DE3130606C2 - Method for the isolation of cells involved in antibody formation - Google Patents

Description

Specific antibodies have long been used in medical diagnostics as essential Part of radioimmunoassays (RIA). Enzyme immunoassay (ELISA). "Solid phase immune tests" (SPIT), etc. found.

Antibodies of this kind are used by the immunocompacter.ten Cells form an organism capable of producing antibodies after the supply of antigen and lie for the most part in its blood plasma. For commercial application of these antibodies isolation from the blood plasma increasingly in favor of the direct isolation of those involved in antibody formation Leaden cells to after their fusion with Tumorzellcn according to Milstcin and Koehler a continuous one monoclonal target formation in vitro / u.

State-of-the-art selective iso-iteration of cells raised against an appli / icrtcs antigen Forming antibodies is done with the help of a so-called FACS ("fluorescence activated cell sorting analyzer"): against The applied antigen has to be formed into antibodies by labeling with, for example, fluorescein conjugated Antibodies in a large number of morphologically identical lines than the specifically antibody-forming cells Cells are first identified so that they can then be selected by machine. This method is labor-intensive and therefore expensive.

According to the invention, the following is proposed for isolating antibody-forming cells: one bclad immunologically inert particles with antigen and applied them in natural and / or artificially created body cavities an organism capable of producing antibodies. except man, and removes them then later together with those in antibody formation involved imri'.unkompctcntcn cells again.

Ti are suitable as immunologically incrie particles. a. Plastic and / or glass and / or ceramics and / or metal particles, which in a variety of forms, such as B. different shapes, surfaces, textures. Dimcnsionicrungcn, porcn size inexpensive are available. Chromatographicmatcrialicn have because of the suitable dimensioning (um range) μ and selectable surface areas (functional groups, charge and pore size).

The loading of the immunologically inert particles mil example

The immunobiologically inert particles were made of divinyl resin Polyslyrole particles for Gclpcrnialionschromatographic (Diameter: 37-74 μm, molecular Exclusion limit c (in benzene): 1400 Daltons. Bctt volume (in benzene): 4.0 ml / g) used (a's Bio Beads SX-4 available from Bio Rad). This raw material was initially offcngelcgt, as in DE-OS 31 26 551, functionalized with phosphatidylserine and phosphaditylethanolamine (approx. 50 mg phosphalidylserine resp. Phosphalidylälhanolamin on 20 g polystyrene). The loading with antigen took place after washing several times in Kinger's solution and after gas sterilization of the particles.

The antigen used was 5% human albumin: 5% by volume Iluman albumin was 1 volume fraction the functionalized polystyrene particles at room temperature with constant mixing for about 72 hours incubated and then washed several times with ring solution under sterile conditions and filtered by filtration concentrated

About 1 part by volume of human albumin-coated polystyrene particles is suspended in 3 parts by volume of 10% dextran 40 in isotonic saline solution and b parts by volume of ring solution.

This suspension was administered intrapcritoncal to rabbits, usually 5 ml o. G. Susperaion / kg body weight. Boosts only became effective when this did not occur, usually after about 3 to 6 days after application occurring Aszilc-s carried out.

By the beginning of the fourth week after the antigen application, the As / itcs had usually increased so much that ascitic puncture was possible without difficulty. The corpuscular ones contained in the as / itcspunklal Components, including the anligen-laden particles were concentrated by graduated centrifugation and analyzed: are found on many analogous particles fcsiiingcliigcrlc / xllcn. Kin GroUlcil of these cells shows again after incubation with FITC-conjugated inli-human albumin antibodies from the rabbit (available from Dako) a clear fluorescence.

Claims (2)

Patent claims: 1. A method for isolating cells involved in antibody formation, characterized in that that one with antigen cladene particles from an immunobiologically inert material in the form of a suspension in natural and / or artificially created body cavities one for antibody formation capable organism, except for humans, applied and then from this later together with the cells involved in the antibody formation of the organism again in vivo removed 2. The method according to claim 1, characterized in that that one cladcnen the cells involved in the antibody formation together with the antigen Particles are removed from the body cavity by puncture Antigen occurs through adsorptive and / or covalent binding of the antigen to the surface of the immunologically inert particles and is sufficiently known to the person skilled in the art under the term "solid phase" techniques, see: Mosbach, Klaus (Ed.): Methods in Enzymology, Volume XLlV: Academic Press, New York, San Francisco, London (1976). The application of the particles loaded with antigen takes place in natural (abdominal cavity, joint space, plcura space) or artificially created body cavities (e.g. pneumothorax). The antignbcladcner particles are used as a suspension applied into the body cavities and after antibody formation has taken place (due to a rise in ticran in the blood plasma of the antibody-forming organism recognizable) then together with the specific antibody-forming immunocompetent Cells (by puncture) wk-vr removed In the puncture then lie on the antigcnbcladcncn Particle-adhering, antibody-forming cells, which are then used for cloning / cleaning permit.