Efficient Purification of Mouse Anti-FGF Receptor IgM Monoclonal Antibody by Magnetic Beads

Abstract

Affinity chromatography has been widely used for the purification of monoclonal antibodies (MAb). Traditionally, activated agarose beads conjugated with specific antisera have been used as a solid support in chromatographic protein purification. Magnetic beads conjugated with various antibodies have recently become an alternative method for the isolation of diverse proteins, nucleic acids, and cell types. In this study, murine anti-fibroblast growth factor receptor 1 (FGFR1) immunoglobulin M (IgM) was isolated from protein solutions to compare immunoaffinity column chromatography and magnetic bead IgM purification methods. Using immobilized rat anti-mouse IgM MAb, an UltraLink 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC)/diaminodipropylamine (DADPA) immunoaffinity column and polystyrene-coated magnetic beads were used for the purification of mouse IgM from bovine serum albumin/phosphate-buffered saline (BSA/PBS) as well as from crude ascites. Protein quantitation and percent IgM yield were determined by reducing SDS-PAGE electrophoresis followed by silver staining, then IgM and protein contaminants were quantitated using densitometry analysis. IgM anti-FGFR1 binding specificity and immunologic activity were determined by enzyme-linked immunosorbant assay (ELISA). This study demonstrates that magnetic bead isolation of IgM from ascites is more effective than traditional affinity chromatography purification as determined by greater IgM yield, purity, and immunologic activity.

Instant on-paper protein digestion during blood spot sampling

“…Antibodies have been shown to be readily immobilized to magnetic beads or other particle shapes suitable for either column packing 110 , precipitation beads 111 or magnetic applications 112,113 . Having antibodies attached to solid supports, moreover, immobilized beads ( Fig.…”

Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies

“…Antibodies are important therapeutic agents and the development of purification schemes for these biomolecules has attracted great interest (Roque et al , 2007), mainly due to the high costs associated with product recovery (Hubbuch and Thomas, 2002; Roque et al , 2004; Ditsch et al , 2006). Packed‐bed chromatography, combined with biological ligands with great affinity and specificity for antibody molecules, has been the workhorse of antibody purification.…”

“…Packed‐bed chromatography, combined with biological ligands with great affinity and specificity for antibody molecules, has been the workhorse of antibody purification. The biological ligands mostly utilized are the bacterial proteins A, G, and L. However, the high cost of these adsorbents as well as stability and leaching issues have triggered the development of synthetic affinity ligands mimicking these biological receptors (Roque et al , 2004). These ligands, mainly those based on the triazine molecule, are known to be low cost and highly resistant to industrial cleaning procedures (Lowe, 2001; Roque and Lowe, 2006).…”

Uso de partículas magnéticas en la purificación de anticuerpos IgM contra Taenia solium

“…Esto es una ventaja de las PM frente al uso de columnas cromatográficas, ya que con el uso de columnas, la preconcentración es necesaria, probablemente debido a que la atracción y la Se agregaron 400 µg de PM-PL al SH en un volumen final de 1500 µl de solución salina tamponada con Tris como un tampón de unión La actividad del anticuerpo se midió mediante ELISA para determinar la capacidad del anticuerpo para reconocer quistes sonicados de T. solium PM-PL: partículas magnéticas acopladas a proteína L; SH: sobrenadante de hibridomas; DO: densidad óptica medida a 590 nm Tabla 1. Optimización del protocolo de purificación de anticuerpos monoclonales IgM a partir de sobrenadantes de cultivos de hibridoma utilizando partículas magnéticas acopladas a proteína L. frecuencia de oportunidades de interacción son menores en comparación a cuando se usan partículas (17)(18) , y además para evitar pasar varias veces el cultivo en la columna sobre todo cuando se trata de volúmenes mayores a 1 ml, lo cual puede ser tedioso. El uso de partículas magnéticas favorece la interacción de mIgM y proteína L en solución en un solo paso, además previene la pérdida de la actividad inmunogénica de los mIgM, ya que esta puede ocurrir durante la unión de IgM a otras resinas (16) .…”

“…En nuestro estudio, el corto tiempo de manipula-ción (dos horas en total) en comparación con métodos de cromatografía de afinidad clásicos (diez horas a dos días) puede haber contribuido al menor riesgo de desnaturalización de las mIgM y aumentar su probabilidad de resistencia a la elución acídica (12) . Nuestro PR (> 90%) es más alto que el reportado previamente usando PM acopladas con anticuerpos anti-IgM (70%), probablemente debido a la mayor afinidad de la PL a la IgM (17) .…”