SensoLyte Anti-MOG (35-55) IgG and Subtypes Quantitative ELISA Kits

Introduction
Myelin oligodendrocyte glycoprotein (MOG), a member of the immunoglobulin superfamily, is expressed exclusively in central nervous system myelin. Studies suggest that MOG may function in the completion, compaction, and maintenance of myelin in the central nervous system.¹ Even though MOG physiological function is not entirely understood, studies have correlated the immune response of MOG to autoimmune mediated demyelination in several species.² MOG is able to induce encephalitogenic T cell response, autoantibody response, and produce relapsing-remitting neurological disease with an extensive plaque-like demyelination.³ Autoantibody response to MOG (35-55) has been observed in multiple sclerosis (MS) patients and MOG (35-55)-induced experimental autoimmune encephalomyelitis (EAE) mice (Figure 1).^4-10 The exact pathological role of anti-MOG (35-55) autoantibody is not known and is currently under vigorous investigation.

Figure 1. MOG (35-55) induction of EAE in mice (n = 10)

AnaSpec is proud to be the first in the industry to develop a series of quantitative anti-MOG (35-55) ELISA kits (see list below). These kits can be used for evaluating the role of anti-MOG (35-55) antibody in the development and treatment of EAE, an in vivo animal model for MS pathogenesis. The SensoLyte^™ Anti-MOG (35-55) IgG Quantitative ELISA Kits (mouse/rat) provide quantitative assays for detecting anti-MOG (35-55) IgG and the different IgG subtypes in a convenient colorimetric read-out system (Abs= 450 nm).

Each kit contains a pre-coated and pre-blocked 96-well plate (strips format). Serum samples from EAE-induced mice are serially diluted from 1k to 125k in sample dilution buffer and 100 ul from each dilution added to the MOG (35-55) pre-coated 96-well plate. A standard curve of anti-MOG (35-55) IgG is run along side the unknown samples. Both the unknown samples and standards are incubated at room temperature for 60 min with gentle shaking. The wells are washed and a secondary antibody, goat anti-mouse IgG added to each well. After incubation with the secondary antibody, the samples are washed. TMB color substrate is added and color development stopped after 5-15 minutes. Absorbance is read at 450 nm, and the concentration of the samples determined.

Examples of absorbance readings (450 nm) from 4 serum samples of EAE-induced mice are shown in Table 1 [Sample#1, 3A-D and 4A-D; Sample#2, 3E-H and 4E-H; Sample#3, 5A-D and 6A-D; Sample#4, 5E-H and 6E-H (at 1:1k, 5k, 25k, 125k dilution in duplicates)]. Columns 1 and 2 are standards from 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 ng/ml in duplicates.

Table 1. OD readings of standards (columns 1 and 2) and serum samples (columns 3-6).

Calculation of linear regression and correlation coefficient for rows A-H, columns 1 and 2 yields an R² = 0.982; since 0.982 is lower than 0.985, the highest concentration (200 ng/ml) standard point (row A) can be disregarded. Recalculate the standard curve and R² for rows B to H only (See Figure 2). The resulting linear regression (R²) is greater than 0.985 (R² = 0.9921). Mouse MOG (35-55) IgG concentration vs. OD₄₅₀ readings between 0.2 and 1.1 can now be used to obtain accurate anti-MOG (35-55) IgG concentration for each sample.

Figure 2. Standard curve of OD readings plotted against Mouse MOG (35-55) IgG concentration.

To calculate the concentrations of all samples, select the highest dilution point that will include all the samples (in this case, OD₄₅₀ readings must be between 0.2 and 1.1). For example, use dilution point 1:25 k to calculate the concentrations for Samples# 1-4 as follows:
Concentration of anti-MOG (35-55) IgG (μg/ml):
Sample x : (Slope x OD_Ave – y-intercept) x dilution factor = final conc.
Sample# 1: (91.406 x (0.246 + 0.223)/2 – 4.3993) x 25 = 425.8 μg/ml
Sample# 2: (91.406 x (0.344 + 0.318)/2 – 4.3993) x 25 = 731.1 μg/ml
Sample# 3: (91.406 x (0.377 + 0.360)/2 – 4.3993) x 25 = 645.5 μg/ml
Sample# 4: (91.406 x (0.660 + 0.643)/2 – 4.3993) x 25 = 1376.0 μg/ml

Using these assay kits, the amount of the different anti-MOG (35-55) IgG antibodies present over a period of 50 days (Figure 3) as well as on Day 35 (Figure 4) were determined.

Figure 3. Anti-MOG (35-55) total IgG and IgG subtype responses overtime.

Figure 4. Anti-MOG (35-55) IgG responses on Day 35

The SensoLyte^™ Anti-MOG (35-55) IgG Quantitative ELISA Kits (mouse/rat) provide ample materials for 80 assays and have the following features:

* Get a 20% discount on the SensoLyte Anti-MOG (35-55) assay kits now through to Mar 31, 2008, (promotion code: MA0308)

To view a poster entitled “Suppressive Role of CD72 in Experimental Autoimmune Encephalomyelitis” presented by scientists from AnaSpec and Stanford University at the 2007American Association of Immunologists at Miami Beach, Florida, please click here.

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