hCG/AFP Multiplexed Assay
Quantitative · Accurate · Sensitive

Introduction

Luminex Corporation has developed an immunometric "capture/sandwich" assay for the simultaneous measurement of human chorionic gonadotropin (hCG) and alpha-fetoprotein (AFP). Each assay consists of a FlowMetrix microsphere set coated with a capture antibody that binds the hormone (target) followed by a green fluorescent anti-target Ig (reporter). The intensity of green fluorescence on the microsphere surface is directly proportional to the concentration of target in the serum sample (Figure 1). Each assay was optimized to provide a limit of detection and dynamic range of physiological significance for each hormone/analyte. Once balanced, the two assays were performed simultaneously using the FlowMetrix System. The results demonstrate that FlowMetrix technology is a uniquely powerful method for immunometric assay in a multiplexed format.

 

Figure 1

Materials

Monoclonal anti-hCG, polyclonal anti-hCG and purified hCG antigen were purchased from Chemicon, Inc., Temecula, CA. Monoclonal anti-AFP, polyclonal anti-AFP, and purified AFP antigen were purchased from Medix Division of Genzyme, San Carlos, CA. EDC (1-ethyl-3-3[3-dimethylaminopropyl] carbodiimide hydrochloride), and Sulfo-NHS (N-hydroxysulfosuccinimide) were purchased from Pierce Chemicals, Rockford, IL. Bodipyâ FL-CASE was purchased from Molecular Probes, Inc., Eugene, OR. RIA grade Bovine Serum Albumin (BSA) and Sorbitol 20 (Tween 20) were purchased from Sigma Chemical Co., St. Louis, MO. Tri-level serum calibrators were purchased from Randox Laboratories, Ltd., Ardmore, UK. hCG/AFP certified negative serum was kindly provided by BioMedical Resources, Inc., Hatboro, PA.

Methods

Antibody labeling: The two affinity-purified polyclonal antibodies (reporters) were labeled with Bodipy FL-CASEâ using methods described by the manufacturer.

Antibody Conjugation to Microspheres: Monoclonal capture antibodies were conjugated to FlowMetrix microspheres with a two-step EDC coupling. twenty microliters of each microsphere set were activated, washed and suspended in a 0.05 mg/ml solution of antibody in PBS, pH 7.4. After two hours, protein-coated microspheres were washed with 0.02% Tween 20, 1 mg/ml BSA in PBS, pH 7.4 (PBSTB) and stored in PBSTB at approximately 3x10⁶ microspheres/ml.

Antigen Titration Assay: Antigen titrations were performed using ten microliters of capture antibody-coated microspheres plus twenty microliters of diluted antigen in PBSTB for a thirty minutes incubation. Microspheres were suspended in twenty microliters of a 25 m g/ml solution of reporter antibody. This mixture was incubated for thirty minutes and assayed using the FlowMetrix System. Microsphere number was adjusted to optimize the limit of detection (LOD) and dynamic range.

Multiplexed assay: Equivalent amounts of the two capture antibody-loaded microspheres were mixed. In triplicate, 10microliters of this microsphere blend was mixed with twenty microliters of serum calibrators or serum samples diluted 1:10 and incubated for thirty minutes The assay was developed by addition of twenty microliters of a blend of green fluorescent-labeled anti-hCG and anti-AFP reporter antibodies at 25 m g/ml. Mixtures were incubated for thirty minutes and assayed using the FlowMetrix System.

Results

AFP/hCG Titration: By controlling the number of microspheres per assay, the dynamic range and LOD for the AFP assay were adjusted to a physiologically relevant range (60 pg/ml with a dynamic range of three logs.) Similar adjustment of the hCG assay resulted in a LOD of 100 pg/ml (1.1 mU/ml) and three logs of dynamic range.

Figure 2

Multiplex hCG/AFP Assay: The two assays were multiplexed and serum calibrators of known hCG and AFP levels were used to generate a standard curve. For each standard curve one serum with unknown levels of hCG and AFP was included to demonstrate how the assay would determine the level of hCG and AFP in an unknown. Coefficients of variation (CV) for the calibrator sera were 4% and for the unknown sample, 3%. R² for both curves was 1.0 using a polynomial equation. The concentration of the unknown was 218 ± 6 mU/ml for hCG and 52 ± 2 U/ml for AFP (Figure 2).

Conclusions

A multiplexed immunometric assay for hCG and AFP in serum has been developed. Assays were individually optimized for sensitivity, dynamic range and cross-reactivity, then multiplexed to quantitatively determine both analyte levels simultaneously from the same sample. Results using commercial calibrator sera demonstrated that FlowMetrix assays provide results in physiologically relevant ranges for this type of quantitative assay. This assay was developed at Luminex Corporation for the purpose of demonstrating the power of FlowMetrix technology. In addition to immunometric assays, this technology can be applied to competitive inhibition immunoassays, enzyme assays and nucleic acid based genetic analyses.

Luminex Corporation provides the 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.

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