for receiving press-fitted plugs 48 and 49, respectively. are doing. Each pubug has an internal counterbore 51, 53 and a passage therethrough as shown. At one end of the plug facing chamber 30, the plug has a diverging conical opening 56;
57 and an annular shoulder for receiving an O-ring seal element as shown, the O-ring elements forming a seal at each counterbore end of support 27. Plug 49 serves to force filters 41 and 43 into position, and screen 41 serves to prevent felt from entering conical opening 57.

Connections to the chamber holder are made using end plugs 48, 49.
and through its terminal bores 51,53.

The chamber holder is therefore removed as a unit and replaced with an immunoadsorbent chamber holder having antibodies specific for the antigen to be subjected to a particular analysis. When not in use, properly identified chamber holders can be stored at 4°C. Chamber holders such as those described above for supporting immobilized immunoadsorbents are used for analysis of the various antigens identified.

The immunoadsorbents of the present invention contain 50% of each immunoadsorbent.
used over 0 analyzes and still obtained results that are better than those obtained with the classical method, i.e. with a standard deviation of 5
Continue working with ~6%. The chamber holder is
The inlet end to the chamber includes a diverging cone 57 whose purpose is to distribute the flow throughout the bed of particulate immunoadsorbent within the chamber 30.

The diverging opening 56 acts as a collector for the exiting material, while the plug 46 has a porous plug 3 in the chamber.
I support 4. Although dextran is mentioned as a polymer that may be used, the invention is not limited to such specific materials.

Other water-insoluble polymeric materials having hydroxyl groups that are susceptible to cyanogen bromide activation may also be used, such as cellulose. However, dextran is preferred. This is because this material, such as dextran-coated charcoal, is used extensively in prior art radioimmunoassays, and its behavior in the environment is not disadvantageous to this method. It will be apparent to those skilled in the art that various modifications may be made to the subject matter disclosed herein.

For example, barrier coatings can also be used to mask other substrates where substrate activity is a potential problem. From the above exemplary description of the invention:
It will be obvious that other modifications, changes and options may be made without departing from the scope of the invention.

[Brief explanation of the drawing]

FIG. 1 is a perspective cross-sectional view with a cut portion;
1 illustrates a substrate that can be used according to the invention. FIG. 2 is a cross-sectional view of a chamber holder according to the invention. In the figure, 10 is a particle forming a matrix, 12
is the core, 14 is the fine particle, 15 is the porous coating, and 2
5 is a chamber holder, 27 is a support, 32 is an immobilized immunoadsorbent, 34 is a porous plug, and 41 and 43 are filter elements.

Claims (1)

Claims: 1. A support structure, a chamber formed within the support structure, and an immobilized immunoadsorbent positioned within the chamber, the immunoadsorbent being resistant to dehydration and disintegration. a matrix of stable refractory particles having a barrier coating of a water-insoluble polymer chemically bonded thereto and an antibody chemically bonded to the barrier coating for binding a specific antigen; a chamber holder for an immobilized immunoadsorbent, further comprising support means cooperating with the chamber for supporting the immobilized immunoadsorbent within the chamber. 2. A chamber holder according to claim 1, wherein the support structure includes means for forming an inlet and an outlet for passage of fluid through the chamber. 3. A chamber holder according to claim 2, wherein the inlet has filter means such that the fluid passes through the filter means before contacting the immunoadsorbent. 4. A chamber holder according to claim 1, wherein the substrate is made of beads having an average diameter in the range of 10 to 500 microns. 5. Chamber holder according to claim 4, wherein the support means is a porous plug. 6. The chamber holder of claim 5, wherein the pores in the plug are smaller than the diameter of the beads. 7. The chamber holder of claim 4, wherein the beads include a surface that is porous and a solid core associated with the porous surface, and the barrier coating is associated with the porous surface. . 8. Claims in which the substrate comprises a core and a population of solid particles having an external surface of small diameter and overall large surface area compared to the core, the external surfaces of the solid particles being coated with a barrier. The chamber holder according to item 1. 9 Claim 8, wherein the polymer is dextran
Chamber holder as described in section. 10. The chamber holder according to claim 6, wherein the pores in the plug are 10 microns or less. 11. The chamber holder of claim 2, wherein the inlet includes means for distributing the flow of fluid as it enters the fluid chamber. 12. A substrate consisting of refractory particles stable against dehydration and decay, a water-insoluble polymer chemically bonded to the substrate, and an antibody chemically bonded to the polymer to bind a specific antigen. An immobilized immunoadsorbent consisting of . 13. The method of claim 12, wherein the substrate has a high surface area external surface, the water-insoluble polymer has hydroxyl groups, and the antibody is attached to the polymer by an imidocarbonate bond. Immobilized immunosorbent. 14. The immobilized immunoadsorbent of claim 13, wherein the polymer is dextran. 15. Claim 14, wherein the particles are impermeable glass spheres coated with an outer porous skin of microspheres.
Immobilized immunoadsorbent as described in Section. 16 The spheres are 5 to 500 microns in diameter and the microspheres are 5 millimicrons to 1 micron in diameter. An immobilized immunoadsorbent according to claim 15.
17. The immobilized immunoadsorbent according to claim 16, wherein the surface area of the substrate is 0.8 to 1.0 m^2/g. 18. The immobilized immunoadsorbent of claim 12, wherein the polymer is attached to the particles by silane bonds and the antibody is attached to the polymer by imidocarbonate bonds. 19. The immobilized immunoadsorbent of claim 12, wherein the antibody is capable of releasably binding thereto an antigen specific for the antibody. 20 To attach a water-insoluble polymeric material, a substrate consisting of refractory particles that is stable against dehydration and disintegration is treated to chemically activate it, and the polymeric material is attached to the activated substrate. and chemically activating the polymer to bind the antibody, and chemically applying an antigen specific for the antibody to the activated polymer matrix. A method of forming an immobilized immunoadsorbent, comprising conjugating a population of bound antibodies. 21 Known amounts of a labeled antigen and an unknown, unlabeled antigen are contacted with an immobilized immunoadsorbent having bound antibodies specific for the antigen to detect the labeled and unknown antigens. binding a portion, thereby forming a bound fraction and an unbound fraction, and measuring the concentration of the unknown antigen as a function of the bound or unbound fraction, or both; A mixture of a known amount of labeled antigen and an unknown, unlabeled antigen is used in radioimmunoassays, in which the bound fraction is released from the immunoadsorbent by washing it with an elution medium. an antibody that is specific for the antigen and that is covalently bonded to a water-insoluble polymer that is chemically bonded to a substrate consisting of refractory particles. A method characterized by continuous flowing. 22. The radioimmunoassay method according to claim 21, wherein the water-insoluble polymer has a hydroxyl functional group. 23. The radioimmunoassay method according to claim 22, wherein the polymer is dextran. 24 Claim 21, wherein the substrate is superficially porous
Radioimmunoassay as described in section. 25. The radioimmunoassay method of claim 24, wherein the polymer is bonded to the substance by a silane bond, and the antibody is bonded to the polymer by an imidocarbonate bond.