Molecular /mmunology, Vol. 16, pp. 483-487 Pergamon Press Ltd. 1979. Prmted m Great Braam.
EFFECT OF AZATHIOPRINE ON THE AFFINITY OF ANTIBODIES AGAINST ACETYLCHOLINE RECEPTOR: ANALYSIS WITH PURIFIED ANTIBODIES* MICHAL
SCHWARTZ,
DORON
LANCET, SARA
Department
of Chemical
Immunology,
(First received 30 October
REBECA
TARRAB-HAZDAI
and
FUCHS
The Weizmann
Institute
of Science.
Rehovot.
Israel
1I December 1978)
1978; in rel’ised fi)rm
Abstract-Rabbit anti-acetylcholine receptor (AChR)t antibodies were purified on an AChR-toxin-Sepharose immunoadsorbent. The immunoadsorbent was prepared by attaching first toxin covalently to Sepharose and then reacting the toxin-Sepharose with AChR. Purified anti-AChR antibodies were utilized for studying the association between the immunosuppressive effect of azathioprine (AZ) on AChR-induced experimental autoimmune myasthenia gravis and on the affinity of the antibodies. Mathematical analysis of the data obtained from binding experiments of ‘ZSI-AChR to the purified antibodies suggest that treatment with AZ decreases the amount of anti-AChR antibodies possessing high affinity values.
INTRODUCTION
Azathioprine
suppressing myasthenia
AChR. Thus, x-bungarotoxin was covalently bound to Sepharose and AChR was non-covalently attached to the toxin-Sepharose, yielding a solid AChR immunoadsorbent for purification of anti-AChR antibodies. The results of affinity measurements obtained with the purified antibodies indicate that anti-AChR antibodies of AZ treated rabbits differ from those of non-treated rabbits in their quantity rather than in their quality. Thus whereas 2.5”,, of the antibodies in the AZ treated rabbits possess an affinity valuein therangeof IO8 AK’, 22”,,oftheantihodiesof non-treated rabbits express this affinity.
was shown to be effective in the onset of experimental autoimmune gravis (EAMG) in rabbits injected with (AZ)
acetylcholine receptor (AChR) from To,p& &[/ovnica (Abramsky et ul., 1976; Tarrab-Hazdai ef ul., 1977).
The suppressive
effect
of azathioprine
was
followed by a decreased cellular reactivity such as lymphocyte transformation and binding of AChR to as well as a decreased humoral macrophages, reactivity to the foreign AChR and to self AChR (Tarrab-Hazdai ef a/., 1977). These two effects may represent a decrease in either the level of the affinity. Moreover, the antibodies, or their immunosuppressive drug may have a selective effect on antibodies of certain specificity resulting in prevention of the disease. In the present work we have studied the effect of immunosuppression with AZ on the affinity of antibodies elicited by AChR immunization. Measurements of the effective affinity could be achieved only by using purified antibodies. Such analysis was performed by using AChR specific antibodies isolated from rabbits injected with AChR, treated or not treated with AZ. Purification of the antibodies was achieved by using AChR-toxin-Sepharose column as the immunoadsorbent. Advantage was taken of the specific high affinity binding of r-bungarotoxin for nicotinic
MATERIALS AND [METHODS
Malerials AChR was isolated and purified from electric organ of Torpedo californica as described by Aharonov et ul.. (1977). a-Bungdrotoxin was prepared according to Clark et al.. (1972). Radioactively labeled AChR (1251-AChR) and rbungarotoxin (‘Z51-n-bun~drotoxin) were prepared and characterized as described previously (Aharonov (‘I cl/.. 1977). Rabbit anti-AChR sera were obtained from rabbits injected intradermally with 100 fig of AChR in the receptor purification buffer (0.01 M Tris-HCI. pH 7.5, 0. I M NaCI. 0.001 M EDTA, O.lS,, Triton-X-100) emulsified with equal volume of Complete Freund’s Adjuvant (CFA) (Difco). The rabbits were bled 30 days following the receptor administration. Administration
oj’aa,-athioprine
Azathioprine suspended in 1.0 ml of sterde phosphate buffer solution (PBS) was injected (5 mg/kg) intramuscularly into rabbits starting on day 0 (day of AChR injection). Such administrations of Az were given daily for I5 days and then every 2-3 days (Abramsky et al., 1976).
*This study was supported by the U.S.-Israel Binational Science Foundation (BSF) and the Los Angeles Chapter of the Myasthenia Gravis Foundation. YAbbreviations used: AChR, acetylcholine receptor; AZ, azathioprine CFA, complete Freund’s adjuvant; CNBr, cyanogen bromide; NRS, normal rabbit serum; SDS, sodium dodecyl sulfate; EAMG, experimental autoimmune myasthenia gravis.
Preparation
o,f AChR-to.rin-Sepharose
CNBr activated Sepharsoe-4B (March et a/.. 1974) (I g) was reacted with r-bungarotoxin (3.8 mg) in 10 ml of 0.05 A4 sodium bicarbonate. Purified AChR was added to the 483
484
MICHAL
SCHWARTZ
toxin-Sepharose solid support (1 mg AChR per 1 g of resin) and it was found that 0.7 mg of the added receptor was attached to the toxin-Sepharose. The AChR-toxin-Sepharose adsorbent was washed in eluting conditions (0.2 M NH,OH) before applying the serum. in order to avoid any AChR release with the antibodies during the elution procedure. Inhrhi/ion of’ binding purified antihodics
’ ‘ jl-cc-hun~rrroro.~in to AChR
of