The document introduces the Agilent Technologies' Multiple Affinity Removal System, which combines the specificity of antibody-antigen recognition with the efficiency of standard liquid chromatography instrumentation. This system effectively removes multiple proteins from human serum, including albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin, in a single step with high specificity and reproducibility. The document also demonstrates several methods to optimize column binding capacities, such as coupling two identical columns to increase capacity, using a custom column with dimensions of 10x100mm to enhance capacity, and achieving optimal binding capacity by injecting samples at a low flow rate.
The Agilent Multiple Affinity Removal System removes high-abundance proteins like albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin from human serum in a single step. This document outlines strategies to increase the system's capacity to handle larger sample volumes, which is beneficial for biomarker investigations.
Three specific optimization methods are detailed: coupling two identical 4.6 x 50-mm columns to double capacity, using a custom 10 x 100-mm column for a tenfold increase, and injecting samples at a low flow rate to achieve optimal binding.
| Targeted proteins removed | albumin, transferrin, IgG, IgA, haptoglobin, and antitrypsin |
|---|---|
| Column capacity (4.6 x 50-mm) | 15-20 uL of human serum |
| Column capacity (4.6 x 100-mm) | 30-40 uL of human serum |
| HSA binding capacity (4.6 x 50-mm) | 0.6-0.9 mg |
| HSA binding capacity (Coupled 4.6 x 50-mm) | 2.0 mg |
| HSA binding capacity (10 x 100-mm) | 10 mg |
Two identical columns can be coupled together to create a tandem column with twice the capacity of a single column.
A custom column with 10 x 100-mm dimensions has a capacity about 10 times that of a 4.6 x 50-mm column.
Optimal binding capacity can be obtained by injecting samples at a low flow rate.