DD145755A1 - ISOLATION PROCEDURE FOR SPECIFIC HLA ANTIBODY PERI - Google Patents

Abstract

The invention relates to an isolation method for HLA antibodies by affinity chromatography with HLA antigens from urine. According to the invention, HLA antigens are obtained from human urine after precipitation with ammonium sulfate and concentration by ultrafiltration and subsequent ion exchange and gel chromatography. The isolated HLA antigens are bound by glutaric dialdehyde to a polyacrylamide-agarose copolymer, which is subsequently loaded with an antiserum for affinity chromatography and eluted with PBS and HCL-glycine buffer. The prepared preparation can be used both for diagnostic purposes (HLA serology) and for therapeutic purposes (avoidance of graft rejection).

Description

Dr ₀ W. Schößler

Isolation method for specific HLA antibodies

Application of bi_et_ of the Er fiiet dang.

The invention relates to a method of isolation of specific HLA antibodies from human antiserum ₀ applies, the method in the pharmaceutical industry and in medicine. , , ,

Characteristics of the known technical solutions The HLA (human leukocyte antigen) antigens are responsible for the rejection of xenogeneic or allogenic transplants. Raise other discussed Llöglichkeiten avoiding the Transplantatabstofiung that have not yet been applied in practice, is the use of HLA antibodies under- use of immunological enhancement (specific immune suppression by specific antibodies) may great importance _o All previous attempts HLA antibodies To separate and isolate with the usual physicochemical methods, failed, since the HLA antibodies are of the IgG type and therefore behave like all existing in the serum IgG-IvIoIeküle when using these methods.

The isolation of specific HLA antibodies has not been described in the literature.

Object of the invention. ,

The object of the invention is to provide a preparation which can be used both for diagnostic purposes (HLA serology) and therapeutic purposes (avoidance of graft rejection). "The invention has

91 5 I

the task of developing an isolation method for this HLA antibody-containing preparation.

Principle of the dung OF ^INVENTION;

According to the invention, from human urine after precipitation "with 80% saturation with ammonium sulfate and subsequent dialysis and ultrafiltration with an SM 12136 membrane and ion exchange chromatography on DEAE-cellulose with trisphosphate buffer. Increasing ionic strength (pH = 8.4) and gel chromatography on Sephadex G .1 00, Iris-phosphate buffer (pH = _8? 4) derived HLA antigens. particularly advantageous is the use of urine of patients with tubular proteinuria, since therein the HLA antigens are enriched _o the isolated HLA antigens are in The following are bound to a polyacrylamide agarose copolymer with 10% glutaric dialdehyde in 0.1 M phosphate buffer (pH = 7.2) After checking the binding strength of the antigens and determining the amount of protein bound to the matrix, the gel becomes a The non-nonspecifically bound proteins of the antiserum are then eluted with PBS buffer (pH = 7.4, 4 ^° C.) and then loaded with an antiserum Finally, the antigen-antibody complexes with HCl-glycine buffer (pH = 2.8 and 2.2 at 4 ⁰ C) cleaved. After immediate neutralization and dialysis against PBS buffer, the antibody-containing fractions are concentrated by ultrafiltration and serologically tested.

The preparation prepared according to the invention can be used for diagnostic purposes (HLA serology) in transplantation immunology, forensic medicine and the like-as well as for therapeutic purposes-to avoid transplant rejection. «

Example '·.

The HLA antigens A 1, A 2, B 8, B 17 were isolated from 8.5 l urine of a transplanted protein having a tubular proteinuria (total protein · 3.8 g / l). The antigenic activities in the various purification steps are determined by the Lymphocytotox Inhibition Test. ,

The urine, which is up to processing. "is stored at 4 C in the presence of 0.02% sodium azide, · is concentrated by adding ammonium sulfate to 30% saturation. The by centrifugation at 5000 g for 20 min in" recovered precipitate is .. in 0.01 m Phosphate Puff he with 0.15 m IaCl (PBS buffer) (pH = 7) 2) suspended. And dialysis against this buffer exhaustively followed by an approximately ten-fold concentration by ultrafiltration mit.einer SM 1213ο membrane to a protein concentration of 31 g / l. The further purification is carried out by ion exchange chromatography on DEAE-cellulose with 0.005 m phosphate buffer with 0.04 m Tris (pH = 8.4) [V "(flow rate 3 ml /

2 cm "h) when using a continuous ionic strength gradient with simultaneous on-line concentration with a Diaflo pM 10 membrane _o The fractions containing the HLA antigens are eluted only at high ionic strengths and are 'largely free of accompanying proteins Purity, by immunoelectrophoresis and disk-electrophoresis ».

As a next step, a gel chromatography on Sephadex G 100 is carried in.0,5 m Tris-phosphate buffer (bed volume (pH = 8.4..): 314 ml, sample volume: 5 _S 5 ml, flow rate: 5 »5 ml / cm h, on-line concentration with PM 10-Ivlembran Ca ₀ 1: 4) «The low-molecular-weight fraction contains the.KLA antigens that are slightly contaminated with albumin _e The yield is 200 - 300 / ug HLA antigens. After dialysis against 0.1 M phosphate buffer (pH = .7.2), the HLA antigens are bound to a polyacrylamide-agarose copolymer (Ultrogel AcA 34). For this purpose, the gel (20 ml) is washed sufficiently with water until no UY absorption at 280 nm is visible and with 10 % glutaraldehyde in 80 ml of 0.1 M phosphate buffer (pH 7.2) (0.02 % hait ₃₎ 24 h at 37 ⁰ C is activated and then washed exhaustively, "the binding of HLA antigens to the activated gel is carried unter.vorsichtigem stirring for 16 Ii at 4 ° 0 and 4 h at room temperature. Subsequent protein determination in the supernatant allows calculation of the amount of antigen bound

rinse again with PBS buffer (pH = 7.4) (UV absorption = 0). Free aldehyde groups ^{: of} the activated gel are blocked with O ₅ Vm lysine solution pH '= 7.4 * After another wash with PBS Buffer is tested for binding strength of the bound HLA antigens with 0.2 M HCl-glycine buffer pH = 2.8 followed by itfeutralization (1m KgHPO ^ solution) and washing (PBS buffer). A determination of the protein in the washing solution gives information about the amount of antigen firmly bound to the matrix _o The gel is now placed in a column (length: 30.mm, diameter: 9 mm) and equilibrated with PBS buffer (pH = 7.4) 5 ml of antiserum containing antibodies B 5 and B 8 are applied at room temperature. "After the antiserum has entered the gel, the outlet of the column is closed for 2 hours."

The subsequent elution of the non-nonspecifically bound proteins of the antiserum is carried out at 4 G with cold PBSt buffer (pH = 7.4, flow rate: 3-5 ml / cm ² h). If the eluate no longer shows UV absorption at 280 nm, the cleavage of the antigen-antibody complexes formed and elution with 0.2 M HCl-glycine buffer pH = 2.8 and pH = 2.2 at 4 C under continuous concentration with one. Diaflo-Hvl .10 membrane. The fractions are immediately neutralized with 0.2 M KpHPO, dialyzed against PBS buffer and concentrated by ultrafiltration with collodion tubes »

While the B 5 antibody is eluted by the PBS buffer because antigen B 5 is absent, B 8 antibody elution is then performed by the HCl reagent. GIycin Buffer _o The protein concentrations of the B 8 primes were 170 mg / l and 90 mg / l _o Thus, by the inventive method, a 400 - 80Ofache enrichment of HLA antibodies according to human serum (total protein about 70 g / l ) ο '.' , ·

Claims (2)

1. A process for the isolation of HLA antibodies, characterized in that from human urine after precipitation with ammonium sulfate and concentration by ultrafiltration ', and subsequent, ion exchange and gel chromatography isolated HLA antigens bound to a matrix and the HLA antibody by subsequent affinity chromatography. isoliert'werden chromatography from human HLA antiserum method according to item 1, characterized _s that the isolation of HLA antigens by precipitation at 80 / Siger saturation is carried out with ammonium sulfate and by subsequent dialysis by ultrafiltration with a SM 1213δ membrane lorienaustauschchromatographie on DEAE -CeI-lulosernit Tris-Piiosphat buffer of increasing ionic strength pH = 8.4 and gel chromatography on Sephadez G 100 with. Tris-phosphate buffer pH = 8 _S 4 is purified _o . 3o method according to item 1, characterized in that the   isolated HLA antigens by Glutardialdehyd to a Po-. lyacrylamide-agarose copolyraer are bound ,, 4. The method according to item 1, characterized in that for affinity chromatography loaded with HLA antigens polyacrylamide agarose Copolynier column, equilibrated with PBS buffer, loaded with an HLA antiserum and then with PBS buffer and HCl glycine Buffer eluted and the eluate is then neutralized, dialyzed and concentrated β