JPS63500893A - Methods for attaching biological materials to solid substrates - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for attaching biological substances to solid substrates The present invention provides methods for attaching biological substances to solid substrates. In particular, but not exclusively, biological agents such as antigens and/or antibodies may be used. Concerning methods for attaching physical substances to solid phase substrates used in solid phase immunoassays . The invention also relates to products produced by use of the method.

Attaching one of the partners in an immunological reaction to a solid support is , facilitating quantitative and/or qualitative measurement of the reaction. In general, par Immunization of one side of the toner facilitates separation of reactive components from unreacted components. . In effect, the immobilized partner in the reaction captures its complementary reactant from solution. and binds it to a solid phase. Once this binding to the solid phase is achieved, the presence of Can be measured quantitatively or qualitatively.

Up until now, immobilization of one of the partners in an immunological reaction has been performed using a variety of different methods. It's done in a way. Support materials used include cellulose, agarose, and dextrose. Thorin, polyacrylamide, glass, rubber, and nylon, polystyrene, Includes plus decks such as polyester. direct, such as those that result in covalent bonds; Chemical bonds can also be used. Binding either antigen or antibody to supporting material The chemical method consisting of glutaraldehyde, cyanogen bromide , carbodiimides and aminoalkylsilanes. these techniques The techniques are well known to those skilled in the art.

Binding of antigens or antibodies to plastic surfaces can be achieved by direct adsorption. Can also be done. Although this is a simple coupling method, it has some drawbacks. antigen or The adsorption of antibodies onto surfaces is achieved through hydrophobic interactions and van der Waals forces. will be accomplished. These may elute from the surface during handling of the comparator support. (1 )reference.

When using a fixed support such as a microtiter plate, binding of various antigens is possible. The ability to combine may vary depending on its biochemical composition.

In addition, the binding strength of the antigen to such a plate is determined by the binding strength of the antigen to the various wells on the plate. can vary greatly between See (2).

Techniques to overcome these problems include preactivation of plastic surfaces. It will be done. Two chemical methods of preactivation are described in European Patent No. 83111144 and and European Patent No. 84103367. These methods include Phenylalanine lysine copolymer and diphenylene-bis-diazonium Preactivation with each compound is included.

Physical method of plastic microtiter plate activation is European patented No. 82102257. This method requires a microtiter plate. It involves exposure to gamma radiation. γ irradiation produces better bonding ( and also sterility) is obtained, leading to the presence of certain free radicals/bonds. (3) Reference Light.

In addition to soluble substances such as antibodies and antigens, they can be used in immunological or biological reaction systems. It is also necessary to immobilize the cell extract or extracts used themselves. . Binding can also be facilitated using methods known to those skilled in the art. See (4). child These technologies generally include glutaraldehyde, poly-L-lysine, and phenylalanine. Lysine copolymer or lectin [Glycine m ax), Limulus Polyester sculinaris) and Phaseolus vulgaris (Phaseolu s vulgari S) and other sources. Pretreatment of the body support is included. In addition, cells are first placed on a solid support and The binding can be achieved by binding a specific antibody to a support.

Binding of antibodies can be accomplished as described above.

The coupling methods described above all rely on the presence of reactive groups on the substrate.

If not included in the binding method, the bound antigen, antibody, cell, etc. Can tolerate non-specific binding of other substances used in subsequent steps of the immunoassay method? Therefore, the reactive groups must be neutralized before use in immunoassays. This non-existence of matter Specific binding leads to high basal readings and therefore limits the sensitivity of the test method. limit The biochemistry of the biological material also dictates the specific binding methods that can be used. determined, and therefore effective binding of all types of biological substances to specific substrates. There is no permanently applicable bonding technique that can be applied.

According to the present inventors, binding of biological materials to solid substrates can be achieved by ion beam or bombardment. It has now become clear that this can be achieved through the use of law.

According to the invention, (i) subjecting the solid phase substrate to ion bombardment; (ii) treating contacting the substrate with biological material to be attached thereto. Provided is a method for attaching a biological material to a solid substrate, the method comprising: be done.

The present invention also provides products produced by the method described above.

In this embodiment, the invention provides a solid phase substrate, a surface treated by ion bombardment and and a solid phase product consisting of biological material attached to the treated surface.

Suitable biological materials for use in the present invention include antigens, antibodies, immunoglobulins, e.g. Protein substances such as IgG, IgM and IgE, and enzymes, as well as red blood cells the cell membrane, the cell itself, e.g. bacteria and viruses, and hormones, e.g. thyroids Cellular substances such as stimulating hormone (TSH) are included. Suitable solid phase substrates include: In particular, but not exclusively, plastic materials such as polypropylene, mylar, polymer Listyrene, Latex and Teflon: Glass and Silica: and Cellulose and cellulose derivatives.

Through the use of ion beam bombardment methods, we have achieved high affinity, stability and It has become possible to reproducibly and specifically bind biological substances to solid-phase substrates.

The ion beam bombardment method is generally well known (see (5)), but this method It is not used to pre-treat substrates for biological attachment.

For example, U.S. Pat. , all due to ion bombardment in the formation or deposition of metal films and similar materials onto substrates. (See also (6) of Bode and Sandgren). (see). However, these prior disclosures do not address the issue of attachment of biological materials to substrates. It does not contain any literature regarding the use of this technique.

The process of ion bombardment of a solid-phase substrate may involve, for example, bombarding the substrate with a beam of energetic Ar+ ions. This can be done by applying a shock with a drum. to such treated substrates. The exact mechanisms leading to strong adhesion of biological materials are currently not fully understood. However, due to ion bombardment, one or more It is thought that the following effects can be obtained.

(a) removal of surface impurities by sputtering; (b) adhesion to biological materials metastable chemical “dumpling” bonds, and CC) Modification of surface composition by preferential sputtering.

The effect of ion bombardment is the effect of mild ionizing radiation (gamma rays, X-rays, high-energy electrons, ・Irradiation, etc.) compared to known interactions with polymeric materials. Very specific. See (7). A large amount of ionization density (blocking) is generated by ion bombardment. Because it is possible to obtain atomic recoil (stopping ability) and atomic recoil, it is possible to use mild ionizing radiation or or create metastable chemical surface states/compositions that cannot be achieved by conventional equilibrium chemistry. be able to. In this sense, the ion bombardment method can be obtained using mild ionizing radiation. Not only similar effects but also a large number of novel metastable chemical states (dumpling bonds) It is considered to be very specific in that it can also generate . This characteristic point can be used for various The binding orientation of several types of biological materials to several different types of plastics The above results seem to bring about universal success with ion bombardment.

Since the ion beam/impact technology itself is well known, this technique in the present invention and method is not applicable. This process can be accomplished using any standard equipment. See (5).

After bombardment with the in-phase substrate material, biological material is then contacted with the treated substrate. Book The inventive specific binding method can be performed at room temperature and does not require any external chemical binding agents. It doesn't even need . In this respect, there is a direct relationship between the biological material and the treated substrate material. Direct binding is obtained, and the biological material is not irradiated or chemically treated during the attachment process. Since it is not chemically treated, it cannot be degraded or destroyed in any way.

As described in detail below, in one embodiment of the present invention, the method of the present invention is used. The red blood cell membrane was transferred to a transparent plastic solid phase substrate chosen as the test substrate, i.e. A first substrate which is polypropylene (which has no reactive groups) and a matrix a second substrate, which has some reactive groups. energy After bombardment of the plastic substrate with the Ar + ion beam, a suitable liquid is then applied. Surface of red blood cell membranes in colloidal suspension in the body treated with ion bombardment in a system (vacuum) Inject on top. The fluid spreads rapidly to form a thin film, and the red blood cell membrane tightens against the substrate surface. Because it is bonded to the force, the film will not peel off from the surface even during long-term ultrasonic treatment underwater. It has no effect at all.

Furthermore, the characteristic points of the present invention will be clarified from the examples shown below, but these are This does not limit the invention in any way. In these examples, polypropylene, Small pieces of Teflon, polystyrene and mylar (1czx, 1.5CIl+) was cleaned with trichlorethylene, then methanol, and air dried. sample Installed in an ion bombardment chamber (Figure 1) evacuated to a pressure <1xlO-5 Torr. I got it. The sample was heated to 60 KeV with a dose range of 5xlO"-5xlO" ions/cIR'. bombarded with Ar+ or He+ ions. Impact on half of the castle owner of the rectangular small piece sample In addition, it was used in the red blood cell experiment described in Example 1. The annular disk covers the entire clay. and used in experiments described in other examples. Some shocks and parameters tested in detail The meter is based on ion dose, draw rate, ion species and the surrounding post-shock storage environment (biological (until contact with target substance). Extensive reproducibility checks were performed.

Red blood cell membranes were prepared from fresh anticoagulated blood by calcium phosphate coprecipitation method. . See (8). Add sodium azide to the sample at a final concentration of 0.1%. Bacterial contamination during the adhesion process to the tick substrate surface was suppressed.

B. Attachment of cell membrane to substrate A piece of polypropylene was treated with Ar+ ions using the apparatus illustrated in FIG. small The sample holder 1 is placed in a target chamber 10 in which the entire system is kept in vacuum. 1 and apply a shock with accelerated Ar+ ions 12 through the aperture plate 13. .

After the ion bombardment of the small piece, the sample holder 11 is placed next to it via the air lock valve 20. It was moved to the airlock 21 in contact without disturbing the vacuum state (Fig. 1). air In Lock 21, a controlled volume of red blood cell membrane/calcium phosphate suspension is then added. The suspension was injected onto the plastic piece through the biological material inlet 22. liquid spreads rapidly to form a coating with impact clay without wetting the non-impact area of the polypropylene. to be accomplished. The plastic piece was removed from the airlock and excess liquid was removed. . After air drying, the adhesion of the red blood cell membrane/calcium phosphate mixture was evaluated. The pieces were evaluated by immersing them in water and then subjecting them to ultrasonic cleaning for 5 minutes. the small piece It was air dried and then examined under an optical microscope. Figures 28 and 2b each show polypropylene Impact and non-impact zone results are shown on Len and Mylar.

The attached film/calcium phosphate mixture appears dark in these micrographs. film/ Calcium phosphate mixture binds to the impact zone and not to the non-shock untreated zone That is clearly shown.

Example 2 IgG was extracted from human serum using Sepharose-Staff A affinity column chromatography. Purified by matography. Aliquots of purified IgG were added +2 according to the Chloamine T method. Radiolabeled with 51. Radiolabeled IgG is added to purified IgG as a tracer. In addition, a ratio of labeled IgG:unlabeled IgG of 1:20 to 1:30 was obtained.

Next, using this radiolabel substance, ion bombardment of IgG on the plastic surface was performed. The effects of IgG concentration, buffer pH, and reaction time on the interaction with were investigated.

In these experiments, IgG was tested in phosphate buffer at pH 7.0 and in phosphate buffer at pH 9.6. diluted in carbonate buffer to concentrations ranging from 10 to 100 μ9/leaf. 40 μg of sample 0.6 CJI diameter wafer molded onto a piece of ion bombarded and untreated plastic. added to the file. After incubation at room temperature, wash the scissors with deionized water and remove the wells with the scissors. The pieces were cut and the radioactivity due to the attached 1gG was counted using a 7 counter.

The buffer p)l has a substantial effect on the amount of IgG bound to the ion bombarded plastic surface. It was found that it had no effect. If the reaction time is increased (18-20 hours) , allowing the attachment of larger amounts of IgG. If the concentration of IgG is increased, the ion bombardment will be increased. A larger amount of IgG adheres to the plastic.

Table 1 shows various forms of ion bombardment and various positive effects on IgG uptake. Showing the effects of tics. As is clear from this table, ion bombardment causes IgG to be It can increase the uptake into the plastic surface and the effect depends on the material used. It was found to vary depending on the species and the type of plastic used.

References Table 1 Ion Beam-Induced Enhancement of IgG Attachment to Various Plastic Substrates Polypropylene 288 349 Teflon 128 42 Polystyrene 11389 The foregoing examples are used only to illustrate the invention and are not intended to be broadly interpreted herein. Various modifications and variations may be made without departing from the scope of the invention as described in It is recognized that what can be done there.

-Titanium (Shield-Holland, Amsterdam, 1973) (1) Engva Lu Yi, Method in Enzymology, 1980, vol. 70, 419- 439 pages (2) Chevsum B Nis and Unmarked JR, Inc. Tanto EL I SA Lancet, 1978, i: 161 pages (3) Du Plessis T.A. and Schnautz N.C., Journal ・Of Oil and Color Chemistry Association (J, Oil Co1. Chem, As5oc, ), 1975, vol. 58, 85-89 page (4) Huuser C.H., Stocker J.D. and Guy Slur R.H., Cell-to-Plastic Attachment Theory, 1 981, vol. 73, p. 406 (5) Darnary C., Freeman G.E. Ichi, Nerman Earl Nis and Stephen Jay, Ion Imblanc Chi(6) Bodeby and Sandgren J.I., evaporated titanium pot. Adhesion to polyethylene: Effect of ion bombardment pretreatment, Journal of Vacuum ・Science and Chikunoroshii A.

Volume 2, Issue 4, to December, 1984 (7) Frank H.B., Polypropylene (McDonald's Chikunoroshii) , London, 1968) Chapter 5 (8) Krakowski D. B., Hemoglos Calcium phosphate coprecipitation method for bottle-free human red blood cell stroma production, Australia Ann Journal of Medical Laboratory Science, 1984 , vol. 5, pp. 124-126. international search report ANNEX To THE I) JTERNATIONAL 5EARCHRE PORT　NINTERNATIONAL　APPLICATION　No, PCT/AU 86100235END OF ANNEX

Claims (9)

[Claims] 1. (i) subjecting the solid phase substrate to ion bombardment; and (ii) subjecting the treated substrate to ion bombardment; the process of contacting the biological material to which it is attached; A method for attaching a biological substance to a solid substrate, the method comprising: 2. The substrates include plastics, glass and silica, and cellulose and cells. The method according to claim 1, wherein the method is selected from the group of substances consisting of rose derivatives. 3. The plastic material is polypropylene, polystyrene, Teflon, Mylar. 3. The method of claim 2, wherein the method is selected from the group consisting of 4. The biological substances include immunoglobulins, allergens, cells and cellular substances, enzymes. and any one of claims 1 to 3 selected from the group consisting of hormones. The method described in. 5. The biological substance is selected from the group consisting of IgG, IgM and IgE. 5. The method according to claim 4, wherein the globulin is a globulin. 6. The biological substances consist of red blood cell membranes, white blood cells, platelets, bacteria and viruses. 5. The method according to claim 4, wherein the cell or cell material is selected from the group consisting of: 7. Claim 4, wherein the biological substance is glucose oxidase. How to put it on. 8. Produced by the method according to any one of claims 1 to 7. product. 9. a solid phase substrate, its surface treated by ion bombardment and the treated surface; A product characterized in that it consists of attached biological substances.