Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/80—Fluorescent dyes, e.g. rhodamine
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/82—Hepatitis associated antigens and antibodies

Definitions

• the invention relates to a method for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescent labeling substance and an excitation agent for this using heterogeneous assays known per se.
• the radioimmunoassay is described in Journal Clinical Endocrinology 21_ (1967), p. 973 and Ibid 2 (1968), p. 343.
• the main disadvantage of this assay lies in the necessary handling of radiation-emitting isotopes and in the complex equipment required to carry out such an assay.
• chemiluminescent substances used up to now for chemiluminescence are those which are very difficult to address to the reaction partners of a serological
• Luminol is specified there as a particularly suitable compound 30 for this purpose. Hydrogen peroxide or calcium hypochlorite should be considered as excitation agents for chemiluminescence. Furthermore, this US patent specification mentions fluorescein isothiocyanate for a homogeneous fluorescence assay.
• the invention has for its object to find from the large number of known chemiluminescent marking substances and excitation means combinations thereof which make it possible to improve the method described in the introduction in such a way that it can be carried out safely, easily and with high efficiency.
• Antibodies is spoken, these terms should be understood as much as possible. Antigens are substances that follow. Introduction to the organism of humans and animals the formation of cause.
• foreign proteins work more animal and vegetable future, especially those of infectious agents, as well as many substances of complex nature with fat, saccharide (or sugar), amine and azo-like structure.
• it can be substances, eg proteins, proteins, polysaccharides, lipids or nucleic acids, which cause the formation of antibodies in the organism of vertebrates and humans and react specifically with them.
• Haptens are also to be counted in general, to which one also says “incomplete antigens", which due to their small size alone do not cause the formation of antibodies, but can form a specific bond with the corresponding antibodies.
• Antigens can be, for example, viruses, bacteria or fungi or parts of these.
• certain hormones, vitamins, enzymes or certain are also be also used to give a specific hormones.
• antibodies are specific products of the immune response which are formed in the vertebrate or in the human organism after contact with an antigen and can react specifically with the antigen.
• specific binding proteins that specifically target one or more substances, such as e.g. Antibodies that can bind.
• An example of this is protein A.
• the antibodies which are subordinate to the immunoglobulins of the IgG, IgM, IgA and IgE classes. These are described in detail in Kabat "Introduction to Immunochemistry and Immunology", Springer Verlag, 1971, pp. 143-197.
• the method according to the invention for the detection of antigens and antibodies in viral is of particular importance and bacterial diseases.
• the detection of the surface antigen of the hepatitis B virus is of particular importance.
• the detection of antibodies against the Herpe ⁇ - or Toga Vire * n succeeds.
• fluorescein or fluorescein derivatives When using fluorescein or fluorescein derivatives, it has been shown that they can be bound particularly well to proteins or proteids. This means that fluorescein is substituted with one or more groups which favor its affinity with regard to coupling with the antigens and antibodies mentioned.
• the isothiocyanate and isocyanate groups have proven to be particularly advantageous as substituents which promote or promote the coupling.
• a particularly preferred position of the isothiocyanate group results from the following formula (FITC).
• the coupling ability of the fluorescein which may also be provided with non-coupling substituents in addition to the coupling-promoting or mediating substituents, can be brought about or improved by adding a suitable reagent, the one Coupling reaction or bridging between the fluorescent base body and the respective antigen or antibody or their equivalents.
• a suitable reagent for coupling to the antigen, antibodies or complexes thereof zu ⁇ no additional coupling group is required.
• carbodiimide derivatives are particularly suitable for this, in particular (N-ethyl-N '- (III-methylamino-propyl) carbodiimide hydrochloride.
• the chemiluminescent labeling substance in the form of fluoro-escein or its derivatives is bound to a protein, in particular an immunoglobolin, in the preparation of the chemiluminescent conjugate.
• the excitation agents to be used according to the invention basically react with the chemiluminescent labeling substance coupled to the antigen, the antibody or their complexes in such a way that the latter emits photons.
• Chloramine T is preferred among the chloramines in question. Chloramine T is the international free name for N-chloro-4-toluenesulfonic acid amide sodium or tosylchloramide sodium.
• Calcium hypochlorite which is used as an excitation agent when using fluorescein isocyanate, shows various remarkable advantages, especially in aqueous solution. In this way, the assay can be reproduced exceptionally precisely.
• concentration of the excitation agent desired in the individual case can be easily determined. In general it can be said that an approximately 0.1 to 20% by weight and in particular a 0.2 to 10% by weight calcium hypochlorite solution leads to advantageous results. An approximately 1.0 to 4.0% by weight solution is very particularly preferred, a 2% by weight solution proving to be very particularly favorable.
• the method according to the invention can be carried out on the basis of heterogeneous assays which are known per se. Such processes are described in a variety of ways in the literature. For this purpose, reference should be made, for example, to US Pat. No. 4,238,195 (see in particular columns 7 to 11, line 15).
• the method according to the invention can consequently be implemented in a variety of ways, which is readily apparent to the person skilled in the art. Basically, what happens in a manner known per se must first of all involve the antigen / antibody complex on the basis of a serological reaction - - - be formed. This can erfol ⁇ gen by first either the antigen and present Anti ⁇ body in solution or a partner, ie the antigen or antibody on a solid phase bound to "in various ways is. This is called either a liquid After the serological reaction has been completed, the antigen / antibody complex is separated from the remaining reaction medium in both cases.
• the solid phase assay in the solid phase assay, this can be done in a simple manner by the supernatant liquid, if appropriate After centrifugation, the liquid phase assay is separated, for example, by centrifugation with subsequent decanting or filtering, in particular by ultrafiltration, chromatography and the like, the antigen / antibody isolated in the manner described or similar Complex is then brought into contact with a liquid medium, which is the contains chemiluminescent conjugate to be interacted with.
• the chemiluminescent conjugate contains one of the aforementioned chemiluminescent labeling substances in addition to the antigen or antibody (or protein).
• a new chemiluninescent complex is formed from the chemiluminescent conjugate and the antigen / antibody complex, which is isolated, for example, using one or more of the aforementioned separation methods. It may already be present in the measuring cell or may have originated in it, or may be transferred into such a cell. In any case, the respective excitation agent is added before the measurement is carried out, in which it is not essential in which type of solvent, if necessary, it is present.
• An aqueous medium, in particular an alkaline medium, in which the excitation agent is contained is preferred.
• chemiluminescent labeling substance is “indirectly” bound to the respective reaction partner of a serological reaction.
• a direct assay Either an antigen or an antibody is specified.
• an antibody or antigen conjugate (with the chemiluminescent marking substance) is added and a chemiluminescent complex is formed, which is then isolated. This is followed by the chemiluminescence measurement after adding the excitation agent.
• the method according to the invention can also be carried out with great advantage on the basis of a sandwich assay.
• a sandwich assay can in principle be used both in the context of a liquid phase and a solid phase assay.
• the essential characteristic of a chemiluminescent conjugate formed in the course of a sandwich assay can be seen in the fact that in the complex measured with respect to chemiluminescence the reactant to be detected in the form of an antigen or an antibody, both with its conjugated and with its conjugate non-conjugated reactant has reacted.
• the coupled labeling substance is disregarded, it is a symmetrically structured complex in which the central reaction component is the antigen or the antibody that is to be detected.
• the predetermined reaction partner or the predetermined reaction component interacts with it on two sides of these central reaction components.
• the first reaction partner (labeled or unlabeled) can thus be an antigen.
• the second reaction partner is an antibody
• the first marked reaction partner and second reaction partner (antigen or antibody) are specified in terms of quantity.
• Chemiluminescence can be measured with commercially available photometers. The procedure can be such that a calibration curve is determined using given known amounts of antigen and antibody. Values determined with an unknown substance (be it an antigen or an antibody) are then quantitatively evaluated on the basis of the calibration curve.
• the advantages achievable with the invention are to be seen in particular in the fact that the chemo-luminescent marking substance used in each case is extremely easy to bind to a reaction partner of a serological reaction, namely in the form of antigens and antibodies or binding proteins.
• the marking substances according to the invention and the excitation agent are of simple construction and are safe for the personnel involved in the method according to the invention, in particular with regard to the release of toxic compounds.
• the conjugates or complexes produced in this way are relatively stable, which is advantageous for carrying out the process.
• the FITC to immunoglobulin G from the Zie ⁇ was ge by the method of BT Wood, SH Thomson and G. Goldstein, in Journal of Immunology 9_5 (1964), p 225. , bound.
• the absorbance at 495 nm of the chemiluminescent conjugate produced in this way was likewise measured in the photometer, and the amount of bound FITC was thus determined.
• a series of dilutions of 10 dilutions was then also made and the chemiluminescence of a 20 ⁇ l sample of each dilution was measured. The result of the series of measurements is shown in the following series of numbers.
• the detection limit for the FITC-protein conjugate was accordingly 0.064 ng / ml. It follows from this that the FITC conjugates are exceptionally well suited for the detection of antigens, antibodies or their complexes via a serological reaction by means of chemiluminescence.
• Methylene blue and thionine were given in a volume of 20 ⁇ l each in the amounts shown in the table below.
• Methylene blue was excited by adding 100 ⁇ l of an aqueous chloramine T solution (16 mg / ml) and 100 ⁇ l of a 10 vol.% Hydrogen peroxide solution.
• Thionine was excited by adding 20 ⁇ l of an aqueous chloramine T solution (20 mg / ml) and 100 ⁇ l of a 5 vol.% Hydrogen peroxide solution.
• the excellent photon yields shown in the table below were measured with a reaction time of 30 seconds.
• BSA was absorbed on a flexible microtiter plate.
• the BSA was added in an amount of 0.1 mg / ml in a solution which was adjusted to pH with a sodium carbonate buffer.
• each well was removed incubated with 100 ⁇ l of a FITC-labeled anti-rabbit serum (90 min at 37 ° C.) After washing again three times, the individual wells were cut out and the chemiluminescence was measured in a commercially available photometer minescence triggered by the addition of 100 ⁇ l HO- (30%) and 100 ⁇ l chloramine (79 mg / ml) The result of the measurement is shown in the attached figure Numerical values on the abscissa express the dilution rate rad of the rabbit hyperimmune serum, while the blank value is a value that was determined in a special way.
• the mean blank values X have been determined and added to this the product of 2.58 x 6 by several blanks, wherein the factor $ 'represents the Standardabwe' ichung.
• the figure shows that the limit titer of rabbit serum is approximately 1: 32788.
• the same rabbit serum had a limit titer of 1: 4096 in the solid phase radioimmunoassay and a limit titer of 1: 512 in the ELISA.

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• 1981
  • 1981-12-21 JP JP57500285A patent/JPS57502137A/ja active Pending
  • 1981-12-21 WO PCT/EP1981/000202 patent/WO1982002252A1/de not_active Ceased
  • 1981-12-21 DD DD81235987A patent/DD202474A5/de unknown
  • 1981-12-21 BR BR8108936A patent/BR8108936A/pt unknown
  • 1981-12-22 ES ES508285A patent/ES8305500A1/es not_active Expired
  • 1981-12-22 US US06/333,407 patent/US4491634A/en not_active Expired - Fee Related

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