CH631549A5

CH631549A5 - Process for the bonding of reactive constituents of immunological complexes in separate layers

Info

Publication number: CH631549A5
Application number: CH1090476A
Authority: CH
Inventors: Ivar Giaever
Original assignee: Gen Electric
Priority date: 1975-08-27
Filing date: 1976-08-27
Publication date: 1982-08-13
Also published as: DE2638207A1; BR7605741A; DE2638207C2

Description

In der vorliegenden Erfindung ist ein Verfahren offenbart, mit dessen Hilfe die Antikörpermoleküle in der zweiten Schicht 12 mit den Antigenmolekülen in der ersten Schicht 11 verbunden sein können und die Antikörpermoleküle gleichzeitig verbleibende aktive Stellen behalten für ein weiteres Verbinden mit anderen Antigenmolekülen in einer weiteren immunologischen Reaktion. In the present invention, a method is disclosed by means of which the antibody molecules in the second layer 12 can be connected to the antigen molecules in the first layer 11 and the antibody molecules at the same time keep active sites remaining for further connection with other antigen molecules in a further immunological reaction .

Die Grundlage dieser Erfindung ist in Figur 2 veranschaulicht und sie ist in dem Verfahren enthalten, mit dem man die anfängliche monomolekulare Schicht aus Antigenmolekülen 11 bildet, die an der Oberfläche des Substrates 10 adsorbiert ist. Zur Veranschaulichung sind die abgebildeten Antikörper von der Ig G-Klase, doch gibt es keine Gründe, weshalb Antikörper der andern vier Hauptklassen, wie sie oben genannt sind, in der vorliegenden Erfindung nicht benutzbar sein sollten. The basis of this invention is illustrated in Figure 2 and is included in the process by which the initial monomolecular layer of antigen molecules 11 is formed which is adsorbed on the surface of the substrate 10. By way of illustration, the antibodies depicted are from the Ig G class, but there is no reason why antibodies from the other four main classes as mentioned above should not be usable in the present invention.

Nach dem Auswählen des Substrates 10, auf dem der multimolekulare immunologisch komplexierte Film gebildet werden soll, wird das Substrat mit einem ersten wässrigen Medium behandelt, z.B. einer Salzwasserlösung, die das Antigen enthält, wie im Falle des zu Figur 1 beschriebenen Verfahrens. Im Gegensatz zu diesem Verfahren ist jedoch vor der Behandlung des Substrates eine immunologisch inerte organische Verbindung zu dem ersten wässrigen Medium hinzugegeben worden, so dass die auf der Oberfläche des Substrates 10 gebildete Adsorptionsschicht aus reaktiven Antigenmolekülen 11 besteht, die voneinander durch Moleküle 20 der inerten organischen Verbindung getrennt und von diesen umgeben sind, wie in Figur 2 ersichtlich. Die Menge der immunologisch inerten organischen Verbindung, die zu dem wässrigen Medium hinzugegeben wird, reicht aus, dass zwischen benachbarten Antigenmolekülen 11 an dem Substrat 10 im allgemeinen ein Abstand von mehreren hundert Angström besteht. After selecting the substrate 10 on which the multimolecular immunologically complexed film is to be formed, the substrate is treated with a first aqueous medium, e.g. a salt water solution containing the antigen, as in the case of the method described in FIG. 1. In contrast to this method, however, an immunologically inert organic compound has been added to the first aqueous medium before the treatment of the substrate, so that the adsorption layer formed on the surface of the substrate 10 consists of reactive antigen molecules 11 which are separated from one another by molecules 20 of the inert organic molecules Connection separated and surrounded by them, as can be seen in Figure 2. The amount of the immunologically inert organic compound that is added to the aqueous medium is sufficient that there is generally a distance of several hundred angstroms between adjacent antigen molecules 11 on the substrate 10.

Die inerte organische Verbindung kann hinsichtlich der chemischen Art variieren, wobei das einzige Erfordernis darin besteht, dass das Protein nicht daran haftet. Beispielsweise kann man Eialbumin, Rinderserumalbumin, Insulin und ähnliche Verbindungen verwenden. Die eingesetzten Konzentrationen liegen im allgemeinen im Bereich von 0,05 bis 100 mg/ml des wässrigen Mediums, wobei die ausgewählte Konzentration von der des Antigens abhängt. The inert organic compound can vary in chemical nature, the only requirement being that the protein not adhere to it. For example, egg albumin, bovine serum albumin, insulin and similar compounds can be used. The concentrations used are generally in the range from 0.05 to 100 mg / ml of the aqueous medium, the concentration selected depending on that of the antigen.

Das mit der monomolekularen Schicht aus Antigen und inertem Bestandteil überzogene Substrat 10 wird dann in ein zweites wässriges Medium eingetaucht, welches die zu dem Antigen des ersten Mediums spezifischen Antikörper enthält. Da der Abstand zwischen den aktiven Stellen eines Antikörpermoleküls 12 (der Ig G-Klasse) etwa 200 Angström beträgt, weist ein grosser Teil der Antikörpermoleküle 12 des zweiten Mediums, die sich mit den Antigenmolekülen 11 verbinden, verfügbare Bindestellen auf (wobei mehr als eine aktive Stelle pro Molekül übrigbleiben kann je nach der besonderen Klasse von Antikörpermolekül) und diese bleiben aktiv für die weitere Kombination mit zusätzlichen Antigenmolekülen bei einer nachfolgenden immunologischen Reaktion. The substrate 10 coated with the monomolecular layer of antigen and inert component is then immersed in a second aqueous medium which contains the antibodies specific for the antigen of the first medium. Since the distance between the active sites of an antibody molecule 12 (of the Ig G class) is approximately 200 angstroms, a large part of the antibody molecules 12 of the second medium, which bind to the antigen molecules 11, have available binding sites (with more than one active one) Site per molecule can remain depending on the particular class of antibody molecule) and these remain active for the further combination with additional antigen molecules in a subsequent immunological reaction.

Irgendein Antikörpermolekül kann sich im allgemeinen nicht mit mehr als einer aktiven Stelle auf irgendeinem Antigen- Any antibody molecule generally cannot have more than one active site on any antigen.

molekül verbinden. Aufgrund des ausreichenden Abstandes zwischen den benachbarten Antigenmolekülen aufgrund der Moleküle der immunologisch inerten organischen Verbindung werden die Antikörpermoleküle 12 an die Antigenmoleküle 11 5 gebunden und im allgemeinen weist jedes Antikörpermolekül 12 noch eine aktive Verbindungsstelle 12a für eine folgende immunologische Reaktion auf. connect molecule. Due to the sufficient distance between the neighboring antigen molecules due to the molecules of the immunologically inert organic compound, the antibody molecules 12 are bound to the antigen molecules 115 and in general each antibody molecule 12 still has an active connection point 12a for a subsequent immunological reaction.

Auf diese Weise ist die Hauptaufgabe der vorliegenden Erfindung gelöst, indem Antikörper in einer solchen Weise an eine io monomolekulare Antigenschicht gebunden sind, dass auf den Antikörpern Verbindungsstellen für weitere immunologische Reaktionen aktiv bleiben. Im Falle des aktiven Antigens aus menschlichem Serumalbumin ist ein Beispiel eines immunologisch inerten Proteins Eialbumin und die Antikörper werden 15 aus Kaninchenserum erhalten. Für den Fall einer 1 %igen menschlichen Serumalbuminlösung liegt die Konzentration des inerten Eialbumin-Proteins in dieser Lösung im Bereich von 1 bis 10%. In this way, the main object of the present invention is achieved in that antibodies are bound to a monomolecular antigen layer in such a way that connection sites remain active on the antibodies for further immunological reactions. In the case of the active antigen from human serum albumin, an example of an immunologically inert protein is egg albumin and the antibodies are obtained from rabbit serum. In the case of a 1% human serum albumin solution, the concentration of the inert egg albumin protein in this solution is in the range from 1 to 10%.

Nachdem das Substrat 10 aus dem antikörperhaltigen wäss-20 rigen Medium herausgenommen worden ist, wird das mit der bimolekularen Schicht überzogene Substrat 10 als nächstes in ein wässriges Medium eingetaucht, welches das gleiche Antigen wie das erste wässrige Medium enthält oder von dem man annimmt, dass es solches Antigen enthält und diese Antigenmole-25 küle 30 werden dann selektiv an die verbleibenden aktiven Stellen 12a der Antikörpermoleküle 12 in einer weiteren immunologischen Reaktion gebunden. Das Ergebnis ist in Figur 3 gezeigt. Diese Prozedur kann daher zur Analyse einer Lösung zum leichten Nachweisen der Anwesenheit eines bestimmten Anti-30 gens darin verwendet werden. Diese Prozedur kann aber auch zum Aufbauen von Ketten abwechselnder Antigen-Antikörpermoleküle verwendet werden, bei denen jede Kette ihren Ausgangspunkt an der Oberfläche des Substrates hat und einen multimolekularen immunologisch komplexierten Film aus meh-35 reren Ketten abwechselnder Antigen-Antikörpermoleküle darauf bildet. Der multimolekular komplexierte Film ist leicht durch Betrachten des überzogenen Substrates mit einem optischen Instrument, wie einem Eilipsometer, überprüft. After the substrate 10 has been removed from the antibody-containing aqueous medium, the substrate 10 coated with the bimolecular layer is next immersed in an aqueous medium which contains the same antigen as the first aqueous medium or which is believed to be it contains such antigen and these antigen molecules 25 are then selectively bound to the remaining active sites 12a of the antibody molecules 12 in a further immunological reaction. The result is shown in FIG. 3. This procedure can therefore be used to analyze a solution for easily detecting the presence of a particular anti-30 gene therein. However, this procedure can also be used to build up chains of alternating antigen-antibody molecules, where each chain has its starting point on the surface of the substrate and forms a multimolecular immunologically complex film of several chains of alternating antigen-antibody molecules on it. The multimolecularly complexed film is easily checked by viewing the coated substrate with an optical instrument such as an egg lipometer.

Man kann den multimolekular komplexierten Film aber 40 auch elektrisch durch Messen der Kapazität eines Kondensators bestimmen, dessen leitende Platten durch das Metall- oder das metallüberzogene Substrat und einen Quecksilbertropfen oder eine andere geeignete Elektrode gebildet werden, wobei das Kondensatordielektrikum durch die Antigen-Antikörperschich-45 ten gebildet wird. However, the multimolecularly complexed film 40 can also be determined electrically by measuring the capacitance of a capacitor, the conductive plates of which are formed by the metal or the metal-coated substrate and a drop of mercury or another suitable electrode, the capacitor dielectric being formed by the antigen-antibody layer 45 ten is formed.

Schliesslich kann man den multimolekular komplexierten Film auch optisch mit dem blossen Auge überprüfen, indem man die Länge der Zeit bestimmt, bevor ein sichtbares Amalgam zwischen einem Quecksilbertropfen und einem auf das 50 Substrat aufgebrachten Metallfilm durch die dazwischen liegenden Schichten hindurch gebildet wird. Finally, the multimolecularly complexed film can also be checked visually with the naked eye by determining the length of time before a visible amalgam is formed between a drop of mercury and a metal film applied to the substrate through the layers in between.

Schliesslich und bedeutsamer kann der multimolekular komplexierte Film optisch mit reflektiertem oder durchgehendem Licht untersucht werden. Hierfür ist eine erste Technik der 55 Untersuchung im durchgehenden Licht, die erfolgreich angewandt worden ist, die folgende: Finally and more importantly, the multimolecularly complexed film can be examined optically with reflected or continuous light. For this purpose, a first technique of 55 light examination that has been successfully used is the following:

Das Substrat 10 muss ein lichtdurchlässiges Substrat sein, wie Glas, Kunststoff, glasartiges Siliciumdioxid, Glimmer, 60 Quarz und ähnliches und es besteht vorzugsweise aus Glas, wobei mikroskopische Objektträger eine bequem erhältliche Quelle sind. Dieses Substrat wird zuerst mit vielen Metallkügelchen durch Aufdampfen eines Metalles, z.B. Indium, überzogen. Das Indium wird z.B. langsam aus einem Tantaldruck in einem übli-65 chen Vakuum von etwa 5 X 10" 5 mm Hg aufgedampft. Da die Indiumatome eine hohe Beweglichkeit auf der Oberfläche des Substrates haben und das Glassubstrat nicht merklich benetzen, agglomeriert das auf das Substrat aufgedampfte Indium zu klei The substrate 10 must be a translucent substrate such as glass, plastic, vitreous silica, mica, 60 quartz, and the like, and is preferably made of glass, with microscopic slides being a convenient source. This substrate is first coated with many metal beads by vapor deposition of a metal, e.g. Indium, coated. The indium is e.g. slowly evaporated from a tantalum pressure in a usual vacuum of about 5 X 10 "5 mm Hg. Since the indium atoms have a high mobility on the surface of the substrate and do not noticeably wet the glass substrate, the indium evaporated onto the substrate agglomerates too small

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nen Teilchen. Jedes Metall mit ähnlichen Eigenschaften, das Kügelchen auf dem Substrat bildet, wenn es auf dieses aufgedampft wird, kann verwendet werden. So sind ausser Indium Gold, Silber, Zinn, Wismuth und Blei erfolgreich eingesetzt worden. Das Aufdampfen des Metalles wird fortgesetzt, bis das Substrat eine leicht bräunliche Farbe zeigt. Zu diesem Zeitpunkt haben die Metallkügelchen Durchmesser in der Grössen-ordnung von 1000 Angström. Die genaue Grösse der Kügelchen ist nicht kritisch, doch müssen sie Durchmesser haben gleich einem grossen Anteil der Wellenlängen des sichtbaren Uchtes. Die nächste Stufe besteht darin, das mit Kügelchen bedeckte Substrat 10 mit einem wässrigen Medium zu behandeln, welches ein erstes immunologisch reaktives Antigen 11 und die inerte organische Verbindung 20 enthält. Das erste reaktive Antigen und die inerte organische Verbindung haften in einer monomolekularen Schicht auf dem Substrat und den darauf befindlichen Metallkügelchen. Hat sich eine monomolekulare Schicht gebildet, dann wird das überzogene Substrat aus dem ersten wässrigen Medium herausgenommen und in ein zweites wässriges Medium eingetaucht, welches den spezifisch reagierenden Antikörper 12 zu dem ersten Antigen enthält und dies führt zur Bildung einer auf dem Substrat und den Metallkügelchen haftenden bimolekularen Schicht ähnlich der in Figur 2 gezeigten, wo lediglich die Metallkügelchen fehlen. Das überzogene Substrat wird dann aus dem zweiten wässrigen Medium herausgenommen und in ein drittes wässriges Medium eingetaucht, welches das gleiche reaktive Antigen wie das erste wässrige Medium enthält oder von dem man annimmt, dass es dieses Antigen enthält und wenn das Antigen vorhanden ist, wird eine dritte Schicht oder Teilschicht auf dem Substrat gebildet. Das überzogene Substrat wird dann mit durchgehendem Licht betrachtet und aufgrund des Aussehens des überzogenen Substrates wird eine Bestimmung der Dicke der daran haftenden Schicht vorgenommen und damit bestimmt, ob das vermutete Antigen vorhanden war oder nicht. Der Nachweis der Schichten entspricht Variationen im Braunton, die in dem überzogenen Substrat beobachtet werden. Diese Variationen sind recht deutlich und der Nachweis der Schichten ist daher eine einfache gerade Prozedur. Die Teilchen allein auf dem Substrat erscheinen als ein erster Braunton. Die mit einer monomolekularen Schicht überzogenen Teilchen erscheinen als ein dunklerer Braunton, die mit einer bimolekularen Schicht bedeckten Teilchen erscheinen als ein noch dunklerer Braunton und die mit einer trimolekularen Schicht bedeckten Teilchen erscheinen als ein noch dunklerer Braunton. Dieses Nachweisverfahren beruht auf der Tatsache, dass elektromagnetische Strahlung durch leitende Kugeln mit Durchmessern gleich einem grossen Anteil einer Wellenlänge des sichtbaren Lichtes zu einem grossen Masse zerstreut wird und dass dieses Zerstreuen stark durch einen dünnen auf die Kugeln aufgebrachten dielektrischen Überzug beeinflusstwird. a particle. Any metal with similar properties that will form beads on the substrate when vapor deposited thereon can be used. In addition to indium, gold, silver, tin, bismuth and lead have been used successfully. Evaporation of the metal continues until the substrate shows a slightly brownish color. At this point, the metal spheres have diameters in the order of 1000 angstroms. The exact size of the spheres is not critical, but they must have a diameter equal to a large proportion of the wavelengths of the visible spectrum. The next step is to treat the beaded substrate 10 with an aqueous medium containing a first immunologically reactive antigen 11 and the inert organic compound 20. The first reactive antigen and the inert organic compound adhere in a monomolecular layer on the substrate and the metal spheres thereon. Once a monomolecular layer has formed, the coated substrate is removed from the first aqueous medium and immersed in a second aqueous medium which contains the specifically reacting antibody 12 to the first antigen and this leads to the formation of an adherent on the substrate and the metal spheres bimolecular layer similar to that shown in Figure 2, where only the metal spheres are missing. The coated substrate is then removed from the second aqueous medium and immersed in a third aqueous medium containing the same reactive antigen as the first aqueous medium or which is believed to contain this antigen, and if the antigen is present, one becomes third layer or partial layer formed on the substrate. The coated substrate is then viewed with continuous light and, based on the appearance of the coated substrate, a determination is made of the thickness of the layer adhering to it, thereby determining whether or not the suspected antigen was present. The detection of the layers corresponds to variations in the brown tone that are observed in the coated substrate. These variations are quite clear and the detection of the layers is therefore a simple straightforward procedure. The particles alone on the substrate appear as a first shade of brown. The particles coated with a monomolecular layer appear as a darker shade of brown, the particles covered with a bimolecular layer appear as an even darker shade of brown and the particles covered with a trimolecular layer appear as an even darker shade of brown. This detection method is based on the fact that electromagnetic radiation is largely scattered by conductive balls with diameters equal to a large proportion of a wavelength of visible light, and that this scattering is strongly influenced by a thin dielectric coating applied to the balls.

Eine zweite Technik für die optische Untersuchung mit Hilfe reflektierten Lichtes, die erfolgreich angewendet worden ist, ist folgendermassen: A second technique for optical inspection using reflected light that has been successfully used is as follows:

Ein Goldsubstrat, das aus wirtschaftlichen Gründen vorzugsweise eine auf ein anderes Metall plattierte dünne Goldschicht ist, trägt adsorbiert eine monomolekulare Schicht des ersten reaktiven Antigens 11 und der inerten organischen Verbindung 20 nach der Behandlung des Substrates mit dem oben beschriebenen ersten wässrigen Medium. Gold weist ein Adsorptionsband innerhalb des sichtbaren Spektrums auf und dies verursacht die charakteristische Goldfarbe und gestattet die Ausführung dieser besonderen optischen Untersuchungstechnik. Das Goldsubstrat kann bequemerweise ein Glastestobjektträger sein, der mit einer dünnen Indiumschicht überzogen ist, die ihrerseits mit der Goldschicht überzogen wurde, wobei die Indiumschicht die Adhäsion sowohl zwischen Glas und Gold verbessert als auch die optischen Charakteristika des Objektträgers. Das relative Reflektionsvermögen des Goldsubstrates als Funktion der Wellenlänge führt zu einem Substrat, welches die charakteristische leuchtendgelbe Farbe des Goldmetalles hat, wenn keine Proteinschicht daran haftet. In Anwesenheit einer s monomolekularen Schicht auf dem Substrat hat der Testobjektträger, das Substrat, eine blassgelbe Farbe. Nachdem der Testobjektträger einem wässrigen Medium ausgesetzt wurde, welches den immunologisch reagierenden Antikörper 12 zum Antigen 11 enthielt, trägt der Testobjektträger eine bimolekulare io Schicht und die Reflektivität ist derart, dass er ein grünliches Aussehen hat. Eine dritte Schicht führt zu einem noch grünlicheren Aussehen. In bisher ausgeführten Tests hat sich gezeigt, dass die optischen Untersuchungen des überzogenen Substrates mit reflektiertem oder durchgehendem Licht bei einem Subis strat, welches Metallkügelchen trägt oder ein Goldsubstrat ist, am brauchbarsten sind. Weiter wurde festgestellt, dass diese beiden Techniken verschiedene Empfindlichkeiten haben in Abhängigkeit von den Dicken der interessierenden Proteinfilme. Im besonderen erhält man die grösste Empfindlichkeit für 2o die Technik mit einem Substrat, das Metallkügelchen trägt, bei Filmen mit Dichen unterhalb von etwa 200 Â. Die Goldsubstrattechnik hat die grösste Empfindlichkeit für Filme, die 30 Â in der Dicke übersteigt. Das im besonderen Falle angewendete Nachweisverfahren bestimmt daher die Art des eingesetzten 25 Substrates 10, d.h. ob das Substrat ein Metall- oder metallisierter Glasobjektträger ist mit einem flachen Metall, beispielsweise Goldüberzug oder mit Metallkugeln, z.B. aus Indium, auf der Oberfläche. Zur Vereinfachung ist das gezeigte Substrat 10 mit einer flachen Oberfläche abgebildet, wobei klar sein sollte, dass 30 die Oberfläche, an der die erste Antigenschicht adsorbiert wird, auch die vorgenannten Metallkügelchen tragen könnte. A gold substrate, which for economic reasons is preferably a thin gold layer plated on another metal, adsorbs a monomolecular layer of the first reactive antigen 11 and the inert organic compound 20 after treatment of the substrate with the first aqueous medium described above. Gold has an adsorption band within the visible spectrum and this causes the characteristic gold color and allows the execution of this special optical examination technique. The gold substrate can conveniently be a glass test slide coated with a thin layer of indium, which in turn has been coated with the gold layer, the indium layer both improving the adhesion between glass and gold and the optical characteristics of the slide. The relative reflectivity of the gold substrate as a function of the wavelength leads to a substrate which has the characteristic bright yellow color of the gold metal if no protein layer adheres to it. In the presence of a monomolecular layer on the substrate, the test slide, the substrate, has a pale yellow color. After the test slide has been exposed to an aqueous medium which contains the immunologically reactive antibody 12 to antigen 11, the test slide carries a bimolecular layer and the reflectivity is such that it has a greenish appearance. A third layer leads to an even greener look. Tests carried out so far have shown that the optical investigations of the coated substrate with reflected or continuous light are most useful in a substrate which carries metal beads or is a gold substrate. It was further found that these two techniques have different sensitivities depending on the thickness of the protein films of interest. In particular, you get the greatest sensitivity for the technique with a substrate that carries metal balls, in films with diches below about 200 Â. Gold substrate technology has the greatest sensitivity to films that exceed 30 Â in thickness. The detection method used in the particular case therefore determines the type of substrate 10 used, i.e. whether the substrate is a metal or metallized glass slide with a flat metal, e.g. gold plating or with metal balls e.g. made of indium, on the surface. For simplification, the substrate 10 shown is shown with a flat surface, it should be clear that the surface on which the first antigen layer is adsorbed could also carry the aforementioned metal spheres.

Das beschriebene Verfahren der Bildung multimolekularer (dreischichtiger) immunologisch komplexierter Filme, wie in Figur 3 abgebildet, ist besonders bedeutsam beim Testen eines 35 wässrigen Mediums auf einen gewissen biologischen Bestandteil, z.B. ein Hormon, da Hormone und Antiseren zu Hormonen allgemein erhältich sind. Wenn daher die dritte Schicht das interessierende Hormon ist, kann es leicht nachgewiesen werden. The described method of forming multimolecular (three-layer) immunologically complexed films, as shown in Figure 3, is particularly important when testing an aqueous medium for a certain biological component, e.g. a hormone because hormones and antisera to hormones are widely available. Therefore, if the third layer is the hormone of interest, it can be easily detected.

Das Bilden der multimolekularen immunologisch komple-40 xierten Filme gemäss der vorliegenden Erfindung ist auch wichtig beim immunologischen Identifizieren von Zellen oder Viren. Wie in Figur 4 veranschaulicht, ist durch das Aufbauen einer Vielzahl von Ketten aus Antigen-Antikörper-Komplexen ausgehend von der Oberfläche des Substrates 10, die Wahrschein-45 Iichkeit relativ hoch, dass mehrere der Antikörpermoleküle am Ende der jeweiligen Ketten eine antigene Stelle auf einer Zelle oder einem Virus 40 finden. Und diese Wahrscheinlichkeit bleibt hoch ungeachtet der Unregelmässigkeit der Oberfläche der Zelle oder des Virus. Wie bekannt, weist die Zellmembran, 50 die jede Zelle umschliesst, sich nach aussen erstreckende Moleküle auf, die als Transplantations-Antigene bezeichnet werden. Diese Antigene sind diejenigen, die zum Bilden der ersten Schicht 11 in einem Zweischichtsystem zum immunologischen Identifizieren von Zellen benutzt werden, wobei die interessie-55 rende Zelle die dritte Schicht bilden würde. Im Falle eines Vierschichtsystems aus Antigen-Antikörper-Ketten, wie es in Figur 4 abgebildet ist, würden die erste und die dritte Schicht (11 und 30) aus den Transplantations-Antigenen bestehen. Es ist glei-chermassen bekannt, dass ein Virus einen Protein-Überzug aus 60 Proteinmolekülen aufweist, der aufgespalten und getrennt werden kann und diese Moleküle bilden die erste bzw. die erste und die dritte in Zwei- bzw. Vierschichtsystemen der Antigen-Antikörper-Ketten, die zum immunologischen Identifizieren eines Virus 40 verwendet werden. Die Antikörper 12 in der zweiten 6S Schicht und 41 in der vierten Schicht wären, natürlich spezifisch zu der jeweüigen Zelle oder dem Virus, nach dem man sucht. Forming the multimolecular immunologically complexed films according to the present invention is also important in the immunological identification of cells or viruses. As illustrated in FIG. 4, by building up a large number of chains of antigen-antibody complexes starting from the surface of the substrate 10, the probability is relatively high that several of the antibody molecules at the end of the respective chains have an antigenic site on one Find cell or a virus 40. And this probability remains high regardless of the irregularity of the surface of the cell or the virus. As is known, the cell membrane 50 that surrounds each cell has outwardly extending molecules called transplantation antigens. These antigens are the ones used to form the first layer 11 in a two-layer system for immunologically identifying cells, the cell of interest forming the third layer. In the case of a four-layer system of antigen-antibody chains, as shown in Figure 4, the first and third layers (11 and 30) would consist of the transplantation antigens. It is equally known that a virus has a protein coating of 60 protein molecules that can be split and separated, and these molecules form the first and the first and the third in two- and four-layer systems of the antigen-antibody chains that are used to immunologically identify a virus 40. Antibodies 12 in the second 6S layer and 41 in the fourth layer would, of course, be specific to the particular cell or virus you are looking for.

Die Konfiguration des verwendeten Substrates ist für die vorliegende Erfindung nicht kritisch. Vorzugsweise hat es je- The configuration of the substrate used is not critical to the present invention. It preferably has

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doch die Gestalt eines Objektträgers, z.B. in Form von metalli- Ein Glasobjektträger wurde mit einer Schicht aus Indium-sierten Glasobjektträgern, da diese leicht erhältlich sind. Das Metallkügelchen metallisiert. Eine Lösung von mit der Hepatitis Substrat kann auch die Form eines Gurtes haben. Die einzige verbundenem Antigen in 0,85 %iger Salzlösung mit einer Kon-Beschränkung bei der Auswahl des Substrates ist die, dass es die zentration von 1 mg des Antigens pro Milliliter wurde zuberei-Bildung einer monomolekularen Schicht darauf gestatten muss. s tet und zu dieser Lösung gab man Rinderserumalbumin, um Die Abmessungen der Form werden durch die Art und Weise eine Konzentration von 1 mg/ml herzustellen. Ein Tropfen der vorgeschrieben, in der der Test ausgeführt wird, einschliesslich Lösung aus Antigen und inertem Protein wurde im Zentrum des der nachfolgenden Analyse und der Zielsetzung des Tests. Soll Objektträgers angeordnet. Der Objektträger wurde in einer ein besonderer immunologisch aktiver biologischer Bestandteil Feuchtigkeitskammer, einem mit feuchten Schwämmen gefüllgesammelt werden, dann ist ein Gurt bevorzugt. io ten Kunststoffbehälter, bei 23 °C inkubiert, bis das Antigen an Der Begriff «immunologisch inerte organische Verbindung» der metallisierten Oberfläche haftete (10 bis 30 Minuten). Der verkörpert jede organische Substanz, z.B. proteinhaltige oder Objektträger wurde dann mit destilliertem Wasser gewaschen nichtproteiniialtige Materialien, die gegenüber den miteinander und mit einem Luftstrahl trocken geblasen. Das inerte Protein in Wechselwirkung tretenden Bestandteilen nicht reaktiv ist und bedeckte etwa 3/4 des kreisförmigen Bereiches der trockenen die keine Wirkung auf die komplexierenden Moleküle hat. Es i s Adsorptionsschicht und das Antigen etwa1U, wobei ein Haupt-rauss jedoch an dem Substrat haften und die Antigenmoleküle teil der Antigenmoleküle frei zugänglich war. Dieser Objektträtrennen und umgeben. ger wurde dann einer Zubereitung von einem gegenüber dem Der durch die Multischicht-Adsorption geschaffene Kon- mit der Hepatitis verbundenen Antigen als Antiserum wirken-trast kann noch verstärkt werden, indem man Antigen und iner- den Kaninchenserum ausgesetzt, welches Antikörper zu dem te organische Verbindung auf das Substrat 10 in Form eines 20 mit der Hepatitis verbundenen Antigen enthielt. Dies führte zur einzelnen Tropfens des wässrigen Mediums aufbringt und Bildung einer bimolekularen Schicht, in der ein grosser Teil der danach das mit dem Tropfen überzogene Substrat in ein wässri- von aussen zugänglichen Antikörpermoleküle freie Bindestellen ges Medium eintaucht, welches nur die inerte organische Ver- behielt. Objektträger und Antikörperlösung wurden 10 Minu-bindung enthält, um eine monomolekulare Schicht aus dem ten bei 20 °C inlcubiert. Danach wurde der Objektträger wieder kleinen Bereich aus Antigen und inerter organischer Verbin- 25 mit destilliertem Wasser gewaschen. Schliesslich wurde der dung zu bilden, der vollkommen von der inerten organischen Testobjektträger einer Lösung ausgesetzt, von der man annahm, Verbindung umgeben ist. Damit kann man das Problem des dass sie das mit der Heptatitis verbundene Antigen enthielte, nicht-spezifischen Haftens lösen, wenn die Antikörper in einem Die Bildung eines scharf kontrastierenden sichtbaren Fleckes Serum vorhanden sind, da die an der übrigen Oberfläche des auf dem Objektträger zeigte eine positive Reaktion. but the shape of a slide, e.g. in the form of metallic glass slides was made with a layer of indium glass slides as these are easily available. The metal ball metallized. A solution with the hepatitis substrate can also be in the form of a belt. The only associated antigen in 0.85% saline with a Kon limitation in the choice of substrate is that it must allow the concentration of 1 mg of the antigen per milliliter to form a monomolecular layer thereon. and bovine serum albumin was added to this solution to measure the dimensions of the mold by the way to produce a concentration of 1 mg / ml. A drop of the prescribed in which to run the test, including antigen and inert protein solution, was at the center of the subsequent analysis and objective of the test. Shall slide arranged. The slide was collected in a special immunologically active biological moisture chamber, one filled with damp sponges, then a strap is preferred. io ten plastic container, incubated at 23 ° C until the antigen adhered to the term "immunologically inert organic compound" of the metallized surface (10 to 30 minutes). It embodies every organic substance, e.g. Protein or slide was then washed with distilled water, non-proteinaceous materials, blown dry against each other and with an air jet. The inert protein interacting components are not reactive and covered about 3/4 of the circular area of the dry which has no effect on the complexing molecules. It is the adsorption layer and the antigen is approximately 1U, but one main surface adheres to the substrate and the antigen molecules are freely accessible. This object race and surround. Then a preparation of an antigen linked to the antigen linked to the hepatitis created by the multi-layer adsorption can be further enhanced by exposing the antigen and inert rabbit serum, which antibody to the organic compound on the substrate 10 in the form of an antigen 20 associated with hepatitis. This led to the individual dropping of the aqueous medium and the formation of a bimolecular layer in which a large part of the substrate subsequently coated with the droplet is immersed in a binding medium which is free of water and accessible from the outside and which only retains the inert organic contents . Slides and antibody solution were included for 10 minutes to incubate a monomolecular layer from the ten at 20 ° C. The slide was then washed again with a small area of antigen and inert organic compound with distilled water. Eventually, the manure was formed that was completely exposed to the inert organic test slide of a solution that was believed to be compound. This solves the problem of non-specific adhesion if it contains the antigen associated with heptatitis if the antibodies are present in a serum which forms a sharply contrasting visible spot, since the serum showed on the remaining surface of the slide positive reaction.

Substrates adsorbierte inerte organische Verbindung solches 30 nicht-spezifisches Haften von im Serum vorhandenen Bestandteilen verhindert und dadurch den Kontrast verbessert. Substrate adsorbed inert organic compound prevents such non-specific adherence of components present in the serum and thereby improves the contrast.

Nachfolgend wird die Erfindung anhand von Beispielen nä- Beispiele 2 bis 6 In the following, the invention is illustrated by examples n- Examples 2 to 6

her erläutert. Nach dem Verfahren des Beispiels 1 wurden diagnostische explained here. Following the procedure of Example 1, diagnostic

Beispiel 1 Tests ausgeführt und dabei für das dort verwendete Substrat Example 1 Tests carried out and in doing so for the substrate used there

Ein diagnostischer Test auf Hepatitis-Antigen wurde fol- Antigen, immunologisch inerte organische Verbindung und An- A diagnostic test for hepatitis antigen was carried out using the following antigen, immunologically inert organic compound and

gendermassea ausgeführt: tikörper und die folgenden Stoffe eingesetzt: gendermassea executed: body and the following substances used:

Bei- Substrat spiel Example substrate

2 Indiummetallisiertes Glas 2 Indium metallized glass

3 Indiummetallisiertes Glas 3 Indium metallized glass

4 Gold-metallisiertes Glas 4 gold metallized glass

5 T antal-metallisiertes Glas 5 T antal metallized glass

6 Indium-metallisiertes Glas 6 Indium metallized glass

Antigen mit der Hepatitis verbundenes Antigen mit der Hepatitis verbundenes Antigen mit der Hepatitis verbundenes Antigen mit der Hepatitis verbundenes Antigen Insulin Antigen related to hepatitis Antigen related to hepatitis Antigen related to hepatitis Antigen related to hepatitis Insulin

Inerte organische Verbindung Eialbumin Insulin Inert organic compound egg albumin insulin

Rinderserumalbumin Rinderserumalbumin Rinderserumalbumin Beef Serum Albumin Beef Serum Albumin Beef Serum Albumin

Antikörperquelle Antibody source

Kaninchenblutserum Kaninchenblutserum Kaninchenblutserum Kaninchenblutserum Kaninchenblutserum Rabbit Blood Serum Rabbit Blood Serum Rabbit Blood Serum Rabbit Blood Serum Rabbit Blood Serum

C C.

1 Blatt Zeichnungen 1 sheet of drawings

Claims

PATENT CLAIMS

1. Method for binding reactive components of immunological complexes in separate layers on a surface so that the components remain active, characterized by the steps:

Coating a metallized substrate with a monomolecular layer of a first reactive component

Adding inert organic compounds so that the first reactive components are separated from each other by the inert organic compounds, contacting the coated substrate with an aqueous medium containing a second immunologically reactive component specific to the first reactive component for an immunological complex reaction in which the second components combine with the first components to form a bimolecular layer on the substrate, the inert organic compound being present in sufficient quantity to maintain a sufficient average distance between adjacent first reactive components in the monomolecular layer , so that the second reactive components keep active sites for another immunological complex reaction.

2. The method according to claim 1, characterized in that the complex reaction between a protein antigen and a protein antibody takes place specifically to the antigen, that the inert organic compound is an inert protein and the aqueous media are solutions.

3. The method according to claim 1, characterized in that the step of coating the metallized substrate with a monomolecular layer of the first reactive component - preferably a protein antigen - and inert organic compound comprises the following steps:

Producing a first aqueous medium from the first reactive constituent,

Adding the inert organic compound in sufficient quantity to the first aqueous medium,

contacting the substrate with the first aqueous medium in order to partially or completely coat it with the monomolecular layer of the first active ingredient and inert organic compound, and

Removing the substrate coated with the monomolecular layer from the first aqueous medium.

4. The method according spoke 1 or 3, characterized in that the first reactive component is antigen and that the second reactive component is an antibody specific to the antigen, or that the first reactive component is an antibody, and that the second reactive component is an Antigen is against which the antibody is specific.

5. The method according spoke 1, characterized in that the metallized substrate also includes a metal oxide layer which lies between the metal on the substrate and the monomolecular layer, wherein the metal is preferably titanium and the oxide is a titanium oxide.

6. The method according to claim 1, characterized in that the metallized substrate is a metal slide or a metallized glass slide, the metal being selected from indium, gold, silver, tin and lead, and wherein the metallized slide indium on a glass slide, gold a glass slide, or a thin layer of indium, which in turn is coated with a gold layer, on a glass slide.

7. The method of claim 1, wherein forming the first layer comprises:

Producing an aqueous medium which contains an immunologically reactive antibody,

Add a sufficient amount of an immunologically inert organic compound to the medium and contact a metallized substrate

2nd

the aqueous medium to coat at least part of it by adsorption with a monomolecular layer of antibody and inert organic compound, the antibody molecules being sufficiently spaced from one another by the inert inorganic molecules, so that the antibody molecules have a larger number of active sites maintained for a further immunological reaction as if the antibody molecules were closely arranged.

8. The method of claim 1, wherein a multimolecular, immunologically complexed layer is formed, characterized by the steps:

Coating a metallized substrate with a monomolecular layer of an immunologically reactive antigen 15 and an immunologically inert organic compound, so that the antigen molecules are separated from one another by the molecules of the inert organic compound and

Contacting the substrate coated in this way with an aqueous medium which contains the immunologically re-20 active antibody specific for the antigen in order to cause an immunological reaction therewith in which the antibody molecules combine with the antigen molecules and form a bimolecular layer on the substrate, the antibody molecules retain a considerably larger number of active 25 sites for a further immunological reaction than if the antigen molecules were only a short distance apart.

9. The method according spoke 1, characterized in that the step of coating the metallized substrate with

30 of a monomolecular layer of an antigen and an inert organic compound comprises the following steps:

Forming a first aqueous medium containing the immunologically inert organic compound,

Immersing the substrate in the first aqueous medium in order to coat the substrate with a monomolecular partial layer made of the inert organic compound,

Removing the partially coated substrate from the first aqueous medium,

Forming a second aqueous medium containing the im-40 munologically reactive antigen,

Immersing the partially coated substrate in the second aqueous medium in order to coat the remaining surface of the substrate with a monomolecular partial layer of the antigen, so that the monomolecular layer consists of antigen 45 molecules which are separated from one another by the inert organic molecules and

Removing the substrate coated with the monomolecular layer from the second aqueous medium.

10. The method according to claim 1, characterized in that the step of coating the metallized substrate with a monomolecular layer of antigen and inert organic compound comprises the following steps:

Preparation of a diluted first aqueous medium 55 which contains the immunologically reactive antigen

Immersing the substrate in the first aqueous medium in order to coat the substrate with a monomolecular sublayer of the antigen,

Removing the partially coated substrate from the first aqueous medium,

Preparing a second aqueous medium which contains the immunologically inert organic compound,

Immersing the partially coated substrate in the second aqueous medium to coat the remaining surface of the substrate with a monomolecular partial layer of the inert organic compound, so that the monomolecular layer consists of antigen molecules which are separated from one another by the inert organic molecules, and

3rd

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11. The method according to claim 1, characterized in that the step of coating the metallized substrate with a monomolecular layer of antigen and inert organic compound comprises the following steps:

Preparing an aqueous medium containing the immunologically reactive antigen,

Adding the immunologically inert organic compound in sufficient quantity to the first aqueous medium,

Arranging at least one drop of the first aqueous medium on the substrate so that antigen and inert organic compound also form a small area of a monomolecular layer,

Producing a second aqueous medium which contains immunologically inert organic compound,

Immersing the drop-coated substrate in the second aqueous medium to form a complete monomolecular layer on the entire surface of the substrate, including the small area of antigen and inert organic compound surrounded by inert organic compound from the second aqueous medium is

12. The method according to claim 1, characterized by the following stages:

Coating a metallized substrate with a monomolecular layer of an immunologically reactive antigen by producing a dilute first solution of the antigen, immersing the substrate in the first solution in order to coat the substrate with a monomolecular partial layer of the antigen,

Removing the partially coated substrate from the first solution and

Immersing the partially coated substrate in a serum solution containing an immunologically reactive antibody and inert organic compound, the inert organic compound adhering to the substrate to complete the monomolecular layer of antigen molecules separated from each other by the molecules of the inert organic compound , the antibody molecules forming a second layer in the serum.

13. The method according to claim 3 or 4, further characterized by the steps:

Removing the substrate coated with the bimolecular layer from the antibody-containing medium,

Exposing the coated substrate to a third aqueous medium which is believed to contain the same antigen as the first aqueous medium to cause an immunological reaction with the antibody in which the remaining active sites on the antibody molecules are in contact with the antigen molecules connect in the third aqueous medium and

Examine the coated substrate to determine if it carries a third layer, indicating the presence of the antigen in the third aqueous medium.

14. The method according to claim 3 or 4, characterized in that the step of producing a first aqueous medium with the immunologically reactive antigen comprises the following steps:

Separating the antigens, which are the outermost parts of a particular virus or cell, and

Prepare an aqueous medium therefrom before adding the inert organic compound and further comprising the steps of

Immersing the coated substrate in a third aqueous medium, which is believed to contain the particular cell or virus, so that the antibody molecules find antigenic sites on the cell or virus to bind to and

Examine the coated substrate to determine if a third layer is present thereon, indicating the presence of the particular cell or virus in the third aqueous medium.

15. The method according to claim 14, further characterized by the steps:

Removing the coated substrate from the third aqueous medium and

Contacting the coated substrate with another aqueous medium containing the same antibody as before to cause an immunological reaction in which the first active sites of the antibody-2o permolecules in the fourth aqueous medium combine with antigen molecules from the third aqueous medium The said antibodies retain active binding sites so that chains of antigen-antibody complexes can be built up from the surface of the substrate.

16. The method of claim 3, characterized in that the step of adding a sufficient amount of the immunologically inert organic compound to the first aqueous medium comprises adding an amount of

30 is sufficient so that the average distance between neighboring immunologically reactive antigen molecules which are adsorbed on the surface of the substrate is in the order of magnitude of several hundred angstroms.

17. The method according to claim 1, characterized in that the antibody is of the immunoglobulin Ig G class,

and that the antigen in the first aqueous medium is an enzyme, the inert organic compound is bovine serum albumin, and the antibody in the second aqueous medium is obtained from a rabbit to which the enzyme has previously been injected.

18. Application of the method according to claim 1 for determining the presence or absence of a suspected component in an aqueous medium, characterized by the following steps:

45

Producing a first aqueous medium containing an immunologically reactive antigen,

Adding an amount of an inert organic compound to the first aqueous medium,

contacting a metal slide or a metallized glass slide with the first aqueous medium in order to coat the slide in whole or in part with a monomolecular layer of antigen and inert organic compound, the amount of the inert organic compound being sufficient for the antigen molecules to pass through separating inert organic molecules from each other,

Removing the substrate coated with the monomolecular layer from the first aqueous medium,

Exposing the coated substrate to a second aqueous medium containing an immunologically reactive antibody specific to the antigen to produce an immunological reaction therewith in which the antibody molecules combine with the antigen molecules to form a bimolecular layer on the substrate and the 65 Antibody molecules retain a significantly larger number of active sites for a further immunological reaction than if the antigen molecules were arranged close to one another,

631 549 4

Removal of the over-characterized with the bimolecular layer in which the bond between the amino acid substrates from the antibody-containing medium, known as peptide bonds, is known. These heavy and

Exposure of the coated substrate to a light peptide chain is in the general form of the Buchdritt aqueous medium, which is believed to orient the letter "Y" and contains the active or binding sites in the same antigen as the first aqueous medium and Ver 5 at the extreme ends of the two arms of the Y-cause an immunological reaction with the antibody, the antibody in the Ig G antibody.

in which the remaining active sites on the antibody moles- in addition to the immunological reaction that occurs between the culminate in the third aqueous specific protein antigens and specific protein anti-media, which combine and form to form a protein antigen / assay of the coated substrate to determine protein-antibody complex are determined by whether it carries a third layer, which indicates the presence of the present invention also other immunologically complexing antigen in the third aqueous medium. Reactions between immunologically reactive antigens and 19. Use according to claim 18, characterized characterized antibodies. Furthermore, by the term “im-net that the examination stage is the visual observation of the munological reaction”, as it comprises in the present application-coated substrate in solid light, using 15 manure, also specific reactions between an-the metal indium and that the visual determination is made by their biological particles, such as enzymes and substrates, in order to distinguish between four shades of brown, the touch. The terms tigen used in the present application are derived from a bacterium, a virus, an "antigen" and "antibody" include enzymes, substrates and fungus, mammalian tissue or mammalian body fluid and -like biological particles.

where the inert organic compound is selected from cattle- 20 So close e.g. the following systems biological partial serum albumin, egg albumin or insulin. Those who can carry out an immunological reaction as described in the present application:

Viruses

Bacteria and bacterial toxins The invention relates to a method for binding fungi of reactive components of immunological complexes in layers separated from parasites. animal tissue

Publications related to the present invention body fluids and the like.

Mainly as background materials. In the case of viruses, the antigens are virus cultures or parts thereof are the following: 30 and the antibody specific thereto can be administered

«Optica! Measurement of the Thickness of a Film Adsorbed the antigens are generated on a living host. In the from a Solution by Irving Langmuir et al, in the Journal of the following virus systems, antigen-antibody com-American chemical society, volume 59 (July to December 1937) plexes are formed which are useful in the present invention page 1406 , are:

"Immunology and Enzymatic Reactions Carried Out at a 35 Rubella Virus Culture (Antigen) - Rubella Virus Antibody, Solid Liquid Interface" by Alexandre Rothen, in Physiological Polio Virus Culture (Antigen) - Polio Virus Antibody, Chemistry and Physics , Volume 5 (1973) pp. 243-258, Culture of inflammation of the vesicular oral mucosa

"Interactions Among Human Blood Proteins at Interfaces" virus (antigen) - antibodies to this virus by Leo Vroman et al, in Federation Proceedings, Volume 30 No. Virus.

5 (September to October 1971) pages 1494-1502, and <to For bacteria and bacterial toxins or poisons

"The Antibody-Antigen Reaction: A Visual Observation", antigens the respective bacteria or bacterial toxins or from Ivar Giaever in The Journal of Immunology, Volume 110 No. parts thereof and the antibody is obtained by injection of Anti-4 (May 1973) pages 1424 -1426. gene generated in a living host. The following are required

Immunological reactions are still specific, descriptive examples of the antigen-antibody pairs which are seen in effects in which an antigen is used with a second biological method according to the invention, which is specific for the antigen and can.

generally known as its antibody, forming tetanus toxoid suspension (antigen) - tetanus antibody,

of an immunological complex interacts. Im- Diphtheria toxin suspension (antigen) - Diphtheria-anti-munological reactions that occur in a biological system, body,

How an animal or human take place are so vital to Neisseria gonorrhea suspension (antigen) - gonorrhea for fighting disease. In a biological Sy antibody,

stem causes the entry of a foreign protein, i.e. of anti-Treponema pallidumsuspension (antigen) - syphilis antigen, the production of the specific antibody protein for the body.

Antigen in a process that is not yet fully understood. In the case of fungi, the antigens are the extracts of fungal suspensions. The antibody protein molecules have chemical 55 and the antibody is the fungal antibody which is complemented by In binding sites which are those which are generated on the antigen molecule injecting the suspension into a living host, so that antigen and antibody form antigen Antibody complexes from fungal systems are linked together by the immunological complex. the following examples illustrate:

Most antigens are proteins or they contain Pro Aspergillus extract suspension (antigen) - Aspergillus-

teine as an essential component, while all antibodies so fungal antibodies,

Are proteins. Proteins are large molecules of high molecular weight Candida extract suspension (antigen) - Candida mushroom anti-weight, and in particular they are made up of chains of ami bodies.

existing polymers. The antigen and antibody antigens and antibodies in parasite systems can each have several binding sites in protein. The five tested similarly to mushrooms. A typical example

The main classes of antibodies (immunoglobulins Ig G, Ig M, 65 and the system Toxoplasma gondii extract (antigen) - Toxo-Ig A, Ig E and Ig D) are evidently each by at least plasma gondii antibodies.

two heavy (long) peptide chains of amino acids and min- The term «polysaccharides» means a system

at least two light (short) peptide chains of amino acids in which the antigen is a hydrocarbon antigen. A

5

631 549

An example of such a system containing antigen-antibodies is Pneumococcus Polysaccharides (Antigen) - Pneumo-coccus-Antibodies.

In addition to the typical enzyme-enzyme substrate reaction that the present invention is intended to encompass, enzymes themselves or portions thereof can be used as antigens and the antibody is the particular enzyme antibody that is formed after injection by a living host. Exemplary antigen-antibody complexes of enzyme systems are:

Trypsin extract - Trypsin antibody Chymotrypsin extract - Chymotrypsin antibody Pepsin extract - Pepsin antibody Ribonuclease extract - Ribonuclease antibody Thrombin extract - Thrornbin antibody Amylase extract - Amylase antibody Penicillinase extract - Pennicillinase. In the case of hormones, the antigenic component is usually found in a hormone extract and the antibody is the respective hormone antibody that is formed by a living organism after the injection. An exemplary antigen-antibody complex is:

Insulin - insulin antibody.

Immunological reactions can be demonstrated using various techniques, including the use of a suitable substrate, such as a metallized glass or metal slide. When the substrate is exposed to an aqueous medium containing antigen, the antigen is physically adsorbed in a dense monomolecular layer on the surface of the substrate. Subsequent exposure of the antigen-coated substrate to a serum which contains antibodies specific to the antigen results in an immunological reaction in which the antibodies selectively bind to the antigens via the binding sites on the antibody molecule which complement those on the antigen molecule adhere and thereby form at least one bimolecular sublayer of immunologically complexed antigen antibody on the substrate surface.

If the coated substrate containing the antigen-antibody complex is then exposed again to an aqueous medium which contains the same antigen or is believed to contain it, the problem arises that no further binding of antigen to the Antibody will take place since all active sites of the antibodies are already bound to the first antigen layer because the antigen molecules are only a very short distance apart. The problem with surface immunology is therefore that the antibodies bound to an antigen surface lose their activity, i.e. the ability to connect to an antigen, a cell or a virus.

The inventive method for binding reactive components of immunological complexes in separate layers on a surface is characterized in the preceding claim 1.

The invention is explained in more detail below with reference to the drawing. In detail show:

FIG. 1 shows a side view of a substrate after it has first been treated with an aqueous medium which contains an antigen and then immersed in an aqueous medium which contains the antibody specific to the antigen,

FIG. 2 shows a side view of the substrate after the two stages have been carried out as in FIG. 1, but the first treatment has been carried out in accordance with the present invention,

Figure 3 is a side view of the substrate of Figure 2 after a subsequent immunological reaction in which the

coated substrate was immersed in an aqueous medium containing the same antigen and

Figure 4 is a side view of the substrate of Figure 3 after it has been further immersed in an aqueous medium 5 containing the same antibody and with the antibody molecules at the ends of the chains of the anti-gene-antibody complexes antigenic sites on a virus or find a cell to identify them immunologically.

Figure 1 shows a greatly enlarged side view of part of the diagnostic device in the form of a thin disk 10 made of a suitable substrate material, which can be metal, glass, mica, plastic, glass-like silicon dioxide, quartz or a similar material, with metal being preferred since it has the greatest difference in refractive index from protein and preferably the metal is used in the form of a metal or metallized glass slide.

If the substrate 10 is treated with a first aqueous medium, which may be an antigen or an antibody biologically dissolved in salt water, then the substrate has an adsorbed monomolecular layer 11 thereof. For the purposes of the present invention, the treatment can take place by applying a drop of the aqueous medium or by partially or completely immersing it in the aqueous medium.

For the sake of simplicity, the layer 11 adsorbed on the surface of the substrate 10 is referred to below simply as the antigen layer. Each antigen or antibody is adsorbed in such a monomolecular layer where no further adsorption takes place, i.e. Antigen or antibodies adhere to the substrate, but not to themselves. The antigen layer 11 can therefore only be monomolecular and not of greater thickness. The time required to completely coat the substrate with the antigen is a function of the concentration of the antigen in the aqueous medium, the strength with which the aqueous medium is stirred and the temperature of the medium. For example, a 1% bovine serum albumin solution completely covers a slide with a monomolecular antigen 40 in about 30 minutes.

After the monomolecular antigen layer 11 has formed on substantially the entire surface of the substrate 10, the coated substrate is removed from the antigen-containing aqueous medium and next immersed in an aqueous medium containing the specifically reacting antibody to the antigen or which is believed to contain it. This second aqueous medium can contain many components in addition to the specifically reacting antibody, the presence of which is to be detected. However, no antibody other than the specifically reacting antibody will adhere to the first layer of antigen on the substrate. Therefore, only when the specifically reacting antibody is present in the second aqueous medium will immunological complexation take place between the antigen 55 and its specifically reacting antibody and the substrate will therefore have a bimolecular layer after a certain time. The time required for the binding of the second (antibody) monomolecular layer 12 is again a function of the concentration of the specifically reacting protein in the aqueous medium, the stirring strength and the medium temperature. For antibodies in the blood serum, this time can take minutes depending on the concentration, but also up to a day. The second layer can be a sub-layer or a substantially complete one, depending on the three factors above.

As illustrated in FIG. 1, in the antigen layer 11, which essentially completely covers the substrate 10, the distance between the adjacent antigen molecules is mini

631 549

mal and the antibody molecules therefore essentially use all of their binding sites to bind to the antigen molecules and thus lose their activity, i.e. essentially all active sites on the antibody molecules 12 combine with corresponding active sites on the antigen molecules 11.

Since the monomolecular antigen layer 11 is adsorbed on essentially the entire surface of the substrate 10 in FIG. 1, the second layer 12 of the specific antibody for such an antigen combines with it to form the bimolecular layer and a further connection with additional antigen is not possible. since there are no active sites on the antibody molecules in layer 12.