Detection of antibodies directed against the cytoplasmic region of the human acetylcholine receptor in sera from myasthenia gravis patients

Abstract

The nicotinic acetylcholine receptor (AChR) is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti-AChR antibodies in MG sera bind mainly to conformational epitopes, therefore the determination of their specificities requires the use of native AChR. Antibody competition studies suggest that most MG antibodies are directed against the extracellular part of the molecule, whereas antibodies directed against the cytoplasmic region of the AChR have not been detected. To determine whether even small quantities of such antibodies exist in MG sera, we performed competition experiments based on the inhibition by MG sera of the binding of MoAbs to the human AChR, rather than inhibition by MoAbs of the binding of MG sera performed earlier. When MoAbs directed against cytoplasmic epitopes on the alpha or beta subunits (alpha 373-380 and beta 354-360) were used as test MoAbs, 17% or 9% of MG sera inhibited the binding of the anti-alpha or anti-beta subunit MoAbs, respectively, by > or = 50%. Non-specific inhibition was excluded. These results suggest the presence, in several MG sera, of antibodies directed against cytoplasmic regions of the AChR; yet these antibodies seemed to represent a relatively small proportion of the total anti-AChR antibodies. The corresponding epitopes may be involved in the inducing mechanisms in certain MG cases, and knowledge of the presence of such antibodies may be useful in understanding the autoimmune mechanism involved in MG.

Fig 1

Myasthenia gravis (MG) mediated inhibition of MoAb binding to two cytoplasmic epitopes of human acetylcholine receptor (AChR). ¹²⁵I-α-bungarotoxin-labelled AChR was incubated with 30 μl of one of 95 sera (proteolysed by papain to Fab fragments), then MoAb 155 (a, directed against the cytoplasmic epitope Very Immunogenic Cytoplasmic Epitope (VICE)-α) or MoAb 124 (b, directed against the cytoplasmic epitope VICE-β) was added. Subsequently, the MoAb-bound labelled AChR was immunoprecipitated using anti-rat immunoglobulin serum. The sera are arranged in order of decreasing titre and the titre, in nm, of the 10th, 20th, etc., sera (plus the first and last) are indicated. Arrows indicate sera from five MG patients with thymoma. The 50% cut-off of inhibition is shown by the horizontal lines. A small negative inhibition (1–10%) exhibited by some sera is represented as 0%. For most sera, s.d. among measurements from independent experiments did not exceed 12%. For a few sera s.d. was between 13% and 20%.

Fig 2

Dose response of the inhibitory effect of three papain-treated myasthenia gravis (MG) sera on the binding of anti-α subunit MoAb 155 to the acetylcholine receptor (AChR). The titres of the inhibitory MG antibodies estimated from 50% to 60% inhibition of MoAb binding are: 36 nm (MG 1989; •), 3.1 nm (MG 3722; ▴) and 0.84 nm (MG 3421; ▪). MG 1989, 3722 and 3421 are the 23rd, 35th and 39th sera in Fig. 1 (left to right).

Fig 3

Myasthenia gravis (MG)-mediated inhibition of binding to the acetylcholine receptor (AChR) of various MoAbs and a Fab fragment. Similar inhibition by each individual serum was seen using different MoAbs directed against a single region (Very Immunogenic Cytoplasmic Epitope (VICE)-α or VICE-β), or using intact and Fab fragments of MoAb 124. MG 1989, 4536, 3722, 3388 and 3421 are the 23rd, 3rd, 35th, 76th and 39th, respectively, sera in Fig. 1. NT, Not tested.

Fig 4

Comparison of myasthenia gravis (MG)-mediated inhibition using soluble or Sepharose-bound MoAb. Soluble MoAbs (with bound ¹²⁵I-α-bungarotoxin-labelled acetylcholine receptor (AChR)) were immunoprecipitated using anti-rat immunoglobulin serum, while Sepharose-bound MoAbs were precipitated simply by centrifugation. The first three sera gave extensive inhibition of MoAb binding, whereas the last two only inhibited to a small degree. Each papain-treated MG serum gave similar inhibition using soluble or Sepharose-bound MoAb-precipitation of labelled AChR. MG 1989, 5137, 3722, 4918 and 6196 are the 23rd, 21st, 35th, 16th and 64th, respectively, sera in Fig. 1.

Fig 5

Effect of increasing volumes of myasthenia gravis (MG) sera on the MG precipitation of ¹²⁵I-α-bungarotoxin (α-BT)-labelled acetylcholine receptor (AChR). The symbols on the right represent the effect of a mixture of 1 μl of a high plateau MG serum and 9 μl of test MG sera or NHS (○). Since the low plateau sera had little effect on the result obtained with the positive control high plateau serum, it is concluded that the low plateau MG sera did not release ¹²⁵I-α-BT from the AChR. MG 5660, 3836, 3775 and 3667 are the 4th, 28th, 30th, and 40th, respectively, sera in Fig. 1.