Jun 6, 1986 - ABSTRACT. Human IgG specific for (8-endorphin was identified by enzyme-linked immunoabsorbent assay and iso- lated by affinity ...
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 8739-8743, November 1986 Medical Sciences
Anti-(3-endorphin immunoglobulin G in humans (neuropeptide/autoantibody/affective disorder)
BENJAMIN F. ROY*tt, JOHN W. RoSE§, HENRY F. MCFARLAND§, DALE E. MCFARLIN§, AND DENNIS L. MURPHY* *Section on Clinical Neuropharmacology, Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892; and §Neuroimmunology Branch, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD 20892
Communicated by Seymour S. Kety, June 6, 1986
Human IgG specific for (8-endorphin was ABSTRACT identified by enzyme-linked immunoabsorbent assay and isolated by affinity chromatography. From a sample of 27 subjects, three individuals with major depression demonstrated plasma IgG highly reactive with human 13-endorphin, while four other subjects (two with depression and two randomly selected blood donors) had intermediate reactivity. The recognition site for fi-endorphin was retained by F(ab')2 fragments.
and Statistical Manual of Mental Disorders III (13) criteria for either major depression (n = 9) or personality disorder (n = 3) who were sequentially admitted over a several-month period to a National Institute of Mental Health Clinical Center psychiatric unit. One of the subjects with major depression (subject 10) had a 1-year history of chronic pain and a prior history of an urticarial reaction to hydromorphone hydrochloride. ELISA. P-Endorphin (10 gg/ml) and substance P or corticotropin (10 ,g/ml) in pH 9.4 coating buffer were pipetted in a 100-,ul volume into an Immulon I polystyrene plate (Dynatech, New York) and allowed to stand at 0C for 48 hr. The plate was washed with 0.5% Tween 20 (Fisher) in phosphate-buffered saline (PBS; pH 7.3, without calcium or magnesium), and nonspecific binding sites were blocked with 100 ,ul of 0.5% bovine plasma albumin in distilled water for 20 min at room temperature. Then 100 41. of human IgG was added and incubated at 370C for 2 hr. The plate was then washed, and 100 ,1u of a 1:200 dilution of an alkaline phosphatase-conjugated goat anti-human Fc IgG (heavychain specific) antibody (Sigma) was pipetted into each well. After 2 hr at 37°C, the plate was washed, 100 ,u1 of the alkaline phosphatase substrate p-nitrophenyl phosphate (disodium salt) dissolved in 10% diethanolamine was added to each well, and the reaction was allowed to proceed at 37°C. Substrate catalysis reflects the amount of goat antibody bound to human IgG which in turn is bound to neuropeptide and was determined by optical density units read at 405 nm on an automatic micro-ELISA spectrophotometer (Dynatech Instruments, Torrence, CA). Values represent the means + SD of triplicate OD405 readings at 30-min reaction time. The IgG concentration of each sample was determined by ELISA. Goat anti-human IgG F(ab')2 at a 1:1000 dilution was attached to the plate, followed by the addition of human samples and standards. Then alkaline phosphatase-conjugated goat antihuman Fc IgG at a 1:1000 dilution was added, and the IgG concentration was determined by linear regression analysis. Preparation of F(ab')2 Fragments. Purified IgG from subject no. 12 (experiment 1) was combined with pepsin in a weight ratio of 100:2, and the mixture was incubated overnight at 37°C in acetate buffer (pH 4.5). The pH was then raised to 8.0 by the addition of 1 M NaOH, the digest was applied to a Sephadex G-200 column, and F(ab')2 fragments were collected and evaluated by diffusion in Ouchterlony plates. The fraction with reactivity to sheep anti-human F(ab')2 but not goat anti-human Fc IgG was evaluated for activity against ,B-endorphin and corticotropin. The ELISA
The central nervous system and neuropeptides have been shown to modulate immune function (1-4). In contrast, the existence of immunoreactivity to peptides involved in synaptic modulation and neuroendocrine function would support the possibility that neuroendocrine and synaptic events might be altered by immune mechanisms. For example, antibodies to neuropeptides could function as part of an immunoregulatory network modulating central nervous system function. To investigate this possibility, we have begun to test for the presence of anti-,B-endorphin antibodies in individuals with disorders that might manifest immune sensitivity to this neuropeptide, such as patients with major depression, including one with the chronic pain syndrome and allergic hypersensitivity to a narcotic analgesic. An enzyme-linked immunoabsorbent assay (ELISA) system was used to detect human and rabbit antibodies to 3-endorphin. In preliminary studies, the sensitivity and specificity of this assay was examined using rabbit anti-pendorphin antiserum (Immuno Nuclear, Stillwater, MN). This antiserum was found to have relatively high specificity for ,-endorphin with negligible reactivity to bovine plasma albumin (
