GAM

Luminex Corporation has developed quantitative competitive inhibition assays for immunoglobulins G, A and M that are performed simultaneously in the same tube. Each assay consists of a FlowMetrix microsphere set coated with the immunoglobulin (Ig) of choice (target) and an anti-human Ig (reporter). In the absence of soluble Ig (inhibitor), the anti-human Ig causes the Ig coated microspheres to emit strong green fluorescence. In the presence of soluble Ig, the green fluorescence signal is reduced relative to the level of soluble inhibitor. Each assay is optimized to provide a dynamic range that includes normal serum levels of each Ig. Once balanced, the three assays were mixed into a multiplexed format and performed simultaneously using the FlowMetrix System. Results indicate that FlowMetrix is a uniquely powerful method for competitive inhibition immunoassay in a multiplexed format.

Human IgG, IgA, IgM, goat anti-human gamma chain, goat anti-human alpha chain, and goat anti-human mu chain were purchased from Cappel Division of Organon Teknika, Durham, NC. EDC (1-ethyl-3-3[3-dimethylaminopropyl] carbodiimide hydrochloride), and Sulfo-NHS (N-hydroxysulfosuccinimide) were purchased from Pierce Chemicals, Rockford, IL. RIA grade Bovine Serum Albumin (BSA) and human serum devoid of IgG, IgA and IgM was purchased from Sigma Chemical Co., St. Louis, MO. Serum calibrators with known levels of IgG, IgA and IgM were purchased from Kamiya Biomedical Company, Tukwila, WA.

Antibody labeling (reporter): The three goat antibodies were labeled with Bodipyâ FL-CASE using methods described by the manufacturer.

Antigen Conjugation to Microspheres (target): Four FlowMetrix microsphere sets were conjugated separately to IgG, IgA, IgM and bovine serum albumin (BSA) with a two-step EDC coupling method. Activated microspheres were suspended in a 0.05 mg/ml solution of Ig protein in PBS, pH 7.4. After one hour, protein-coated microspheres were washed and stored in 0.02% Tween 20, 1 mg/ml BSA in PBS, pH 7.4 (PBSTB).

Multiplex assay: Equivalent amounts of the 4 protein-loaded microspheres were mixed. Ten microliters of the microsphere blend was mixed with ten microliters of serum calibrators or serum samples diluted 1:500. Immediately, ten microliters of a cocktail of the three reporter antibodies was added and mixed well. Reporter antibody concentrations were 30.0, 8.0, and 2.5 m g/ml for anti-gamma, anti-alpha and anti-mu respectively. Mixtures were incubated for thirty minutes and assayed in fifteen to twenty seconds using the FlowMetrix System.

Multiplex Analysis: Figure 1 shows the three standard curves resulting from one assay. For each inhibition curve, a polynomial trendline was used for non-linear regression analysis. The fit of this trendline to the data was demonstrated by the R² correlation factor. Coefficients of variation (CV) between the triplicate data points indicated that the assay was highly precise with CVs of approximately 5%. The dynamic ranges for each assay were 300-3000 mg/dl for IgG, 60-522 mg/dl for IgA, and 36-305 mg/dl for IgM. By altering reporter concentration the dynamic range can be adjusted.

Fifty clinical samples were assayed using the Beckman Array nephelometry system and the levels of the three Ig isotypes recorded. The same fifty samples were assayed using the FlowMetrix multiplexed assay. Samples were diluted 1:500 in PBSTB and ten microliters assayed as competitor in the same thirty microliters assay format as the standard curve. Results are seen in Table 1. The correlation coefficient for IgG, IgA and IgM were .920, .982 and .982 respectively.

FlowMetrix vs. Rate Nephelometry	Sample Range (mg/dl)	No. of Observations	Intercept (mg/dl)	Slope	Correlation Coefficient
IgG	505-2720	50	98	0.74	0.920
IgA	62-475	50	4.9	0.94	0.982
IgM	31-302	50	8.5	0.91	0.982

A multiplexed, competitive inhibition assay for quantitation of human serum IgG, IgA, and IgM levels has been developed. It allows simultaneous assay of three Ig isotypes from serum samples diluted 1:500. The assay demonstrated excellent sensitivity, precision and accuracy as determined by comparison with an FDA-approved nephelometric method. This assay was developed at Luminex Corporation for the purpose of demonstrating the power of FlowMetrix technology. In addition to competitive inhibition immunoassay, this technology can be applied to immunometric and enzyme assays as well as nucleic-acid based genetic analyses.

Luminex Corporation provides the FlowMetrix system including microspheres in a format ready to apply to your specific application. Using this Assay Development Kit, your research group can develop multiplexed assays of interest in a matter of days. For further information in the US call toll free 1-888-219-8020, outside the US call 1-512-219-8020 or email scombs@luminexcorp.com.