DK147809B - IMMOBILIZED IMMUNOADSORBENT - Google Patents

Description

147809

The invention relates to an immobilized immunoadsorbent, in particular to radioimmunoassay, which immunoadsorbent comprises an adsorbent and antibodies attached thereto.

A radioimmunoassay is an analytical method based on an antigen's competition for (affinity for) antigen binding sites on antibody molecules. In practice, standard curves are determined from data obtained on the basis of a series of samples, each having (a) the same known concentration of labeled antigen and (b) different, but known, concentrations of unlabeled antigen. Antigens are labeled 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 attached thereto, and then determined by a suitable detector, such as a gamma or beta radiation detector, the percentage content of either bound or freely labeled antigen or both. This procedure is repeated for a number of samples with different known concentrations of unlabeled antigens and the results are plotted in a diagram. The percent content of bound tracer antigens is plotted as a function of antigen concentration. Typically, the relative amount of the tracer antigen bound to the antibody decreases with increasing total antigen concentration. After a standard curve is prepared, it is used to determine the concentration of antigen in samples subjected to analysis.

In the actual analysis, the sample whose antigen concentration is to be determined is mixed with a known amount of trace antigen. The trace antigen is the same antigen known to be present in the sample but which has been labeled with an appropriate radioactive isotope. The sample with the tracer is then incubated in contact with the antibody. Then, a count can be made by a suitable detector which, by counting, determines the free antigen remaining in the sample. The antigen bound to the antibody or immunoadsorbent can also be determined similarly by counting. Then, from the standard curve, the concentration of the antigen in the original sample is determined. Finally, the antibody or immunoadsorbent mass is discarded.

To determine the percent content of antigen bound to the antibody (bound antigen) and / or percent content which remains free or unbound, it is first necessary to separate the sample into a bound antigen-containing fraction and a fraction containing only free antigen. A well-known method for this is to add dextran-coated charcoal to the mixture. The dextran allows unbound antigen of lower molecular weight than for 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 consists in introducing to the mixture another antibody which selectively precipitates the first antibody (with bound antigen) and thus leaves only free antigen in solution. A classification for obtaining appropriate fractions of free and bound antigens is then carried out by separating the precipitate from the supernatant by centrifugation or other appropriate means.

Some researchers have used a method whereby the antibody binds to the inner walls of a plastic container, where the container is filled with the antigen-containing sample, where the container is placed for an incubation period typically between 4 and 72 hours and where the free antigen is separated from the bound. antigen by draining and rinsing the container, leaving only here? the antibody is left and bound antigen. A later developed method consists of preparing the immunoadsorbent by binding the antibodies to soluble crosslinked dextran. The immunoadsorbent and the antigen-containing sample are then incubated and the dextran with bound antigen is appropriately separated from the solution.

By all of the aforementioned methods, the percentage of labeled antigen is determined in either the bound antigen-containing fraction or the free antigen-containing fraction or both, and the standard curve is used to determine the antigen concentration.

Then the immunoadsorbent is discarded.

Although the above-mentioned radioimmunoassay methods have proven to be valuable tools and have gained widespread acceptance, they still leave something to be desired as the antibody (immunoadsorbent) is consumed in each assay and therefore must be discarded. Furthermore, the known batch-type methods are and several reagents are added to the antibody in reagent tubes in which the various steps, such as incubation, rinsing and the like, are carried out, resulting in a slow and expensive operation.

It has been proposed to use the same immunoadsorbent for multiple assays by releasing from the immunoadsorbent antigen bound to the antibody mass, the latter being immobilized in the adsorbent, i.e. that a selective and stoichiometric release of all the antigen into the immunoadsorbent is made after the assay is completed. The invention relates to such a reusable immunoadsorbent.

It is well known from the literature that antibodies can be isolated using immunological adsorbents, and that this method is useful for isolating and purifying antibodies, rather than for quantitative determination thereof, see Campbell et al., Proc. National.Acad.Sci. U.S. 37 (1951) 575.

The use of an antibody coupled to an insoluble polymer for extraction of specific antigens for the purpose of isolating and purifying the same is described in Weetall et al., Biochem.Biophys. Acta. 107 (1965) 150-152.

Porous glass has been described as adsorbent for the immobilization of enzymes, see Weetall, Biochem.Biophys.Acta. 212 (1970) 1-7. According to this literature, glass was treated with gammaaminopropyltriethoxysilane and the isothiocyanate derivative was prepared by treatment with thiophosgene. The enzyme was coupled to the isothiocyanate derivative. The literature site also describes the preparation of an arylamine derivative by reaction of alkyl amine glass with p-nitrobenzoyl chloride followed by the use of sodium dithionate to reduce the nitro groups. The arylamine glass was then diazotized and the enzyme coupled thereto.

Weetall has in Biochem.J. (1970) 117, 257-261, also disclosed the use of the antibody bound to porous glass by a silane coupling agent, the immunoadsorbent being used to isolate and purify specific antigens. However, the data cited show that a recycled column in which the antigen was eluted from the immobilized antibody immunoadsorbent gave erroneous results, with the recovery of released antigen varying between 74 and 100%.

Also, reference is made to the disclosure to U.S.A. patent no.

3652761. Although the described system has been useful as an isolation system, it suffers from considerable disadvantages as a useful tool in quantitative assays, thereby causing substantial stoichiometric release of the antigen.

In the description to U.S.A. Patent No. 3,555,143 discloses radioimmunoassay methods whereby an immobilized immunosorbent is used only once and then discarded. The immunoadsorbent is a dextran ("Sephadex" G 25 superfin) crosslinked with glycerin ether bridges and substituted with p-nitrophenoxyhydroxy 4 propyl ether groups. The nitro groups are reduced to amino groups using sodium dithionite. "Sephadex" substituted with p-aminophenoxyhydroxypropyl groups is then treated with thiophosgene to form "Sephadex" substituted with p-isothiocyanate - phenoxyhydroxypropyl groups and the antibody binds to the latter substituted product.

One reaction which is widely used to render a protein insoluble consists in providing a covalent bond between the protein and a cyanogen bromide-activated cellulose matrix material. The mechanism of such activation is disclosed in Bartling et al., Biotechnology and Bioengineering, Vol. XIV (1972) 1039-1044.

The descriptions of U.S.A. patents Nos. 3,502,888, 3,639,559 and 3,720,760 also disclose methods of interest.

When an immobilized immunoadsorbent is to be used only once and discarded, the long-term properties of the adsorbent are not essential. Thus, materials such as "Sephadex11 (dextran) or" Sepharose "(particulate agarose product) satisfactorily act as adsorbents for antibodies bound thereto, as described in the specification of the above-mentioned United States Patent No. 3,555,143. repeatedly, certain problems arise.

One of these problems is the tendency of "Se-phadex" and "Sepharose" type products to dehydrate, i.e. the gel collapses and packages to such an extent that the flow through the mass is substantially reduced and the ability of the antibody to bind the antigen is altered. Hereby, the reproducibility and stability of the immunoadsorbent for repeated use is affected.

Glass and other solid inorganic materials constitute a desirable alternative as they can be tipped into bead-like bodies, thereby providing a better flow and lighter gasket to form a column-like arrangement. Such materials do not collapse and are not subject to dehydration during long periods of use. Although glass is a desirable alternative, glass-type products also have certain disadvantages. One of the problems is getting the antibody to bind to the adsorbent sufficiently. Either an insufficient initial binding appears to elicit the activity required for quantitative analysis, or the activity changes over the life of the immunoadsorbent by unwanted release of antibodies.

When glass is strongly porous as the glass used in the method discussed in connection with the above-mentioned Weetall article, there is such a large active glass surface area that an abundant binding of the antibody takes place, but at the same time a non-specific binding of the antigen. Thus, the antigen bound to the glass is not completely released. That is, instead of a stoichiometric release occurring with each application, as is necessary in a quantitative analysis, the release properties are variable and unpredictable. This is corroborated by the findings in the said Weetall article. Since such glass generally has a void fraction of 96%, there is a significant active surface area in the glass that is not occupied by the antibody and serves as an antigen binding site.

Another difficulty with highly porous glass products is that there are a large number of countersinks in the pores, which results in a containment of the countersinks and a slow release due to a slow diffusion in the countersinks. When a rapid reaction is required, e.g. In automated equipment, the diffusion of the reactants is a rate limiting factor and, as you know, the diffusion can be a relatively slow process. Thus, although not bound to the substrate, the diffusion of the antigen is relatively slow, and in the case of fast automated assay equipment, effective binding of the antigen rather than a rapid and stoichiometric release occurs effectively.

Porous support materials in the surface are known for use in chromatography, cf. eg. the description of U.S.A. patent no.

3,505,785, which describes a product that is traded under the trademark "Zipax". Such beads for use as chromatographic column packing consist of a series of separate macroparticles having impermeable cores and to which are irreversibly bound a coating of a series of successively absorbed monolayers of one's colloidal microparticles. Spherical micro glass beads with a diameter of approx. Thus, 30 microns comprise an outer porous surface shell having a thickness of approx. 1 micron. Such material will be used as an adsorbent to provide a substantial surface area available for the desired activity, but the adsorbent must be appropriately prepared to ensure a suitably rapid reaction, as well as a stoichiometric release.

The provision of a reusable immunoadsorbent, which stoichiometric releases the antigen after each assay, is thus highly desirable. If such an immunoadsorbent is substantially also free to dehydrate and settle so that the flow conditions through the pulp are satisfactory over the life of the immunoadsorbent, a substantially improved reusable immobilized immunoadsorbent is provided.

According to the present invention, there is provided an improved immobilized immunoadsorbent for use and renewed use by radioimmunoassay methods. This immunoadsorbent is characterized in that the adsorbent consists of a mass of solid particles that are resistant to dehydration and collapse, each particle having an outer surface having a large surface area, that a water-insoluble polymer is chemically bonded to the outer surface of the solid. particles and that antibodies are bound to the polymer with covalent bonds to bind a specific antigen.

A preferred embodiment of the immunoadsorbent according to the invention is characterized in that the solid particles are surface porous, heavy-melting particles, each particle comprising an impermeable core to which sufficient layers of microparticles are bonded to form an outer porous coating on the core.

As mentioned, a water-insoluble polymer is chemically bonded to the outer surface of the solid particles. The polymer may be bonded to this surface by treating the solid particles for the purpose of forming an aminoalkylsilane derivative followed by a treatment to form an isothiocyanoalkylsilane derivative to which the polymer attaches. A typical example of useful polymeric materials is dextran.

The dextran acts as a barrier covering the active sites on the adsorbent to which the antigen can be bound in a side-way that interferes with subsequent assays. Since the immunoadsorbent of the invention, using an elution medium which separates the antigen from bound antibodies, is used and reused, the release of any antigen which may be bound to the adsorbent causes errors in subsequent analyzes. Such errors occur as a result of the unpredictable and unknown amount that is retained or released. Thus, the polymer effectively acts as a barrier preventing the adsorbent from binding antigen.

The polymer is then activated to bind the antibody through a covalent bond by treatment with cyanogen bromide. Reaction 147809 7 the mechanism for this is described in the above article by Bartling and co-workers.

The immobilized immunoadsorbent formed can then be placed in a chamber holder.

The invention will now be described in more detail with reference to the drawing, in which the figure is a perspective view which partly in section schematically illustrates an adsorbent according to the present invention.

The improved immobilized immunoadsorbent of the invention is in principle intended for use in radioimmunoassay methods.

Typical examples of materials that can be quantitatively measured using the adsorbent of the present invention are the following: estriol, digoxin, digitoxin, testosterone, estradiol, aldosterone, progesterone, cortisol, 11-deoxycortiosterone, 11-deoxy-cortisol, thyroid hormones, such as thyroxine (T ^), triiodothyronine (Tg), polypeptides such as angiotensin, TSH (thyroid stimulating hormone), ACTH, GH (growth hormone), HP (human placental lactogen), parathormone, calcitonin, insulin, glucagon, polypeptide proteins such as CEA ( carcino-embryonic antigen), alphaphetoprotein, interferon, viruses such as Australian antigen, vitamins such as D and B 2, folic acid and drugs such as dilantin and barbiturates, to name but a few.

Antisera for the above antigens are well known, as are also labeled antigens which occur in the form of materials 125 labeled with radioactive isotopes, generally in the form of I isotopes 3 or H isotopes.

The immobilized immunoadsorbents of the invention comprise an adsorbent with which the antibodies are relatively permanently linked. During use, a sample containing unlabelled antigen with a known concentration of labeled antigen is contacted with the immobilized immunoadsorbent placed in a chamber holder. Hereby, part of the mixture of labeled antigen and unlabeled antigen is bound to the specific antibody bound on the adsorbent. Then, the unbound antigen or bound antigen or both are measured and the concentration of unlabeled antigen is determined from standard data.

Then, the immobilized immunoadsorbent is rinsed with an appropriate aqueous solution containing solvents such as methyl alcohol, isopropyl alcohol or ethyl alcohol, and dimethylformamide to induce a stoichiometric release of the bound labeled and unlabeled antigen from the immobilized immunoadsorb. The rinsing or elution operation effectively regenerates the immunoadsorbent for reuse, and then the same immunoadsorbent can be used repeatedly to determine the antigen to which the immobilized antibody is specific, with leaches as described above between each use.

Since the antigenic material is flowed into a chamber supporting the immunoadsorbent to be reused, the flow character of the adsorbent should be such that contact between the antibodies bound to the adsorbent and the antigen mixture is achieved. Furthermore, the adsorbent must be of such a type that it does not interfere with the release of bound antigen while retaining the bound antibody. Reproducibility, stability and speed are some of the advantages associated with the improved method, and the adsorbent must therefore be such that sufficient antibody binding activity can be obtained with the antigen binding sites present. The adsorbent is therefore particulate and formed from a mass of separated particles, thereby achieving an improvement in the desired flow properties not only for the sample mixture containing labeled and unlabeled antigen, but also for the rinse and elution medium.

Particulate materials capable of providing the necessary antibody activity are well known, e.g. "Se-phadex", "Sepharose" and porous glass. Materials such as "Sephadex" and "Sepharose" are gel-type materials, and over extended use, they tend to dehydrate, resulting in a coincidence with resulting gasket, which impedes flow.

Materials such as porous glass are so active that the antigen binds to the glass and is not released from it.

Thus, an important feature of the immunoadsorbent of the invention is the formation of a barrier coating over each particle, the barrier coating serving to provide a mask which prevents the potentially active sites on the particle from irreversibly binding the antigens.

The barrier also acts as an immobilized component of the adsorbent to which the antibodies can be attached.

Since assays are performed in aqueous and non-aqueous solvents, the barrier coating is preferably insoluble and is not adversely affected by the solvents and the solutions used in the process. Water-insoluble polymeric materials, such as dextran, are preferred for use with the adsorbent of the invention.

The adsorbent itself is resistant to dehydration and collapse. A rapid mass transfer at relatively high flow rates is a function of the shape of the adsorbent and the packing conditions of the chamber holder. A preferred material has a controlled surface porosity comprising surface porous, highly fusible particles formed from discrete macroparticles with impermeable non-porous cores and to which is coated a series of successively adsorbed, monolayers of uniform, inorganic body particles.

In the figure illustrating the invention, a surface porous, heavy-melting particle 10 forming the adsorbent comprises a core 12 in the form of a macroparticle forming an impermeable, non-porous core. The core 12 is shown to be spherical in shape. is preferred for the sake of the gasket. The core in the form of a sphere has a diameter of between 5 and 500 microns and is composed of glass but may also consist of sand, ceramic materials and the like.

The cores are preferably of uniform size, i.e. that they are all within approx. 50% of the average diameter. Adhered to the core 12 are a series of layers of body parts 14 which form an outer porous coating. The size of the body types can be between 5 millimicrons and 1 microns, and the number of layers can be between 2 and 30.

The body types may consist of amorphous silica, alumina, thorium oxide and the like.

The adsorbent has a relatively large surface area due to the porous coating 15 and is relatively free of pores in the core material. With spherical bodies with a total diameter of 30 microns and a porous shell of 1 micron, a surface area of p between 0.8 and 1.0 m / g is obtained with a density for a packed layer of 1.5 3 g / cm. The regular geometric structure, the stability to dehydration and coincidence, and the voluminosity make the above material exceptional as adsorbent.

However, if used in the form described as directly adsorbent to the antibody material, such material tends to contain active sites which tend to bind the antigen mixture or a component thereof in such a way that it does not can be released. This problem can be quite troublesome when the immobilized immunoadsorbent is reused. Since the accuracy of assay and the rate at which the assay is performed depends in part on the antibody's ability to bind the antigen and stoichiometric to release the same when rinsed, any undissolved antigen may adversely affect the accuracy of a subsequent assay.

Although a background count could be made, this is not entirely satisfactory as the detention-release phenomena appear to be disjointed and unpredictable.

According to the invention, this tendency can be eliminated by using a barrier coating bonded to the adsorbent by means of a silane coupling agent, i.e. the polymer is bonded directly to the outer portions of the adsorbent surface with silane bonds. The polymer is then activated by treatment with cyanogen bromide, which covalently binds the protein (antibody) to the polymer-activated particulate adsorbent. Not only does the polymer coating act as a barrier that effectively masks latent active sites on the subject adsorbent, but also provides an active surface to which the antibody can covalently bind, obtaining a bond which is considered to be relatively strong.

In a typical process for preparing the adsorbent of the invention, 12 g of a particulate material (surface porous, heavy-melt particles of 30 microns diameter, as described above) was added to a 500 ml flask to which 20 ml (18.84 g) was added. 3-aminopropyltriethoxysilane and 180 ml of toluene.

The mixture was refluxed for 22 hours to form the aminoalkylsilane derivative of the particulate material. The derivative was filtered off, washed with 200 ml of toluene while it was on the filter carrier material and air dried. This treatment was followed by another wash with 100 ml of chloroform and another air drying.

The isothiocyanoalkylsilane derivative was prepared by treating the prepared aminoalkylsilane glass derivative with 16.6 ml (25 g) of thiophosgene and 150 ml of chloroform. The reaction vessel was protected from light and a reflux was carried out for 18 hours to give the described derivative which was filtered off, washed in chloroform and air dried.

The result of the described measures was that an "activated" adsorbent was prepared to which the water-insoluble polymer can be bonded.

U7809 11

According to the invention, it is preferred to use dextran having a molecular weight of 70,000, although other materials may also be used.

200 ml of a 1% solution of dextran in 0.1 M sodium hydrogen carbonate with a pH of 9.0 was added to the "activated" adsorbent. The mixture was stirred for 3 hours, filtered, washed with 300 ml of water and then with 100 ml of acetone and air dried to give 11.6 g of polymer-coated particulate adsorbent.

The remaining steps include the activation of the polymer-coated adsorbent and, optionally, the purification of the antibody and the binding of the antibody to the coated surface. This is sometimes referred to as the conjugation of the antibody to the adsorbent produced.

To activate the dextran, 20 g of cyanogen bromide was dissolved in 200 ml of water. Cyanogen bromide is quite toxic and therefore standard safety precautions are taken. The dextran coated adsorbent (11.6 g) was then added and the mixture stirred. The pH was increased from 3.6 to 10-11 using 23 ml of 6N sodium hydroxide. The pH was kept between 10 and 11 by the addition of 6N sodium hydroxide for 2 minutes. The activated dextran-coated adsorbent was then washed with 400 ml of water, 400 ml of 50% (by volume) water and acetone, 400 ml of 25% by volume of water and acetone and finally 400 ml of acetone. The product was then air dried.

The treatment of the polymer coated adsorbent with cyanogen bromide results in a reaction with adjacent hydroxyl groups on the polymer to form an imidocarbonate which couples with the nucleophilic groups (amino groups) on the antibody to form the carbonic ester by hydrolysis. A rapid hydrolysis of the imidocarbonate in acidic media results in the formation of a cyclic carbonate which is not as effective in bonding as the imidocarbonate. Care must therefore be taken to avoid conditions which promote the formation of a cyclic carbonate.

A simple purification of the antibody prior to conjugation may optionally be carried out as follows: 1 ml of 18% sodium sulfate solution was added to 0.1 ml of the antisera used. The solution was stirred and incubated for 1 hour to precipitate gamma globulins. The resulting mixture was then centrifuged for 5 minutes at 1000 g at room temperature followed by decantation and disposal of the supernatant liquid. The obtained pellets were suspended in 10 ml of 18% sodium sulfate solution with the addition of 0.10 ml of water. The lower mixture was then stirred and centrifuged again. The supernatant was removed by decantation while the pellets formed were dissolved in 0.8 ml of 0.1 M sodium hydrogen carbonate solution.

It is preferred to conjugate the antibody ten! the adsorbent while the antibody is saturated with the antigen to which it is specific. The purpose of this is to achieve improved activity by protecting the active sites on the antibody during the conjugation procedure and thus ensuring that the antibody occupies such a position relative to the adsorbent that active sites are available. The binding of the active sites with antigen allows to some extent an orientation of the antibody which reduces the coverage of the binding sites by the conjugation method.

Thus, a 0.5 ml aliquot of a solution containing 0.1 mg / ml of antigen specific to the antibody was dissolved in an ethanol-aqueous solution (two parts of ethanol and one part of water) and dried in a test tube with nitrogen gas. . The purified antisera dissolved in 0.8 ml of 0.1 M sodium bicarbonate solution or 0.1 ml of antisera in 0.8 ml of 0.1IV sodium bicarbonate solution were added to the test tube containing the dried antigen followed by an incubation for one hour in a closed culture glass. Then, 300 mg of the cyanogen-activated polymer-coated adsorbent was added to the glass containing the antibody solution and incubated for 1 to 3 days at 4 ° C with stirring. During the incubation, a conjugation occurs with the antibody which has the antigen binding sites protected by bound antigen.

After incubation, the suspension was centrifuged at 1000g for 5 minutes and the supernatant was decanted and discarded. The obtained pellets were washed twice with 10 ml of 0.5 M sodium hydrogen carbonate solution. After each washout, the suspension was centrifuged and the supernatant was discarded. The antibody-coated adsorbent was then washed twice with 10 ml of 0.1M acetate buffer with a pH of 4. The antibody-coated adsorbent was then washed with 10 ml of 0.05M phosphate buffer with a pH of 7.5 containing 0.05M sodium chloride and 0 , 5% bovine serum albumin and 0.02% sodium azide as a preservative. The resulting product was then resuspended in 10 ml of the last wash solution and stored at 4 ° C.

The resulting product is rinsed prior to use by assaying one of the above-described solutions to release the antigen bound to the immobilized antibody.

However, during storage, it is preferred that the antigen remains bound to the antibody.