In Vitro Binding ANTI-GM1 IGM To GM1 Inhibition Pattern By Three Intravenous γ-Globulin Preparations In Patients With Distal Lower Motor Neuron Syndrome
Aim of the study: To evaluate the in vitro inhibition of different Intravenous γ-Globulin (IVIgG) preparations and of their F(ab′)2 fragments on the anti-GM1 IgM binding to GM1. Methods: Amounts of three commercially available IVIgG preparations (Sandoglobulin, Ig Vena and Flebogamma) were digested by 10 mg/mL pepsin acetate buffer. Dialyzed digests were gel filtrated by a Sephadex G-150 column and PBS to obtain purified F(ab′)2 fragments. Two patients with a distal lower motor neuron syndrome (A.B., female with motor conduction blocks at the electrophysiological examination; P.M., male without motor conduction blocks) with high anti-GM1 IgM titer (1:84,000 and 1:52,000) were studied. Serum amounts of these patients were mixed with increasing amounts (from 0.25 to 5 times IgG patient molar concentration) of the three IVIgG preparations or their gel filtrated F(ab′)2 fragments, or with 2% bovine serum albumin. One hundred μL of each mixture was incubated in microtiter wells coated with 500 ng/mL GM1 methanol solution. Peroxidase conjugated goat anti-human IgM and O-phenylenediamine solutions were added according to the ELISA procedure and anti-GM1 IgM titer was calculated by absorbance at 490 nm, extrapolating the value from a standard curve. Each mixture percent of inhibition of anti-GM1 IgM to GM1 was expressed as a ratio between the difference of anti-GM1 titer before and after IVIgG or F(ab′)2 incubation and the baseline anti-GM1 titer. Results: Intact IVIgGs were able to inhibit binding of anti-GM1 IgM to GM1 in a quite dose-dependent manner, but at a different extent either between patients than among different IVIgG preparations: in patient A.B. the inhibition ranged from 8% to 71% using Sandoglobulin, 2% to 54% using Ig Vena and 9% to 65% using Flebogamma, while in patient P.M. the inhibition ranged respectively 0% to 35%, 3% to 62% and 8% to 74%. Similar results were reached using F(ab′)2 fragments: in patient A.B. the inhibition ranged respectively from 7% to 76%, 5% to 68% and 8% to 59%, while in patient P.M. the inhibition ranged respectively from 1% to 40%, 5% to 59% and 10% to 81%. Conclusions: IVIgG preparations inhibit the in-vitro binding of anti-GM1 IgM to GM1 supplying a possible explanation of IVIgG therapy in-vivo efficacy. This inhibition is due to F(ab′)2 fragments and probably involves an idiotypic - anti-idiotypic interaction, because the same IVIgG or F(ab′)2 preparations give different percent inhibition of anti-GM1 to GM1 binding according to the treated patient anti-GM1 idiotopic pattern.
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Document Type: Abstract
Affiliations: Division of Neurology and Dept. of Pathology, “San Giovanni Bosco” Hospital and C.M.I.D. “Luigi Einaudi” Hospital, Torino, Italy.
Publication date: March 1, 2001