Antibody-coated columns

Wigzell, H., Sundquist, K. G., Yoshida, T. O. Separation of cells according to surface antigens by the use of antibody-coated columns. Fractionation of cells carrying immunoglobulins and blood group antigen. Scand. J. Immunol. 1, 75-87 (1972). 

Technologies to purify cells from white cell concentrates are in the research stage. Principles used include antibodies covalently bound to a surface, antibody-coated microbeads in a column, magnetic microparticles that have been coated with antibodies, and hoUow fibers that have been coated with antibodies. 

It has to be taken into account that microorganisms can also be separated, identified, and characterized by CE without the necessity of using PCR. A comprehensive review of Desai and Armstrong [71] gives details about these possibilities, such as the recent paper of Gao et al. [72] who detected Staphylococcus aureus by a combination of monoclonal antibody-coated latex and CE. CZE separations were performed on a Beckman P/ACE MDQ System equipped with aphotodiode array detector. A27-cm-long capillary column was used for the separation (20 cm to the detection window) applying 215 kV at a constant temperature of 25°C. 

FIGURE 3.4 Schematic diagram of an ABICAP column and binding of target analytes to primary (capturing) antibody and secondary-antibody-coated magnetic beads. 

The borderline between immunoassays performed in the flow injection analysis format and those achieved in the immunochromatographic format is sometimes difficult to establish. Flow injection renewable surface immunoassay (FIRSl) avoids the desorption step of immunochromatographic assays by using a layer of antibody-coated beads held in a jet ring cell instead of the chromatographic column. The coated beads are discarded after each analysis. To ensure assay reproducibility, antibody-coated beads with constant antigen capacity should be obtained [129,130]. 

The column technique has been applied to lymphocytes from non-im-munized mice as well (Wigzell and Makela, 1970). A mixture of normal lymph node, spleen and bone-marrow cells was passed through an OA-coated column. Column passed cells as well as control cells were transferred to lethally irradiated mice (10 cells per mouse) and followed by immunization with OA and BSA. The antibody content in the sera was detected by the Farr assay. There was no response to OA, while the response to BSA was normal. Control cell suspensions which were not subjected to the column passage resulted in a good response to both OA and BSA (Table 5). 

Wigzell, H., Andersson, B. Cell separation on antigen-coated columns. Elimination of high rate antibody-forming cells and immunological memory cells. J. exp. Med. 129, 23-36 (1969). 

Wigzell, H., Andersson, B., Makela, O., Walters, C. S. Characteristics of surface-attached antibodies as analyzed by fractionation through antigen-coated columns. In Cell interactions and receptor antibodies in immune responses (O. Makela, A. Cross, T. U. Kosunen, Eds.), p. 231-242. London and New York Academic Press 1971. 

Wigzell, H., Makela, O. Separation of normal and immune lymphoid cells by antigen-coated columns. Antigen binding characteristics of membrane antibodies as analyzed by hapten-protein antigens. J. exp. Med. 132, 110-126 (1970). 

Figure 16.5 Immunostained peptide arrays after various treatments of fixation, protein cross-linking, and antigen retrieval, as indicated at the top. Each row has a different peptide that is immunoreactive for the antibody denoted to the left. Column A represents the baseline condition, without any treatment whatsoever. Column B shows immunoreactivity of each peptide after overnight formalin fixation. Column C shows the immunoreactivity after first coating the array with an irrelevant protein (casein) followed by overnight formalin fixation. Column D illustrates the immunoreactivity of the peptides after the treatment of column C, and then antigen retrieval. Reproduced with permission from Reference 15, 2006 American Society for Clinical Pathology.

Initially, the antibodies should be purified prior to prepare the immunoaffinity column. Precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration chraoma-tography or affinity chromatography may be employed with the aim of antibody purification. Activated beads which are coated with bacterial proteins A or G may be used as the support material. Some parameters may be changed for the elution of the sample solution for example the ionic conditions of mobile phase may be changed or chaotropic buffers may be used [11]. 

For high-performance lAC, the preferred solid support is a glass bead solid support coated wilh either protein-A or protein-A covalently linked with the antibody through a carbodiimide bond (165, 166). In either case, protein-A binds to the Fc portion of the antibody so that the combining sites are oriented to the mobile phase. Once the protein is attached, the lAC matrix is packed into the column either as a slurry or dry. Pump-slurry techniques use buffers with a low salt content, such as Tris or 0.01 M phosphate buffer to minimize friction and denaturation of the immobilized antibody (16). If the solid support consists of glass beads, the packing can be freeze-dried after antibody attachment and packed dry. 

Other than protein, histamine in human blood samples was determined by the immunoassay method. It was detected by its binding with anti-histamine IgG, which was coupled to ferrocene (Fc-IgG). Separation of histamine and its complex with antibody was based on p/ differences, and it was achieved in a PMMA chip consisting of a multichannel matrix column coated with a cation-exchange resin (Nation). Histamine was detected electrochemically a decrease in the current due to Fc-IgG occurred after its binding to histamine [1012],... 

Another approach to increase the loadabihty is to enrich the analyte at the capillary inlet by means of an adsorptive phase, as reviewed in detail in Ref. 10. Re-versed-phase materials such as octadecyl-bonded silica have regularly been used in the form of membranes or column materials, thus, in principle, performing miniaturized SPE ( SPE) in-line with CE, allowing injections of 10-15 /uL. More selective sample concentration is obtained when using antibodies or Fab fragments for coating the inner wall of the capillary inlet [3,5]. 

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