DD214695A1 - SOLID PHASE IMMUNOASSAY FOR THE DETERMINATION OF CIRCULATING IMMUNOMCOMPLEXES - Google Patents

Abstract

The invention relates to a solid-phase immunoassay for the determination of circulating immune complexes from human sera by immobilization of the immunocomplexes-binding protein C1q to siliceous form bodies by means of silane coupling agents and hetero- or homobifunctional reagents. The solid phase prepared in this way can be used repeatedly in the immunoassay by using non-denaturing media to switch off the bound immunoreactants. The method is suitable for the determination of circulating immune complexes in medicine as well as life science research.

Description

"Solid Phase Immunoassay for Determining Circulating Immune Complexes"

Field of application of the invention

The invention relates to a solid phase Iramunoassay for the covalent bonding of proteins to siliziunidioxidhaltige moldings, preferably made of glass, and their repeated use in the (enzyme) immunoassay for the sensitive and specific detection of antigens »antibodies, viruses, etc., in particular of circulating immune complexes. The assay is used in the pharmaceutical industry, medicine, life science research and biotechnology.

Characteristics of the Known Technical Solutions The use of irra-sonoassays for the specific and sensitive determination of haptens, anti-genes and antibodies has become very important in recent years both in clinical routine diagnostics and in life science research. The most frequently used method is the solid phase technique ( ^lf oiid-phase technique), in which one of the immunological reaction partners is bound to a solid phase and thus easy separation of the formed antigen-antibody complexes from the free, unbound reaction partners is possible. Hereby, this was based on K, O. Gatt and G, W * Tregear (Science 158 (1967), 1570) a widespread method of adsorptive binding of proteins to solid custody phases (polystyrene, polyethylene, PVC, etc.) (review: 3, E. Herrmann in: Methods in Enzymology 72 »(1981) • 3, 239).

Disadvantages of this method are:

- The loading density of the plastic materials and thus the sensitivity of Imtnunoassays are limited by the plastic material and the available surface,

During the individual incubation and washing operations by immunoassay, considerable quantities of protein (68 % in virus antigens: 3. E, Herrmann, RM Hendry, MF Collins: 3rd Clin. Microbiol. IO (1979) 210, 50 % in the case of antibodies:

W. Schößler, G. Wegner: Z. med. Labordiagn. in pressure) desorbed. Such desorptions, in turn, adversely affect the sensitivity and decisively determine the error of the method.

- The loaded with proteins plastic materials can be used only once after the immune reaction », so that very valuable and expensive to be prepared proteins are lost.

- The use of these plastic materials as EimveggefäSe represents a not inconsiderable economic factor for larger numbers of samples in the clinical routine

To circumvent these difficulties, various authors have described the use of chemically modified (plastic) materials for covalent coupling of proteins in the immunoassay (supervisor: CU E. Herrmann in: Methods in Enzymology 73 (1981) p. 239). Of such materials, in particular cellulose, Oextrane, agar and its derivatives in addition to polyacrylic acid derivatives, substituted polystyrenes or nylon have been used as matrix · However, these materials have not been able to prevail in the desired extent, once, as the. On the other hand, because these materials bind proteins nonspecifically, are thermally and chemically relatively unstable or microbially easily attacked.

It has been known for some years that the OH groups on SiO 2 surfaces are available as individual chemical reaction sites. Among the most significant reactions is the reaction with Qrganosilanen that have different functional groups · While the silicofunctional group in the presence of water on glass surfaces (or similar substances) is bound, the organofunctional group is the usual chemical reactions using amino, carbonyl -, carboxy, isocyano, diazo, isothiocyano, sulfhydryl or nitroso groups available so that these siliziuraorganischen compounds act as a bridge between the inorganic material and the organic / biochemical compound · In this way, the covalent Binding of enzymes (RA Messing, HH Weetall: U.S. Pat. No. 3,519,538; RE Miller: US Pat. No. 3,669,341) and antigens and antibodies (H.H.Wetall, N.Y. Elmirai US Pat 652,761). These preparations were used exclusively as immobilized enzymes for preparative and analytical purposes as well as immobilized antigens / antibodies for affinity-chromatographic purposes (s.'hierzu also: F. danowski, W "Heyer: Porous glasses, Dt · Verlag für Grundstoffindustrie, Leipzig 1982) In US Pat. No. 3,975,511, WP Vann and S. Yabaumi have described the coupling of antibodies to porous glass and their use in radioimmunoassay for the determination of digoxin, insulin and estriol. This method has two major drawbacks that severely restrict widespread application in practice, if not impossible. Although the use of porous glasses with particle sizes between 0.05 and 12, inter alia, and a mean pore size between 30 A and 1 200 A guarantees a large surface area, but on the other hand leads to significant nonspecific reactions, since serum proteins invade almost exclusively into these pores and on Reason for the molecular sieve effect of the porosity glasses only with great effort

Furthermore, the use of porous glasses requires extensive and extensive centrifugation steps for the separation of the bound reactants from the free and between the individual washing processes. This effort precludes a rational determination of large numbers of samples in the clinical routine and severely restricts the possibilities of a (semiautomatic determination.) In addition, the reuse of the antibodies bound in this manner by spiking the immune complexes formed after the end of the immune response The formation of iratin-corplexes provides a physiological defense mechanism for the rapid elimination of endogenous and exogenous antigens in the body. However, in a variety of diseases, the circulating immune complexes are at elevated levels in the serum detectable and may cause pathological sequelae (see: A. N · Theofllopoulos, F .. O * Dixon: Adv. Immunol 2B (1979) 89) .Therefore, the determination of circulating Iramunkoraplexe in clinical practice is becoming increasingly important (EV Ritzmann, D. G, Daniels: Clin. formerly 28 (1982) 1259). Methods that are generally recognized as specific include those that detect immunodoaplexes because of their ability to bind CIq, a subunit of the first complement component. The Clq solid-phase immunoassays described for this purpose work exclusively with polystyrene adsorptively bound CIq (Zubler, R. et al.: 3. Immunol 116 (1976) 232; DA Pohl et al.: O. immunol. Methods 40 (1931) 313 Ch. Wehler et al .: Mol. Immunol., 18 (1981) 157; W. Schößler et al., Biomed., Biochim * Acta in press), which can only be used once for the reasons described.

Object of the invention

Thus, the object of the invention is to provide a solid-phase immunoassay for the determination of circulating imbrounous complexes using generally accessible and inexpensive support materials which can be regenerated easily and quickly for reuse practically after the determination has been made To significantly improve the economics as well as the accuracy of the assay.

Explanation of the essence of the invention

The object of the invention is to provide a solid-phase irmunoassay based on silica-containing material which has been surface-modified in _a manner known per se with silane coupling agents "for the determination of circulating immunoarplexes."

The object is achieved by a solid-phase immunoassay for the determination of circulating Imtaunkoroplexsn »The inventive assay is characterized in that are used as the solid phase insoluble siliceous Forakörper, which according to known prior chemical surface modification with Silanhaftraitteln and hetero- or horaobifunctional reagents with the Protein CIq are loaded and that after cleavage of the bond between CIq and the applied Imraunreaktanten by non-denaturing media iait with CIq loaded moldings-again be used in immunoassay. The shaped bodies used as a solid phase have a different geometric shape, Particularly suitable are spherical, planar or cylindrical bodies, but also cubic or conical can be used «The volume of the body is at least 0.1 era and should not exceed 15 cm. The moldings are compact or hollow, with cavities closed or open. The silicon dioxide-containing material is in the main

glass, the composition may vary. "It must only be ensured" that there are enough Si-OH groups on the surface. * The non-denaturing media for the cleavage of the bond between Clq and the Itnmunraaktanten are made up of buffered solutions which are between 0.5 m to 3 ra NaCl contain "together" The surface modification for the purpose of creating conditions for the entry of covalent bonds with the protein CIa is carried out by so-called silane coupling agents such as X- (CH ₂ ) -Si ₂ QR

^ OR,

wherein R is alkyl radicals and X amino groups, diazo groups u. a, are * and by reacting the remaining amino group with hetero or homobifunctional reagents such as glutaraldehyde. According to the invention, the protein CIq (a subunit of the first complement component) is bound to the thus activated glass surface in the known manner. Important here is »; the glass bodies to be used according to the invention are easy to handle and have a sufficiently large surface area.

The glass matrix thus loaded with the protein GIq is then used as a solid phase in the (enzyme) immunoassay. "After the immune reaction has ended, the bound immune reactants are cleaved off by suitable, non-denaturing media such as 1 M NaCl solution, so that the covalently bonded CIq for a renewed use in the immunoassay is available. The immunoassay according to the invention is characterized by a number of advantages:

- The covalent bonding of CIq to glass surfaces allows higher loading layers to be achieved. These lead to a higher sensitivity in the immunoassay as well as the improved star-like accessibility of the protein - due to the spacer.

Immobilization does not cause any desorption of the imide reactant during incubation and washing in the immunoassay. This significantly reduces the methodological error.

- The non-specific binding to the glass moldings is negligible

The immunoassay according to the invention leads to a considerable reduction of costs in clinical practice. Once, since the used as disposable containers Plastemaferialien be replaced and on the other hand, since this immunoassay can be easily automated by eliminating elaborate Zentrifugationsschr.itte.

- By covalent binding of the protein CIq to the glass surface, this can be repeatedly used in the immunoassay using non-denaturing media. As a result, the protein is saved consuming only to be prepared, which in turn favorably affects the cost of the assay.

In the following, the invention will be elucidated by some examples. tert ·

Exemplary embodiments Example 1

500 ml of a 1% strength solution of the silane coupling agent N3 1114 (aiainopropyltriethoxysilane, VEB Chemiewerk Nünchritz) in ethanol / water (.1: 1) are added to commercial glass tubes (10 mm in diameter) and heated for 4 hours at elevated temperatures (37 ⁰ C - 6O ⁰ C) incubated * After washing three times with P8S buffer, pH 7.4, are added per 500, ul 2% glutaraldehyde in PBS buffer and incubated at 37 ⁰ C for 2 h. After washing again three times, the thus activated tubes are mixed with 500 μl of CIq in PBS buffer and incubated for 4 h at room temperature. After further washing, any remaining aldehyde groups are blocked with 0.2 M lysine solution. After repeated washing with 0.01 μl phosphate buffer, pH 7 * 4, 0.05 % NaCl, 0.05 % Tween 20 and 0.01 % Contains NaN ₃ , the execution of the Enzyrairarounoassays (EIA) in the usual manner ·

CIq, a subunit of the first complement coronucleotide, binds circulating Iramunkomplexe or aggregated IgG in vivo and in vitro »To prepare the calibration curve appropriate dilutions (7.8 ng / rnl to 7.8, ug / ml) of aggregated IgG with 0, 01m phosphate buffer, pH 7.4, containing 0.05M NaCl, 1.5 % Tween 20 and 0.02 % NaN ₃ . The human sera to be determined are first diluted with 0.2 M EDTA 1: 3 and subsequently with the same dilution buffer such that a final dilution of 1: 100 results. In each case 0.5 ml of these sera dilutions or standard dilutions are transferred to the loaded glass tubes and incubated for 4 h at 37.degree. After washing three times with the above washing buffer, 0.5 ml of a conjugate consisting of a rabbit anti-huraan IgG antibody and alkaline phosphatase are added at a concentration of 15 ng / ral and incubated at 37 ^° C. for 4 h. After washing again, the enzyme activity of the alkaline phosphatase is determined by hydrolysis of 4-nitrophenyl phosphate in 1 ml of diethanolamine buffer pH 9.8 and photoradical measurement of the enzyme product at 405 nm after quenching with 1 N NaQH (W. Schößler et al.Meded.Siochira.Acta , in print),

After execution of the enzyme immunoassay (EIA), the tubes are washed twice with 1 ml of washing buffer and incubated with 1 ml of PBS buffer containing 1 M NaCl, and allowed to stand for 2 h at 37 ° C. This establishes the circulation of circulating immune complexes or interrupted by aggregated IgG at GIq, so that the rait CIq loaded tubes can be reused in the EIA »

Example 2

* "-" - ~ "~~ *" ".... .p

Glass balls of 5.5 mm diameter (frosted, F rJ 50 am) are, as described in Example 1, treated with the silane adhesive NB 1115 (Glycidoxypropyltriäthoxysilan, VEB Chemiewerk Nünchritz) and then activated with glutaraldehyde and bonded with Clq. To carry out the EIA, the

Place glass beads in commercial microtiter plates containing 96 samples of a 300 yUl, and subsequently add with jo 200, μl solution of aggregated IgG or Humanserura according to Example 1, respectively. The sequence of the individual steps is analogous to Example 1, the washing operations being carried out by covering the microtiter plate with 200 μl of washing buffer after each displacement and emptying them by simply turning them over. Thus, compared to Example 1, in which each tube must be aspirated by vacuum in the usual manner, the washing process is greatly simplified. After incubation with the conjugate and displacement with the substrate, the enzyme activity is evacuated by aspirating the formed dye with a suction cuvette (VEB Carl Zeiss CJena) determined · According to Example 1, the bound Imraunkocnplexe and the conjugate with PBS buffer, pH 7.4, containing 10 mM EDTA and 1 ra NaCl, cleaved and eluted, so that the glass beads used again in ΞΙΑ can · be

Example 3

Zylinderförraige shaped body made of glass (outside diameter 6 mra

 Internal diameter about 4 ram, height about 3 mra) are activated and loaded as described in Example 1 or 2 with silane coupling agents, glutaraldehyde and CIq. These glass moldings are introduced into polystyrene microtiter plates and subsequently carried out the EIA in the manner described. The enzymatic processes are also carried out by simply filling the sample wells with washing buffer (or tap water) and inverting the covered microtiter plates. After the enzymatic reaction, the activity of the enzyme is determined by photometric determination of the reaction product perpendicular to the clear microtiter plate with a suitable instrument as described in Examples 1 and 2 by elution with reactivated in NaCl and used repeatedly.

Claims (3)

, 4 (Γ
invention claim 1. Solid phase Imnjunoassay for the determination of circulating Immunkckaplexsn based on siliciurndioxidhaltiger materials, characterized in that are used as the solid phase siliceous moldings which loaded after previous, known chemical Qubflächenraodifizierung with silane coupling agents and hetero or homobifunctional reagents with the protein CIq and that after cleavage of the bond between CIq and the applied immune reactants by non-denaturing media such as buffered solutions containing 0.5 to 3 ra NaCl, the CIq-loaded molded bodies are reused in the immunoassay · 2. Solid phase immunoassay according to item 1, characterized in that the moldings used in the immunoassay as a solid phase spherical, planar, zylinderförraige, cube-shaped and conical shape that they are both compact and hollow body, that the hollow bodies are open or closed and that the volume dsr shaped body OyI cm is ⁰ to 15 cm, 3. FSstphasen-Iramunoassay according to item 1, characterized in that the shaped siliceous material is glass ·