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Detection of human leukemia inhibitory factor by monoclonal antibody based ELISA
K J Kim1, M Alphonso, C H Schmelzer
1Department of Medicinal Analytical Chemistry, Genentech Inc., South San Francisco, CA 94080.
Insights
This study developed a sensitive and specific double monoclonal antibody ELISA to measure Leukemia Inhibitory Factor (LIF). The assay can detect low levels of LIF in biological samples, aiding research into its diverse cellular functions.
Area of Science:
- Biotechnology
- Immunology
- Cell Biology
Background:
- Leukemia inhibitory factor (LIF) regulates critical cellular processes, including growth and differentiation in both normal and malignant cells.
- Accurate quantification of LIF in biological samples is essential for understanding its multifaceted roles.
Purpose of the Study:
- To develop a highly sensitive and specific assay for quantifying human Leukemia Inhibitory Factor (LIF).
- To characterize novel monoclonal antibodies (mAbs) for potential use in LIF detection.
Main Methods:
- Production and characterization of four murine monoclonal antibodies (mAbs) against recombinant human LIF (rHuLIF).
- Affinity determination, epitope mapping, and cross-reactivity testing of the generated mAbs.
- Establishment of a double mAb-based ELISA using selected mAbs for rHuLIF detection.
Main Results:
- Four high-affinity IgG1 mAbs against rHuLIF were generated, recognizing distinct epitopes.
- The mAbs demonstrated high specificity, with no cross-reactivity to several other cytokines (IL-1, IL-3, IL-6, TNF-alpha, GCSF, GMCSF).
- A double mAb ELISA achieved a sensitivity of 100 pg/ml for rHuLIF, further enhanced to 10 pg/ml with an amplification system.
Conclusions:
- A robust and specific double mAb ELISA for human LIF quantification has been successfully developed.
- This assay provides a sensitive tool for measuring LIF levels in biological samples, supporting further research.
- The characterized mAbs offer valuable reagents for LIF-related diagnostic and research applications.
Abstract:
Leukemia inhibitory factor (LIF) is known to exhibit multiple functions by regulating the growth and differentiation of multiple normal cell types as well as malignant cells. To have a better understanding of the role of LIF, it is important to determine the level of LIF in various biological samples by developing an easy, sensitive and LIF specific assay. In this study, we have established a double monoclonal antibody (mAb) based ELISA. Four hybridoma cell lines (D3.14.1, D4.16.9, D25.1.4 and D62.3.2) secreting murine monoclonal antibodies (mAbs) against recombinant human leukemia inhibitory factor (rHuLIF) were produced by immunization of BALB/c mice with rHuLIF and by fusing immune spleen cells with P3X63Ag8U.1 myeloma cells. These mAbs each belong to the IgG1 isotype and have unique isoelectrofocusing point patterns. All four mAbs were shown to have high affinities for rHuLIF (Kd = 7 x 10(-10) to 6 x 10(-11) M) and were able to recognize the native as well as the reduced rHuLIF in an immunoblotting assay. All these mAbs showed no cross-reactivities to IL-1, IL-3, IL-6, TNF-alpha, GCSF and GMCSF. MAb D3.14.1 showed a weak binding to Oncostatin M but not to rMuLIF whereas the other three mAbs D4.16.9, D25.1.4 and D62.3.2 showed cross-reactivity to rMuLIF but not to Oncostatin M. Data obtained from a competitive binding enzyme-linked immunosorbent assay (ELISA) suggested that these four mAbs recognized different epitopes on rHuLIF. Using mAb D4.16.9 as coat antibody and horseradish peroxidase (HRP) conjugated mAb D3.14.1 as the conjugate antibody we established a double mAb based ELISA specific for human LIF which could detect as little as 100 pg/ml and 10 pg/ml of rHuLIF in the absence and in the presence of the ELAST ELISA amplification system, respectively. The addition of serum had very minimal effect on this ELISA.
