FI58404C - FOERFARANDE FOER VISUELLT PAOVISANDE AV ANTIKROPPAR I ETT VATTENHALTIGT PROV - Google Patents

Description

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Patent · And registered (44) NIhtlylWp * »» J. moonLullulam p * m.-

Patent and registration authorities Antöion uthfd oeh uti. * KrHtM puWk * Nui 30.09. o0 (32) (33) (31) Pyy4 «tty« tuolkws — fefird prior It * t 29.05.7 **

Sweden-Sweden (SE) 7 ** 07139 ~ 0 (71) Pharmacia Diagnostics AB, Box 17, 751 03 Uppsala 1, Sweden-Sweden (SE) (72) Ulf Ragnar Svante Jonsson, Lund, Sweden-Sweden (SE) ( 7 * 0 Berggren Oy Ab (5 * 0 Method for visual detection of antibodies in an aqueous sample - Förfarande för visellt pävisande av antichroppar i ett vattenhaltigt prov

The present invention relates to a method for detecting the presence of antibodies in an aqueous sample, which antibodies are specifically directed against an antigen.

Several methods for detecting and quantifying specific antibodies in biological samples are already known. According to one such method, antigen-coated surfaces of glass, polymer, metal or the like are used. Samples optionally containing antibodies specifically directed against a surface antigen are added to that surface, after which the antibody-antigen complex can be detected in various ways. Thus, radiolabeled antibodies can be allowed to compete with the antibodies in the sample and the radioactivity that is specifically bound to the antigen surface can be measured. A disadvantage of such a technique using radioactivity is e.g. the high time required and the need for expensive and complex equipment, as well as the health risks associated with the use of radioactive materials.

2 58404

It has also been suggested (Adams et al., Journal of Immunological Methods 3, (1973), pp. 227-232) to show an antibody-antigen complex 1) by observing an increase in wettability by applying the action of water vapor to the surface, 2) by staining with protein dyes (such as Coomassie Brilliant Blue R "), and 3) by detecting interference dye transfers on certain reflected surfaces. A common disadvantage of these methods is, among other things, that they cause inaccurate reactions, since a false positive reaction cannot be prevented by adding a sample with a high protein concentration, such as undiluted serum. In addition, it is not possible to perform any analysis of reactive specific antibodies based on their immunoglobulin class, which would be very important in diagnosing e.g. infectious diseases.

It has now surprisingly been shown that the disadvantages of the detection methods previously used can be eliminated by the present invention.

The present invention relates to a method for detecting the presence of antibodies (I) in an aqueous sample, which antibodies are specifically directed against an antigen, the sample being contacted with a flat, preferably planar or tubular surface coated with said antigen to bind antibodies to the surface. to the antigen, after which the antibodies thus bound on the surface are detected, and the invention is characterized in that for said detection of antibodies, the surface is contacted with a suspension containing small, water-insoluble particles to which the biopolymer is directly bound, which biopolymer is capable of specifically binding to antibodies in a non-antigen binding construct, preferably an Fc portion thereof, whereby said particles accumulate in the portion of the surface to which the antibodies specifically bind, thereby forming a clearly detectable effect with the naked eye; va, coating of particles.

The coating of the antigen on said flat surface can take place either by using adsorption forces or by a covalent bond between the reactive groups of the antigen (hapten) and the surface, possibly by functional reagents. As the reaction surface, a flat surface of glass, polymer, metal or similar material may be used, optionally covered with a substance which facilitates the binding of the antigen to this surface.

3 58404

The surface may first be covered with a layer of antibodies specifically directed against the antigen, and the antigen is then adsorbed on that antibody surface. The surface can then preferably be flat or tubular, alternatively formed into small pieces or rods. The surface material may also be formed by thin foils of polymer, metal, etc., optionally fitted to a support of paper, polymer, glass, and the like. One advantage of the reaction surfaces in the form of thin foils is that they allow simple storage and transport of the antigen-coated surfaces, whereby the antigen can be protected against possible denaturation, e.g. by a thin film of a suitable liquid. By "antigen coating" on a surface is meant in this context and in the claims that the antigen is adhered to the surface so that it does not detach from this surface upon contact with the aqueous sample to such an extent that the reactions are disturbed.

As antigens that can be adsorbed or covalently bound to surfaces of glass, polymer, metal, or the like, polypeptide antigens comprising proteins, glycoproteins, lipoproteins such as serum albumin, immunoglobulins, other plasma proteins can be particularly mentioned. microbial polypeptides, etc., polysaccharide antigens such as dextran, other capsular polysaccharides, lipopolysaccharides, lipopolysaccharide protein complexes derived from bacteria and elsewhere, nucleic acids such as DNA and RNA, haptens such as small molecule hormones and viral antigens such as Hepatitis B antigen (HBAg, formerly known as Australia antigen, Au-ag), particulate antigens such as whole bacteria and other microbes. robit unicellular or multicellular parasites, animal cells or suitable (sub-) cellular fragments, exemplified by whole Salmonella, Treponema palladium (syphilis) and Chlamydia bacteria.

The term "biopolymers" as used above and in the claims preferably also means polypeptides, proteins, glycoproteins, polysaccharides, lipoprotein polysaccharide complexes, regardless of which part of the molecule has an affinity for the structure of immunoglobulin non-antigens.

“58404

The biopolymer may be derived from microbes, e.g. bacteria, or may have some other biological origin, and the biopolymer may also be formed by an immunoglobulin (antibodies (II)) directed against an immunoglobulin (actual antibodies (I)). A very suitable example of a biopolymer in the above sense is the so-called protein A derived from Staphylococcus aureus, or a portion of this protein that is polypeptide in nature and capable of binding itself to the non-antigen binding structure of antibodies. Another example of biopolymers having similar properties is a polypeptide derived from Staphylococcus epidermis, as well as biopolymers having the surface structure of certain streptococci. The biopolymer may be bound to the surface of the microorganism that formed it. When the biopolymer is formed by protein A or a part thereof, the particles are very preferably formed from staphylococci, preferably killed staphylococci. -30 (197 * 0) to give, for example, a 10% stock suspension lasting at least one year, which must be diluted to only about 1/2% of the suspension in order to detect IgG antibodies bound on the antigen surface. in phosphate-buffered table salt while adding a suitable surfactant.

According to the invention, the biopolymer may also be bound to small particles of the polymer which are insoluble in water. Examples of such polymers are gel particles containing copolymers of polyhydroxy compounds and bifunctional substances, e.g. copolymers derived from dextran and epichlorohydrin (Sephade®, manufactured by Pharmacia Pine Chemicals, Uppsala), gel particles containing acrylate, etc., gel particles containing acrylate, etc. however, be swellable in water. Binding of the biopolymer to the moieties can also occur by polymerizing the biopolymer, optionally by copolymerization with other polymers, e.g., by rendering insoluble a serum containing antibodies specifically directed against a particular immunoglobulin class or classes. If desired, these small particles can be colored. Preferably, particles of the order of 0.1 to 20, e.g. 0.2 to 10 micrometers, are used.

According to the invention, the biopolymer may be bound to the surface of small particles by bonds of a covalent nature or by adsorption.

5 58404

According to the invention, the bacterial bodies in the surface layer may contain as biolopolymer a biopolymer derived therefrom or a biopolymer derived elsewhere which is bound to the surface of the bacterial bodies due to the natural bonding of the biopolymer and the bacterial bodies and / or by a bond artificially provided. Bacterial bodies with their biopolymers may be treated with an aldehyde, e.g. formaldehyde and / or glutaraldehyde. They may also be heat treated to kill and / or sterilize them.

In particular, staphylococci in protein A can be used to detect IgG antibodies.

If, instead, it is desired to detect antibodies which are incapable of binding to protein A, particles to which other biopolymers which specifically react with these antibodies are attached may be used in accordance with the invention.

According to another embodiment of the invention, antibodies that are unable to bind to protein A can also be detected by exposing a surface antibody-antigen complex to a solution of FgA antibodies reactive with protein A that are directed to some of the antibodies. or against some (sub) classes (e.g., IgA, IgE, and IgM) that do not react with protein A. Protein A-containing staphylococcal reagent is then added and the possible reaction is monitored in the same manner as described above.

According to another embodiment of the invention, the surface of the antigen-bound antibodies (I) can be contacted with a suspension of bacterial bodies on the surface of which antibodies (II) have been directly bound (via the Fc portions) directed against said antibodies (I). ) to see the surface covered with antibodies (I).

Various methods can be used to semi-quantitatively determine antibodies in a sample. For example, a series of different dilutions of a sample can be tested and the result compared with analogous experiments performed on a reference sample. In this case, determine the maximum dilution of the sample which causes a positive reaction.

6 58404

According to a highly preferred embodiment of the invention, the presence of antibodies in a sample can be quantified by adding it to the sample surface outside the antigen-covered part and causing the sample to pass in a certain direction in a zone, preferably a narrow zone, over the antigen-coated surface. After the transfer of the sample, which can be detected, for example, by coloring the sample, etc., biopolymer-containing particles are added to the suspension. The surface which then becomes covered by these particles forms a measure of the antibody content in the sample and can be compared with the corresponding surface of a known reference sample.

To effect the passage of the sample over the antigen-coated surface, the method used in the thin layer gel chromatography technique can be applied, e.g., by adding a thin layer (possibly several adjacent layers) of capillary medium, e.g., particles such as Sephade® G-75 Superfine (cross-linked dextran gel). Chemicals, Uppsala). Fibers of spun or non-spun fibers can also be used as the liquid transfer medium. Of course, prior to consideration, using biopolymer-bearing particles, said medium must first be removed.

It is also possible to provide a linear fluid flow or multiple parallel linear fluid flows across the plate without any solid material support on the antigen-covered surface if the surface is first wetted using a linear line or parallel linear lines and the fluid is flushed in and out slightly suitable capillary material.

The migration of antibodies in the sample can also be effected in the direction over the surface covered by the antigens using electrophoretic technique.

The method of the present invention can also indirectly semi-quantitatively or quantitatively determine the concentration of antigen in a sample by testing mixtures of antigen-containing samples and doses of a standardized amount of antibody for that antigen and then detecting any excess antibody that the antigen in the sample has failed to inhibit.

The method according to the invention can be used to determine antibodies 7 58404 in principle for different types of antigens. Thus, both soluble and insoluble (e.g., in particulate form) antigen can be used as the surface antigen. This is a very great advantage over the prior art that antibodies can be detected for insoluble (e.g., particulate) antigens in a simple manner and using a very small amount of antigenic material.

The invention is described in more detail below with reference to the following non-limiting embodiments.

Example 1

The glass surface 76 x 26 mm in the form of a so-called slide is washed with bicromate sulfuric acid, rinsed well with water and blown dry with compressed air. For about 5 minutes or more, the surface is in contact with a 1 # solution of a dextran having an average molecular weight of 2 x 10 6 ("Dextran 2000", manufactured by Pharmacia Fine Chemicals, Uppsala) in phosphate buffered saline (hereinafter referred to as PBS and containing 0, 12-m NaCl, 0.03-m phosphate, pH 7j4), after which it is rinsed with well-distilled water and blown dry with compressed air. The glass was then similarly treated with a 1 JS solution in the test system of an inert protein, e.g., serum albumin from the species from which the sample was derived. The sample is added in drops of 3 to 10 microliters in rows, each containing 8 drops each, using an automatic pipette. Dilution of the sample substantially greater than 1/100 occurs with antibody-free serum from the same species diluted 1/100 or in 0.1% serum albumin. After 5 ~ 10 minutes, the drops are rinsed off with PBS containing 0.1% serum albumin solution. After 5 minutes, the drops are rinsed off with PBS containing $ 0.1 Tween ^ 20 (polyoxyethylene sorbitan monolaurate, manufactured by Atlas Chemie GmbH) (hereinafter abbreviated as T-PBS), after which the glass is placed horizontally and covered with 1 / A 2% suspension of formalin-treated heat-killed protein A-containing staphylococci (S. Jonsson, G. Kronvall, Europ, J. Immunol. 4, p.

29, 1974) in T-PBS. After 5-10 minutes, the glass is allowed to drain and rinsed with T-PBS to remove excess staphylococcal reagent from the glass surface. The glass is then immersed in cuvettes containing T-PBS. The result can be read either from such wet glasses or after further rinsing with distilled water, possibly with the addition of Tweer after drying with blotting paper and air. Reading is best done in oblique light against a dark background. A positive reaction is formed by a yellow-white staphylococcal spot in the area where the sample drop is located, and a negative reaction is caused by the adsorption of staphylococci on the surface. Using this method, using a rabbit anti-dextran serum containing 1.3 mg of specific antibody per ml, a positive reaction was obtained after dilution up to 1 / 10,000. Most human and rabbit sera can be added undiluted and cause a negative reaction.

Serum samples from which only highly sensitive radioimmunological techniques could detect antibodies to dextran give positive reactions in the actual experiment, even when the sample is diluted 25-125 times.

Similarly, rabbit or human IgG antibodies have been demonstrated using antigens of a different chemical structure, including haptens and complex particulate antigens. Examples of such antigens (antibody combinations) which have been studied with good results are bovine serum albumin, egg albumin, kentamycin (antibiotic with e.g. hapten), after prior covalent binding to cyanogen bromide-activated dextran 2000, "Hepatitis B" antigen (HBAg, formerly referred to as Australia antigen, Au-ag), Salmonella bacteria, Chlamydia bacteria are in all cases well suited to the more complex or considerably more time-consuming methods used in the past.

Example 2

The procedure of Example 1 is repeated using samples forming mixtures of a specified volume of solutions containing dextran in weighed amounts and diluting a specified volume of dextran antibodies which has been allowed to react at room temperature for 5-10 min. The anti-dextran dilution 1/2000 used after mixing with the antigen-containing solution has been chosen so that, in the absence of antigen in the sample, it causes a clear staphylococcal coating on the surface without a strong excess of antibody. Under these conditions, even a small minimum amount of 958404 inhibits the binding of the latter to the surface antigen by reaction with a small amount of antibody, thus leaving a staphylococcal coating as an indication of the presence of the antigen. Experimenting with solutions containing weighed concentrations of dextran mixed with equal volumes of the above anti-dextran antiserum dilutions provided complete inhibition of staphylococcal adsorption using an anti-dextran solution with a concentration of 0.1 / ug / ml and a positive staphylococcal. 0.01 / ug / ml. Thus, it has been shown that the sensitivity of this technique is at a level achieved by radioimmunological experiments.

Example 3

A 20 x 20 cm glass plate, washed with dichromate sulfuric acid according to Example 1, is treated with a 1% solution of bovine serum albumin in PBS, allowing about 1 ml of the solution to spread between two equal glass plates, which are pushed on top of each other leaving a border of about 3 cm. against the antigen. The rinsing and dry blowing are performed according to Example 1, however, taking care that the liquid and air are blown away from the antigen-free rim. A thin layer of Sephade® G-75 Superfine gel (Pharmacia Fine Chemicals, Uppsala, Sweden) is applied to the entire plate. The disk is then fitted to a device with which the so-called thin layer gel chromatography, and contacting the upper and lower vessels containing PBS via filter paper, between which vessels the liquid will flow according to the suction pad principle. The sample is added in the form of 5-10 μl of serum drops or dilution thereof to the upstream antigen-free rim. The height difference between the liquid baths is adjusted so that the sample moves over the plate in about 3 hours. The gel is then rinsed off with PBS, the plate is further rinsed with T-PBS, then treated for about 5 min with standard rabbit serum diluted 1/100, rinsed again with T-PB5 and finally treated with staphylococcal reagent and with rinsing liquid according to Example 1. The result is best manifested in diagonally transmitted light against a dark background as triangular surfaces with the base at the boundary line between the antigen surface and the boundary region, and in the flow direction of the peak liquid. The surface area is directly proportional to the amount of antibody in the sample.

ίο 58404

By analogy with the above method, spun and non-woven fibers have been tried to support fluid transport, providing two advantages: partly avoiding lateral diffusion of the sample during transfer, partially eliminating the need to treat the plate with e.g. normal rabbit serum diluted in a ratio of 1/100 before the staphylococcal reagent.

It is also possible to provide several parallel liquid flows without any solid phase support on the glass sheet if the sheet is first moistened along parallel lines e.g. by moistened filaments and the flow is arranged through a plurality of strands a few degrees obliquely arranged on a vessel about 1 cm above the top of the glass plate. The drainage of the liquid is arranged through strands which extend into the bath just below the lower edge of the plate. The latter method provides a sharper definition of the surface bounded by the staphylococcal reagent.

Example M

According to Example 1, rabbit anti-dextran antibodies were added to the antigen adsorbed on a glass plate. Antibodies that were specifically bound to the antigen were detected by then contacting the glass slide with a suspension of reagent portions obtained by treating polymethyl methacrylate moieties having an average size of 1 μm with guinea pig anti-rabbit immunoglobulin antiserum diluted 1/25. , or immunoglobulin fractions of such serum. The particles were washed with phosphate-buffered saline and adjusted to a 0.5-1% suspension before use. During contact between this reagent and the glass surface, the suspension was gently passed back and forth over the glass surface to accelerate the specific attachment of the reagent particles to the antibodies bound to the antigen adsorbed on the glass plate. The results obtained turned out to be completely equivalent to those obtained using the staphylococcal reagent of Example 1.

Claims (9)

A method of visually demonstrating the presence of antibodies (I) in an aqueous sample, which antibodies are specifically directed against an antigen, wherein the sample is contacted with a smooth, preferably peel or tubular surface coated with said antigen for binding. of the antibodies to antigens on the surface, after which the similarly bound antibodies (I) are detected on the surface, characterized in that for contacting the antibodies, the surface contacts a suspension of small in water-insoluble particles, to which a biopolymer is directly bound, which biopolymer has the ability to specifically bind to the non-antigen-binding structure of antibodies (I), preferably to their Fc portion, said particles accumulating on the portion of the surface where antibodies specifically bound 58404 formation of a clearly observable coating of particles to the naked eye. Process according to claim 1, characterized in that the particles are bacterial bodies or other microbes or fragments thereof, which contain in their surface layer said biopolymer or to which said biopolymers are bound by adsorption forces or by covalent bonds. 3. A process according to claim 1, characterized in that the particles are made up of artificially made particles of synthetic or biological material which contain in their surface layer said biopolymers or to which said biopolymers are bound by adsorptive forces or with covalent bonds. Method according to claim 1 or 2 for the detection of antibodies belonging to the IgG class, characterized in that the particles are protein A-carrying, preferably killed, staphylococci. Method according to claim 1, characterized in that the surface is contacted with antigen-bound antibodies (I) with a suspension of bacterial bodies, the surfaces of which antibodies (II) against said antibodies (I) are directly bound. Method according to Claim 1 or 2, characterized in that the surface of antigen-linked antibodies (I) is contacted with antibodies (II) which are directed to said antibodies (I) and whose Fc parts specifically bind to protein A and then adds a suspension of protein A-bearing staphylococci to visualize the double antibody layer-coated surface. Method according to one or more of claims 1-6, characterized in that the sample is passed in a direction in a zone, preferably a narrow zone, over antigen-coated surface. Method according to one or more of claims 1-7, characterized in that the antigen-coated surface is placed over the