METHOD FOR THE DETERMINATION OF BIOLOGICAL SUBSTANCES BY DIFFUSION IN A POROUS MATRIX OR BY ELECTROPHORESIS

Abstract: A method for the determination of the quantity and type of a biological component A in a sample which is added to a basin in a liquid-saturated, porous matrix covering a thin layer of a biologically active component B bound to the solid surface of a carrier. A further biologically active component C included in the matrix is capable of reacting biospecifically with component A when the sample is allowed to diffuse or migrate electrophoretically in the matrix forming a zone containing a precipitate of components A and C. This precipitate will adsorb with biospecificity to component B, bound to the solid surface of the carrier and is visualized in a suitable manner on the surface after removal of the said porous matrix, eg. as a change in the surface tension angle observed with the aid of vapour condensation on the surface. Components B and C or B and A may be the same as long as the remaining component is capable of reacting with both the others. The components A, B and C may be antigens and antibodies. The carrier is suitably made of plastic material.

Description: A biological indicator system The present invention relates to a biological indicator system based on a two-phase system, in which a first biologically active component is bound to the surface of a solid phase, and a liquid- saturated immobilized phase covering said surface containing a second biologically active component, capable of reacting with onunknown third biologically active component added to the immo¬ bilized phase and permitted to diffuse or migrate electrophore- tically during formation of a zone containing a precipitate, and where the first biologically active component is capable of reacting by absorption with said precipitate formed in the im¬ mobilized phase. The presence of the third component is visua¬ lized in a suitable manner on the surface, after removal of said immobilized phase. Serologic testing methods have a central position within the diagnostic techniques of medicine and applied biochemistry in general. This is predominantly due to the fact that such methods afford the possibility of determining minute quantities of the specific reactants in question in complex systems. Within sero- logy there is utilized the basic fact that antibodies (ab) can specifically react with antigen (ag)„ When indicating ag-ab-reactions, there are used different secondary manifestations capable of being indicated in various ways. One common method of indicating these reactions is the pre¬ cipitation technique, in which precipitates are formed under cer- OMPI tain conditions when soluble antigens and anti-bodies come int contact with one another. In certain cases, for example when t antigen is in particle form, agglutination reactions can be ut lized, these reactions occurring when antibodies come into con tact with the antigen. In recent years, different types of markers have been use for determining ag-ab-reactions. The markers comprise radioact isotopes, enzymes or fluorescent substances which are bound either to the antigen or to the antibody. Indication is then e ted by means of the markers in the antigen-antibody-complex, s sequent to separating said complex from non-bound antigen or a ti-bodies and accompanying markers, A specific position is held by the biological determinati methods, in which a natural biological effector mechanism is u lized to indicate the antigen-antibody reaction. Among these i dication methods can be mentioned complement-binding reactions and different types of neutralizing reactions of biologically tive antigenic substances by blocking, for example with virus, toxins and the lil^e. Antigen-antibody reactions can be quantified in different ways, by using different indication principles. The most simpl of these is the end-point titration method, in which one.part of an antigen-antibody system is diluted to a limit at which t indicator system can no longer indicate the reaction. Another method is one in which indication by means of mar kers is used, it being necessary to separate the antigen-anti¬ body complex * The amount of labelled reactants in the antigen- antibody complex or in the residue of the system separated the from is then determined, According to one conventional method, one of the reactant in an antigen-antibody system is incorporated, for example, in agargel. The other reactant is supplied in basins disposed in gel. After some time has lapsed, a radial diffusion gradient is formed by the supplied reactant. The diffusion gradient thus for¬ med is indicated, and the obtained quantification is determined by measuring the area of the circular -indication thus formed. It is also known to have the known reactant in the ag-ab-system added to a surface below the gel phase. After diffusion and migration the unknown reactant will.react with the known reactant on the surface, where it can be visualized after removing the gel phase. Indication and quantification of antigen, utilizing known anti-bodies are carried out within the field of medical diag¬ nostics and in the follow-up various diseases. The quantification of an unknown antigen with the . aid of known antibodies is effected by immunizing a living organism having the ability to form antibodies against said antigen, where¬ after it is possible, with the aid of these antibodies, quanti¬ tatively to determine the antigen with which the organism has been immunized. This method has been widely used when quantifying different human serum proteins of the type im unoglobulin and en- zymes. In latter years it has also been possible to follow up and diagnose tumouroυs diseases by determining antigens speci¬ fic to tumour cells. It has also been found that antibodies can be produced against small molecules having a molecular weight of less than 1000, enabling different types of medicaments and hor- mones to be shown in serum with the aid of antibodies, for example. The present invention is suitable for the detection of a.-fe- to-protein, IgG, IsM, IgA, IgE, hepatitis antigen, acute-phase protein, HCG (pregnancy test) and serum proteins in general. It has now been discovered that an extraordinarily sensitive and readily applied method for determining the quantity and type of biologic substances of the kind in which a component selected from the groups proteins, polysaccarides, nucleic acids, lipides or complexes thereof reacts ahd where the result of the reacti is visualized it^ situ with the aid of a component bound to a surface of -a solid phase, which is. characterized by a system, >- co prising a first biologically active component bound to the solid surface, and a liquid-saturated immobilized phase locate at said surface, said phase containing a second biologically a tive component, to which is added a test aliquot possibly cont ing a third biologically active component, which is to be. de¬ termined, said third component being capable of reacting with the second biologically active component during formation of a precipitate existing of equivalent concentrations of the secon and the third component, said precipitate being capable of rea ing with the first biologically active component and by the th biologically active component being permitted to diffuse or migrate electrophoretically and form a precipitate with the se biologically active component, said precipitate reacting with the first biologically active component by removing the immobi lized phase, and by reaction between said precipitate and the first component thus being arranged for observing an indicatio of the presence and quantity of the third component on said so surface in any suitable manner. The proteins may comprise immunoglobulins such as antibod and antigens and other antigenic substances, such as enzymes, toxins, lectines and hormones or systems which form precipitat of the type defined as an equivalent zone precipitate, i,e. where a precipitate is formed when concentration of said secon component and said third component are substantially equivalen and therefore forms a moving front of precipitate which is re- split when the concentration of the third biologically active component is increased. Examples of the polysaccarides include lipopolysaccarides such as gangliocides, endotoxin, mucopolysaccarides, glycoprot Among the lipides, different lipide complexes can be used, such as cardiolipides. Among the " nucleic acids, desoxyribonucleic acid has an antigen activity and can be used as a component on " the plastics surface. The thin layer of said first component is suitably applied to the plastics surface in the following manner: a liquid medium, suitably based on water, is first dispensed onto the plastics surface, whereafter a solution containing the component is added to the liquid medium to diffuse therein, whereafter the compo- nent is permitted to deposit itself onto the plastics surface, "becoming boand to the plastics surface with a force of such mag¬ nitude that said surface can be washed without the layer being removed therefrom, subsequent to the aqueous ' medium having been removed. The solid surface is suitably a transparent material, such as glass, or a plastics material, e.g.' polystyrene, polyacrylo- nitrile, polyolefines and copolymers thereof. It has been found in recent years that plastics surfaces ad¬ vantageously adsorb macro-molecules to form very uniform and re- producable layers. A technique for visualizing antigen-antibody reactions is one in which a thin layer of indium particles is vapor-deposited on a glass surface. The antigen-antibody reactions are carried out on the indium layer, whereafter the reactions can be observed as a light-propagation phenomena on the indium surface. The most serious disadvantages with this technique appear to be the requirement of advanced apparatus for producing a uni¬ form and reproducable layer of indium on large surfaces. This < < restricts the rational use of such surfaces. Furthermore, it is difficult to classify a reaction as a positive or a negative one, in " borderline cases, since this indicator system has a flat amp- litude and the indication can only be judged subjectively. 1 Anothe ' r, much simpler technique for visualizing adsorbed precipitates on solid surfaces is one employing the condensation of water vapor. This technique involves exposing the dried sur¬ face to vapor, whereupon it is possible to determine whether a reaction has taken place and the extent of any such reaction fro the pattern formed by the condensation. The principles of this technique were described by Langmuir in 1936, This method is as sensitive as the method employing an indium layer, but has a steeper indication-amplitude. Moreover, it permits the objective analysis by contact-copying of the condensation pattern on the surfaces by irradiation of photographic paper and development thereof. The indication of ag-ab-reactions is thus best effected wit vapour condensation on the plastics surface (Vapour condensation on surface, VCS, see Adams, Klings, Fisher and Vro an, Journal of Immunological Methods, 3, (1973) pages 227-232), which, be- cause of its simplicity, is the preferred method. Other known methods can also be used, such as the' so-called ELISA-method (Enzyme-linked im unosorbent assay, J. Immun. 109:129. (1972), mixed haemadsorption (immunology 9:161 (1965) ), particle adsorp tion technique, using a slurry of barium sulphate,for example immunofluorescense, various colouring techniques and auto-radio¬ graphy with isotope-labelled serological reagents. The reason why it is possible to observe a change as a re¬ sult of adsorption of precipitates on the molecular layer on the plastics surface as a result of vapour condensation is due to the fact that the so-called Zeta-potential or surface tension against condensed drops of water is changed on the surface when an adsorption reaction has taken place. Such changes are macro- scopically visible in the light-scattering phenomena of the όon- densation pattern. In principle, all hydrophobic surfaces have a surface-tension angle of from 90 to 170 , Those plastics sur¬ faces which normally hve such properties include polystyrene, polyacrylnitril, polyethylene and copolymers thereof. An immobilized matrix through which the unknown substance shall diffuse is then applied to the surface containing a bio¬ logically active component. Such immobilized matrices are well known within the technique of analysis, and may comprise aqueous gels or various types of sediment or- fibrous substances. The most conventional method is one in which a gel is used, in particular an agar gel, suitably comprising a buffered 1^-solution of agar which is permitted to solidify, A basin is then formed in the matrix, there being supplied to the basin a solution containing the unknown component. The unknown component may also be supplied in cellulose plates or the like which have been saturated with the solution containing the unknown component. The system is left at a suitable temperature of between 5 and 50 C, for the unknown substance to diffuse from the basin, Compared with previously known methods of obtaining quan¬ titative measurements of minute quantities of biological mate¬ rials, the novel method exhibits a simplicity which has not pre¬ viously been achieved, and therewith a subsequent increase in capacity and decrease of costs. Furthermore on plastics surfaces the precipitation is very uniform and thus suitable for diag¬ nostic tests with regard to reproducibility , which is not the case with glass to the same degree. A further advantage afford¬ ed by the use of plastics is that the protein nature of the absorbed component is not denatured or chemically converted, as often happens with glass surfaces. The application of a compo¬ nent fraction by first applying an aqueous medium and then, adding the component fraction to said medium ensures that a much more uniform layer is obtained than would be the case when a protein suspension or solution is applied to the dry surface, Moreover, the amount of component fraction consumed is con¬ siderably lower when a plastics surface is used than when a glass substrate is used. Furthermore, when using a glass substrate it is normally necessary to treat the layer with formalin in or¬ der to denature the same so that it does " not loosen from the gla surface. Finally, the diffusion image obtained on the plastics surface can be treated with antigen-immunoglobulin, thereby to visualize even the most minute reactions or reaction quantities • This method is based on the experience that immunological precipitation reactions take place when the quantities of antig and antibody are in a specific relationship to one another and are redissolved when one part or the other is present in large surplus quantities. In those instances in the aforedescribed method where the matrix contains the said second component in the antigen-antibo reaction, said first component having been added to the plastic surface, and the quantity of the third component is to be deter mined, it is important that said first component is not added t the surfaces in a pure form,_ otherwise an excessively large qua tity of the second component present in the matrix will bind directly to said first component on said surface as soon as the matrix is applied. In turn, this means that it is impossible, o extremely difficult to read the precipitation-print reaction wi any method. It has been found that this problem is removed by mixing said first component with unrelated substances in suitab ratios prior to adding to the plastics surface and thus thin ou the layer of said first component, The method is highly sensitive, and quantities as small as, or less than magnitudes of 10 ug/1 can be detected. It is also possible to obtain multi-precipitating systems in which classes of antibodies can be determined. The system enables less pure a tigen-antibody substances to be used. It is extremely surprisin that precipitation adsorption on the plastics surface is obtain with this technique. This is probably due to the fact that the antibodies have two binding functions, and that there is obtain OMPI <fy W1PO α bond with said first component located on.the solid surface and a further agglomerating and cross-linking bond with the antigens in the matrix. Thus, the matrix contains one or more antibodies against the antigen to be determined, and the solid surface is provided with this antigen or another antigen, whereby complex re¬ actions can be obtained. It is also possible to obtain multiple precipitation zones if said third component includes more than one biologically ac¬ tive" element and if the immobilized phase containing said second component consisting of more than one element, which reacts with the respective element in the third component. The multiple zones thus obtained can be selectively combined with said first component if the latter consists of separate areas each covered with a different compound reacting with the respective preci- pitate to provide individual detection. From the above description it will be evident that the said first and said third component can be the same if the second is different and the said first and said second component can also be the same if the said third component is different, Of course, all three could be of different kinds. Thus, if said second compo¬ nent is an antibody said third component can be an antigen and said first component can be either an antibody like or unlike said second component or an antigen like or unlike said third com¬ ponent. As an example, said first component is the antigens IgG and IgA, each on its own surface area. Said second component consists of anti-IgG, anti-IgG and πnti-IgM and said third component con¬ sists of a human serum containing IgA, IgG and IgM, The serum is placed in a basin on the borderline between the. two . different surface areas of said first compound. After a suitable diffusion period three concentric precipitation lines can be observed and measured in the gel. The gel is then removed and it is possible to record the respective positions of the IgA and IgG precipitate The third precipitate is identified by exclusion as the IgM pre¬ cipitate. In another embodiment according to the invention, said first component in anti-a-feto-protein. Anti-a-feto-protein is include in the gel in such a low concentration that no'-visible precipitat can be obtained with said third component, which is a-fet -protei in human serum, a-feto-protein is an indication of primary hepa- thomas, which are types of tumours, a-feto-protein is present in very low concentration in early cases of tumours, and can thus b observed by a precipitation reaction at the surface area, where said first and said second components together form the precipit The use of low concentrations of anti-a-feto-protein in the gel make this method more sensitive. There will only be precipi- tation if the original concentration of said third component in the sample is higher than the concentration of said second compo nent. OMPI

Claims: CLAIMS j- 1, A method of determining the quantity and type of biologic substances of the kind in which a component selected from the groups proteins, polysaccarides, nucleic acids, lipides or complex¬ es thereof reacts and where the result of the reaction is visu- alized iji situ with the aid of a component bound to a surface of a solid phase, characterized by a system, comprising a first biologically active component bound to the solid surface, and a liquid-saturated immobilized phase located at said surface, said phase containing a second biologically active component, to which is added a test aliquot possibly containing a third bio¬ logically active component, which is to be determined, said third component being capable of reacting with the second biologically active component during formation of a precipitate existing of equivalent concentrations of the second and the third component, said precipitate being capable of reacting with the first biolo¬ gically active component, and by the third biologically active component being permitted to diffuse or migrate electrophore- tically and form a precipitate with the second- biologically ac¬ tive component, said precipitate reacting with the first biolo- gically active component by removing the immobilized phase, and by said reaction between said precipitate and the first compo¬ nent thus being arranged for observing an indication of the presence of quantity of the third component on said solid surface in any suitable manner, 2. A method according to claim 1, characterized in that the first biologically active component and the second biologically active component are the same. 3, A method according to claim 1, characterized in that the first biologically active component and the third biologically active component are the same, 4. A method according to claim 1, characterized in that the solid surface constitutes a plastics material selected from thegroups polystyrene, polyacrylnitrile, polyolefines and copolymers thereof, 5, A method according to claim 1, characterized in that the re tion on the solid surface an the resultant change " in the surfa tension angle are observed with the aid of vapour condensation on the surface, 6„ A method according to claim 1, characterized in that the first and the second biologically active components are anti-a- feto-protein and that * the third biologically active component is a-feto-protein contained in human serum, 7, A method according to claim 1, characterized in that diffe¬ rent sectors of said solid surface are covered with different components acting as the first biologically active component and that more than one component is incorporated in the immobilized phase acting as the second biologically active component and that different reactions are indicated ai different surface sec¬ tors after removing the immobilized phase, 8, A method according to claim 7, characterized in that IgA and IgG are used as the first biologically active component on different sectors of said surface and that the immobilized phase contains anti-IgA, anti-IgG and anti-IgM and that the third component is human serum containing at least one of IgA, IgG and IgM. - β ϋRE O P R

Inventors: ELWING H
Applicants: ELWING H
Application year: 1978
Main classification: G01N33
IPC codes: C12Q1, G01N27, G01N33