DE4424275A1 - Detecting lectin(s) in plant extracts and their specific antibodies in body fluids - Google Patents

Abstract

In the detection of lectins (L) in plant extracts and of L-specific Ig in body fluids, using enzyme immunoassay, the new feature is that all immunological reagents used are monoclonal antibodies (MAb). The bound antibody, for L-capture/presentation, is one of SIFIN anti-ML-A-5H8 and anti-ML-A-5F5, and the other of these 2 MAb is used as detector antibody (for the L-determinants, or in competition with naturally expressed Ig in animal or human body fluid). Each of the MAbs can bind to determinants on the A chain of mistletoe lectins (ML) I, II and (III). Also new are kits contg. MAb for detecting L or its specific Ig.

Description

The invention relates to a method for quantitative detection of mistletoe lectins I and / or mistletoe lectin II and / or mistletoe lectin III in mistletoe extracts.

Likewise, the invention relates to a method that Presence and / or concentration of mistletoe lectin-specific Immunoglobulins in human or animal body fluid skills. Field of application of the invention is Drug validation and clinical diagnostics.

The lectins of mistletoe (Viscum album) are considered to be immune most biologically potent substances related to the expanding therapeutic use of mistletoe extracts for the treatment of human tumor diseases in increasing scientific and public interest.

The in very low concentrations both immunostimulating and also cytotoxic to different cell systems Mistletoe lectins consist of two intertwined via disulfide bridges Chains, whereby after their pure representation (patent, U. Pfüller et al. Process for obtaining A and B chain of mistletoe lectin I, 296 703 BC 12.12.91) determined for the toxic subunit (A chain) that it inhibits protein synthesis as RNA N-glycosidase (Y. Endo u. a. FEBS Katt. 231, 378 (1988)). The B chain realizes with their sugar-binding specificity to terminal galactosyl structure turen (T.L. Reiko et al. J. Biol. Chem. 267, 23722 (1992)) necessary cell contact. The division of mistletoe lectins into the dimeric mistletoe lectin I and the monomeric mistletoe lectins II and III was carried out according to characteristic galactosyl structures for the B chain binding and according to the electrophoretic mobility of the Chains under reducing conditions (H. Franz, Oncology 43, 23 (1986)).

The clinically relevant examinations carried out so far favor the use of mistletoe lectin I (T. Hajto et al. Cancer Res. 49: 4803 (1989); Hajto et al. a. Cancer Res., 50 3322 (1990); I. Beuth Clin. Investigious. 70, 658 (1992)), wherein Presence in mistletoe extracts only indirectly by comparison with  a cleaned mistletoe lectin (patent, P. Ziska, H. Franz, Process for the production of lectins, 144 356 v.15.10.80) without Differentiation was specified. In contrast, the "in vitro "Cytotoxicity of mistletoe lectin II and especially of Mistletoe lectin III compared to mistletoe lectin I as significantly higher (I.B. Dietrich et al. Anti Cancer Drugs 3, 507 (1992)). The previously published methods for the detection of mistletoe lectins in mistletoe extracts with an enzyme immunoassay (P. Ziska et al. in: T.C. Bog Hansen et al. a. Lectins vol IV, 473 (1985)) or one Lectin binding enzyme immunoassay (O. Vang et al. In: T.C. Bog- Hansen et al. a. (1989)) describe the use of polyclonal Mistletoe lectin-specific antibody preparations, their limited Sensitivity and reproducibility due to the different Antibody Repertoires of individual Antigen Determinants from Mistletoe lectin I-III no differentiated analysis of the content Enable mistletoe lectins I, II and III. The standardization of therapeutically used mistletoe extracts and their Medicinal product control with regard to the quantitative Assessment of mistletoe lectins I-III has so far not been possible (H. Lentzen, Therapeuticon 10, 436 (1992)).

In addition to the detection of immunostimulating and cytotoxic Mechanisms triggered by the therapeutic parenteral Application of mistletoe extracts, is the assessment of humoral Immune response to the ingredients mistletoe lectin I, II and III of particular clinical importance. With the previously used direct enzyme immunoassay using anti-human Immunoglobulin detection antibodies are only very insensitive and undifferentiated based on the lectin pattern of the extracts, to register an immune response (A. Stettin et al. Klin. Weekly 68, 896 (1990)).

The aim of the invention is to provide a method develop exclusive use of monoclonal antibodies, with the highly sensitive reproducible concentrations of Mistletoe lectin I and / or mistletoe lectin II and / or mistletoe lectin III in plant extracts, preferably from mistletoe or with which the detection of mistletoe lectin-specific  Immunoglobulins in body fluids, preferably from humans, differentiated for the mistletoe lectins I-III is made possible.

The process according to the invention is characterized in that monoclonal antibodies of the mouse hybridoma cell lines SIFIN Anti ML-A-5H8 (AC-1) and SIFIN Anti ML-A-5F5 (AC-2) are used as capture antibodies for mistletoe lectins I, II and III. be used. The monoclonal antibodies react preferentially with epitopes on the A chain of the native mistletoe lectins I, II and III (Tab. 1, Fig. 1). The immobilized monoclonal antibody is brought into contact with a sample of the liquid which contains mistletoe lectins in addition to other plant constituents, extracted from mistletoe plant material or mistletoe lectins I, II and III purified using protein-chemical methods, each separately as the standard reference substance. The solid-phase bound antibody-antigen complex which forms is reacted with either the enzyme-labeled monoclonal antibody of the mouse hybridoma cell line SIFIN Anti ML-A-5F5 (AC-1) or the cell line SIFIN Anti ML-A-5H8 (AC-2). After washing out the substances not bound to the solid supports, the solid phase-bound enzyme activity is determined in a known manner. The technique of fixing the Fanganti body to the relevant carrier materials is known to the person skilled in the art. The monoclonal antibodies used in the method according to the invention are released from the hybridoma cell lines mentioned using methods of cell culture technology in serum-free cell culture media and then concentrated, purified and also covalently linked to enzymes such as peroxidase, alkaline phosphatase and others.

The advantages of the method according to the invention for the differentiated detection of mistletoe lectin I and / or mistletoe lectin II and / or mistletoe lectin III in mistletoe plant extracts are based on the different binding behavior of the monoclonal antibodies to accessible epitopes on the mistletoe lectins I-III in the combinations AC-1 and AC-2 ( Fig. 1). The mathematical evaluation (Tab. 2) of the preferred (AC-1) or equivalent (AC-2) binding of the monoclonal antibodies to mistletoe lectin II and III in comparison to binding to identical concentrations of mistletoe lectin I enables a differentiated lectin analysis under different therapeutic Mistletoe preparations (Tab. 3).

The method according to the invention is further characterized thereby records that the monoclonal mistletoe lectin-specific Antibodies in the combination AC-1 or AC-2 for competitive Detection of mistletoe lectin-specific immunoglobulins in human body fluids differentiated for Mistletoe lectins I-III are used.

The immobilized capture antibodies SIFIN Anti ML-A-5H8 and SIFIN Anti ML-A-5F5 are used for the separate native presentation of mistletoe lectins I-III. The antibody-antigen complex which forms is brought into contact either sequentially or simultaneously with diluted body fluids from patients who have been treated therapeutically with mistletoe extracts and with the respective enzyme-labeled monoclonal SIFIN Anti ML-A-5F5 or SIFIN Anti ML-A-5H8 . After washing out the substances not bound to the solid support, the remaining enzyme activity is determined in a known manner. In this assay, the z. B. in human body fluids marked mistletoe lectin-specific immunoglobulins concentration-dependent epitopes on the natively presented mistletoe lectins I-III, to which the respective monoclonal detection antibody binds. In comparison to a control (human body fluid free of mistletoe lectin-specific immunoglobulins), the reduction in the detection of the solid phase-bound lectin concentrations is chosen as a measure of competition ( Fig. 2) and defined as lectin-binding units (LBE).

The amount of the LBE

correlates directly with the mistletoe lectin-specific immune globulin titer in human body fluids (Tab. 4). The advantage of the method according to the invention lies in that the specificity of the monoclonal antibodies used gently exceptionally high sensitivity of detecting one  Mistletoe lectin-specific humoral immune response in body fluids from people using for therapeutic reasons Mistletoe extracts are treated. This is of particular importance differentiated detection of human immunoglobulins against the native mistletoe lectins I-III, which is a comparison with that Lectin patterns of the preparations used in the sense of a dose-we principle.

Embodiment 1. Preparation of the mistletoe lectin-specific monoclonal antibody

The hybridoma cell lines SIFIN Anti ML-A-5H8 and SIFIN Anti ML-A-5F5 and the SIFIN Anti cell lines used for characterization ML-B-1C6 and SIFIN Anti ML-B-3B11 were developed after spleen fusion cells of Balb / c mice that are linked to the A chain of mistletoe lectin I (MLI) or were immunized with the B chain of mistletoe lectin I. and cells of the mouse myeloma cell line P3X63 Ag8, 653 per se established methods (J.H. Peters and H. Baumgarten, Monoclonal antibodies: production and characterization, Springer Verlag Heidelberg, New York, London, Paris, Tokyo, Hong Kong (1990)).

The monoclonal released under serum-free culture conditions Antibodies (mAbs) were enriched by known methods, cleaned and also conjugated with horseradish peroxidase (POD).

2. Characterization of the mistletoe lectin-specific monoclonal Antibodies SIFIN Anti ML-A-5H8 and SIFIN Anti ML-A-5F5

The results of a concentration-dependent recording of Mistletoe lectin I (MLI) and the A or B chain of the MLI (MLI- AC, MLI-BC) with the known method of two-sided Binding assays using mistletoe lectin only specific mAbs are summarized in Tab. 1.

The mAbs SIFIN Anti ML-A-5H8 and SIFIN Anti ML-A-5F5 can be used as Solid phase antibodies in addition to the MLI also the MLI-AC and MLI-BC  present. The effectiveness of binding the corresponding Detection antibody SIFIN Anti ML-A-5F5 or SIFIN Anti-ML-A-5H8 (each present as a POD conjugate) is compared to the native ML I for the MLI-AC or the MLI-BC by a factor of 30 or 50 reduced.

The presented antigens are detected with a mAb that in addition to the MLI only with the MLI-BC but not with the MLI-AC reacts (SIFIN Anti ML-B-1C6), the detection effectiveness remains for MLI and MLI-BC of the same order of magnitude, but is for the MLI-BC by a factor of about 55 (SIFIN ML-A-5F5) or 22 (SIFIN ML-A-5H8) increased. This serious difference remains also received when the presentation of MLI and MLI-BC through a MLI-BC-specific mAb (SIFIN Anti-ML-B-3B11) is made.

It is concluded that the binding of mistletoe lectin specific mAb pairs SIFIN Anti ML-A-5H8 / Anti ML-A-5F5 (AC-1) or SIFIN Anti-ML-A-5F5 / Anti ML-A-5H8 (AC-2) preferably not competing epitopes on the A chain of native mistletoe lectin that may be adjacent regions on the MLI-BC include. The mAbs listed in Tab. 1 are not in the Able to inactivated (95 ° C / 5 min) MLI, or inactivated MLI-AC and to bind MLI-BC, d. H. they only bind accessible epitopes that must be in native conformation.

3. Two-sided binding test for the cleaned mistletoe lectins I-III

Into the wells of immunoassay microtitration plates 110 µl of the dissolved in carbonate buffer (0.05 mol / l, pH 9.6) monoclonal antibody (2.5 µg / ml) SIFIN Anti ML-A-5H8 (AC-1) or SIFIN Anti ML-A5F5 (AC-2) pipetted. After a The wells were shaken in for 30 min / 35 ° C a machine 5 times with 275 µl phosphate buffer (pH 7.4), containing 0.05% Tween 20 (PBS-Tw), washed. All further Antigen-antibody incubation steps were carried out for the antibodies combination AC-1 with 30 min / 35 ° C and for AC-2 with 30 min / RT performed shaken.  

100 μl were placed in the washed wells of the microtest plates PBS-Tw submitted and in each 4 wells of the series A 100 ul one to 20 ng / ml (AC-1) or 100 ng / ml (AC-2) in PBS-Tw diluted solution of MLI, MLII and MLIII pipetted. After a serial dilution in 100 µl steps from A to H. incubated and washed (5 times with PBS-Tw). To prove the bound mistletoe lectins were diluted in PBS-Tw POD-labeled monoclonal antibody SIFIN Anti ML-A-5F5 (1: 4000) for the AC-1 combination and the antibody SIFIN Anti ML-A-5H8 (1: 2000) for the AC-2 combination.

After incubation and washing, the relevant development of the enzyme activity bound to the solid phase was carried out with a ready-to-use solution of tetramethylbenzidine (100 μl / well) with shaking (15 min / RT). The photometric evaluation (450/630 nm) after stopping the reaction with 100 µl 0.5 N sulfuric acid led to the concentration-extinction relationships in Fig. 1.

These relationships make it clear that for the antibodies combination SIFIN Anti ML-A-5H8-Mistletoe lectin-SIFIN Anti ML-A-5F5-POD (AC-1 ELISA) for the detection of mistletoe lectins the average factor of 2.8 differentiating recovery (ng / ml) occurs between MLI and ML II or ML III.

The reverse antibody combination SIFIN Anti ML-A-5F5 - mistletoe lectin - SIFIN Anti ML-A-5H8-POD (AC-2-ELISA), however, gives for the mistletoe lectins I-III almost identical recovery rates. The extremely reproducible difference in the binding of the mAb Mistletoe lectin I-III in the antibody combinations AC-1 and AC-2 implies a mathematical evaluation (Tab. 2) differentiated lectin analysis of aqueous mistletoe extracts with regard to their content of MLI and ML II or MLIII.

4. Detection of mistletoe lectin I as well as mistletoe lectin II and III in preparations made from aqueous mistletoe extracts

According to the test conditions listed under 3 Samples of therapeutically used mistletoe extracts in AC-1 and AC-2 ELISA examined. On each micro test plate also a sample of a control standard from MLI, ML II and ML III  carried along. The ones obtained compared to the MLI control standard Concentrations resulted from the mathematical evaluation (Tab. 2) the results summarized in Table 3 for ML I and for ML II and ML III.

With the detection limit of 2 ng / ml in the AC-2 ELISA there are differences adorned lectin analyzes of mistletoe extract preparations in all doses that are therapeutically feasible.

5. Detection of mistletoe lectin-specific antibodies in sera from Patients with mistletoe extracts for therapeutic reasons were treated

Analogous to 3. the wells of immunoassay microtitrations plates with the mistletoe lectin-specific mAb SIFIN Anti ML-A-5H8 loaded adsorptively.

In row 1 of the subsequently washed and with 100 ul PBS-Tw filled wells were 100 ul one in PBS-Tw to 2000 ng / ml diluted, purified mistletoe lectin I sample, which after serial dilution in 100 µl steps (from 1-12) was incubated shaken (30 min / 35 ° C).

After washing (5 times with PBS-Tw), 100 μl of a 1:50 dilution (in PBS-Tw - 10% FcS) of a control serum which did not contain any antibodies against the mistletoe lectins I-III was pipetted into each well of the A series. Rows B to H received 100 µl of a 1:50 diluted patient serum sample in the same way. After a shaken incubation (30 min / 35 ° C.) and subsequent washing (5 times with PBS-Tw), 100 μl of the labeled (POD) monoclonal antibody SIFIN Anti ML. Diluted in PBS-Tw (1: 5000) was added to all wells of the plate -A-5F5 pipetted. The subsequent incubation and washing process was identical to the previous one. The development and evaluation of the enzyme activity bound to the solid phase was carried out analogously to point 3 and resulted in the competition shown in FIG. 2 of the binding of the mistletoe lectin-specific mAb SIFIN Anti ML-A-5F5 to mistletoe lectin I by mistletoe lectin-specific immunoglobulins in patient sera. The evaluation of the different competition of SIFIN Anti ML-A-5F5 epitopes on the mistletoe lectin I or III presented natively via the solid-phase-bound mAb SIFIN Anti ML-A-5H8 by mistletoe lectin-specific immunoglobulins in 10 different patient sera is summarized in Table 4.

Table 1

Reaction of mistletoe lectin-specific monoclonal antibodies with mistletoe lectin I and the A and B chain of mistletoe lectin

1A. Figures in ng / ml, which correspond to OD 450 = 1.0

1B. Correction to 100 ng / ml ML I

Table 2

Calculation of ML I and ML II / III in different mistletoe preparations according to AC-ELISA in ng / ml

Table 3

Calculation of ML I and ML II / III in different mistletoe preparations according to AC-1 and AC-2 ELISA

Table 4

Lectin binding units of antibodies in patient sera competing for SIFIN-Anti-ML-A-5F5 epitopes

Claims (10)

1. Method for the detection of lectins in plant extracts and lectin-specific immunoglobulins in body fluids, in particular for the quantitative determination of mistletoe lectin I or mistletoe lectin II and / or III in aqueous mistletoe extracts and for the detection of their specific antibodies in human and animal body fluids with an enzyme immunoassay on the Basis of antibodies, characterized in that the antibodies used as immunological reagents in the respective assay are all monoclonal, the antibody SIFIN Anti ML-A-5H8 bound to a solid phase or the antibody SIFIN Anti ML-A-5F5, each of which has receptors, preferably to antigen determinants on the A chain of mistletoe lectins I, II and III, as monoclonal lectin capture or lectin presentation antibodies and the antibody SIFIN Anti ML-A-5F5 or the antibody SIFIN Anti ML-A-5H8 as a monoclonal detection antibody for free or in competition lectin determinants accessible to naturally expressed immunoglobulins from human or animal body fluids are used. 2. The method according to claim 1, characterized in that the quantitative detection of mistletoe lectin I and / or mistletoe lectin II and / or mistletoe lectin III, in which at least one epitope per molecule (monomer) or molecular association (dimer) is detected by the antibody binding, comprises the following stages:

• a) implementation of the sample with the mistletoe lectin I and / or mistletoe lectin II and / or mistletoe lectin III to be detected and the purified lectin standards (mistletoe lectin I, II and III) with the immobilized monoclonal antibody SIFIN Anti ML-A-5H8;
• b) removal of unbound components and exposure to the enzyme-labeled monoclonal detection antibody SIFIN Anti ML-A-5F5 or its enzyme-labeled antibody fragments;
• c) Removal of unbound constituents, manifestation of the bound enzyme portion and calculation of the concentration in comparison with the standard lectin concentration (mistletoe lectin I) in a manner known per se.

3. The method according to claim 1 and 2, characterized in that for quantitative detection of mistletoe lectin I and / or mistletoe lectin II and / or mistletoe lectin III, in which the antibodies binding is detected regardless of the molecular lectin structure Capture and detection antibodies in reverse order be used. 4. The method according to claim 1-3, characterized in that for differentiated detection of mistletoe lectin I and / or mistletoe lectin II and / or mistletoe lectin III in mistletoe extracts different binding of the antibody combination purified mistletoe lectins II and III compared to mistletoe lectin I is used. 5. The method according to claim 1-4, characterized in that the quantitative detection of mistletoe lectin-specific immunoglobulins in body fluids comprises the following stages:

• a) implementation of the immobilized monoclonal antibody SIFIN Anti ML-A-5H8 or SIFIN Anti ML-A-5F5 with defined concentrations of mistletoe lectin I or mistletoe lectin II or mistletoe lectin III;
• b) removal of unbound constituents and exposure to the diluted sample (body fluid) whose mistletoe lectin specific immunoglobulins react with determinants of the natively presented mistletoe lectin;
• c) removal of unbound components and exposure to the enzyme-labeled monoclonal detection antibody SIFIN Anti ML-A-5F5 or SIFIN Anti ML-A-5H8, which reacts in competition with the mistletoe lectin-specific immunoglobulins already bound with the available epitopes of the mistletoe lectins;
• d) removal of unbound constituents and manifestation of the bound enzyme portion in a manner known per se;
• e) Calculation of the lectin recovery after competition in comparison to a control body fluid as lectin binding units.

6. The method according to claim 1 and 2, characterized in that Mistletoe lectins that solidify via lectin binding fixed glycoconjugate (e.g. asialofetuin) are bound, by an enzyme-labeled monoclonal antibody (e.g. SIFIN Anti ML-A-5F5) or by its enzyme-labeled fragments be detected depending on the concentration. 7. The method according to claim 1-6, characterized in that the monoclonal antibodies SIFIN Anti ML-A-5F5 and SIFIN Anti ML-A-5H8 are labeled with an enzyme. 8. The method according to claim 6, characterized in that the enzyme Horseradish peroxidase is. 9. Test pack for the determination of mistletoe lectin I or mistletoe lectin II and / or mistletoe lectin III in plant extracts according to Claims 1 to 4, characterized in that the immuno logical reagents all acting mono are clonal antibodies. 10. Test pack for the determination of mistletoe lectin-specific Antibodies in human and animal body fluids according to claims 1 and 5, characterized in that they Contains components for competitive immunoassays and which as immunological reagents all act antibodies are monoclonal antibodies.