Immobilized monoclonal antibodies

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins, using a carbodiimide reaction with EDC. A similar protocol to that previously described for coupling to carboxylate polymer particles may be used. The following protocol is based on the method of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli 0157. 

Binding of the monoclonal antibody to the protein A domain would ensue and the immobilized monoclonal antibody would dictate the cell type targeted. 

Proteins are frequently powerful immunogens and the availability of specific antibodies, particularly monoclonal antibodies, makes the technique of affinity chromatography very useful in the separation and purification of individual proteins. The technique has been used to purify a wide range of proteins such as hormones, membrane receptors and complement proteins. However, it is not restricted to proteins and is potentially applicable to any immunogenic substance. The availability of suitable antibodies is essential and these may be raised by whole animal polyclonal techniques or by monoclonal cell culture. The former antibodies may need some prior purification before being immobilized. 

The effect of the amount of monoclonal antibody immobilized to the Sepharose matrix was further investigated by Fowell and Chase [54]. Batch experiments were used to determine the apparent adsorption rate constant k. d. A marked increase in the fc value is observed as the amount of antibody immobilized is decreased. The values of fc vary from 1.3 x 103 dm3 mol l-s l for the support of high capacity to 7.0 x 105 dm3-moM s l for the support of low capacity. A marked increase of the efficiency of the adsorption process was also observed when using the frontal analysis techniques. This effect has been explained by the improved adsorption properties of the low-capacity immunoadsorbent. 

For this study. Vidal-Madjar et al. [22—25[ used different immobilized anti-HSA polyclonal and monoclonal antibodies. The matrix for antibody immobilization is an HPLC silica (10 gm particle diameter and 6 nm mean porosity). The large antibody molecules are attached to the external surface of the particles, and this support behaves as a pellicular immunoadsorbent. The monoclonal antibodies used differ by their specificities toward several HSA fragments and by their affinities for HSA. This high affinity support strongly retains HSA when a phosphate-buffered saline eluent (pH = 7.4) is used. The regeneration of the immunoadsorbent was generally possible by desorbing the protein under acidic conditions. The column can then be reused several times. 

The repeated pulse injection method was applied to determine the association rate constant of the antigen-immobilized monoclonal antibody reaction 124.25). These monoclonal antibodies differ by their specificity and affinity for HSA. The epitope recognized by the HA6 antibody is located between residues I and 128. The epitope of the commercial antibody (m-anti-HSA) is located between residues 124 and 298 of the HSA molecule. They differ by their affinity for HSA. with equilibrium binding constants of respectively 6.7 x 108 mol-dm and 1.6 x 10K dm3 mol-1 for the HSA/HA6 and the HSA/m-anti-HSA association in solution 25). Close binding rate constants are obtained on both immobilized monoclonal antibodies with Aa = 2.5 x I0 i dm -moC -s-1. 

A biomolecule with specificity for a special molecule (which is used as a ligamd on an immobilization matrix) is modified by an affinity tag. The tag may be a synthetic antigen, such as Flag, which consists of eight amino acids and binds to an immobilized monoclonal antibody (72). The interaction of Flag with the monoclonal antibody is Ca -dependent and reversible at low pH. These features constitute an undesirable constraint when applying this fusion to affinity immobilize some enzymes. 

Immobilized monoclonal antibodies represent yet another useful matrix for reversible immobilization. Because these antibodies are monospecific and can be chosen so as to correspond to epitopes on enzyme surface and not directed against enzyme active site, the immobilization does not result in significant loss of activity. Solomon et al. (45) describes immobilization of carbodypep-tidase A. 

An indirect amperometric immimosensor for the detection of HCG was described by Chetcuti et al. [97]. The assay consisted of an anti-HCG monoclonal antibody immobilized on a GCE and the use of a sandwich assay with HRP conjugated to a second anti-HCG monoclonal antibody. Electrochemical detection of the enzymatic reduction of benzoquinone to... 

Immobilized monoclonal or polyclonal antibodies have been nsed to selectively enrich specific proteins or protein complexes (Ch. 18.4.5 and 18.5.1). 

Fig. 1 Analysis of the oligosaccharide (Glc)4 in (a) human urine and (b) serum by weak affinity chromatography. The affinity column was 5 mm LD. x 100 mm and contained immobilized monoclonal antibodies against (Glc)4. Elution was carried out under isocratic conditions at a flow rate of 0.2 mL/min using a pH 7.5, 0.02 M phosphate buffer that contained 0.1 M sodium sulfate. (From Ref. [1].)...

The measurement of holotranscobalamin II is potentially useftil as a specific marker of biologically available vitamin Bi2, because only cobalamin bound to Tell is specifically available for uptake by aU cells. Other methods have been described for the measurement of holotranscobalamin in serum, one using an immobilized monoclonal antibody to human transcobalamin, followed by measurement of released cobalamin by CPB, This method is currently available as a commercial kit. The other method uses magnetic beads coated with cobalamin to precipitate apotranscobalamin followed by measurement of the holotranscobalamin in the supernatant by ELISA. Though these methods are claimed to be precise and simple to perform, there remains doubt over the interpretation of the measured concentrations, and over their sensitivity and specificity in the diagnosis of vitamin B deficiency. ... 

Protein blots can be used (i) to identify various constituents of a mixture and to establish their relationship(s) by EIH (epitope mapping) (ii) to localize these constituents by the sensitive immunostain after their haptenation and subsequent reaction with labeled anti-hapten antibodies and, (iii) to elute antibodies immobilized to a certain protein band ( poor man s monoclonals ). 

Similarly, Coutlee et al. (1989a) developed a sandwich assay in which (i) hybrids formed in solution between the biotinylated hapten probe and the target were collected by anti-biotin antibodies immobilized in wells of a microtiter plate and (ii) detected by /3-galactosi-dase-Fab fragment (of monoclonal antibody to RNA DNA) by conversion of a substrate to a fluorescent product. Optimum conditions included hybridization for 16 h at 75°C in 2 X SSC, 10 mM HEPES, using 0.1 xg/ml probe with 7% biotinylation. 

After the appropriate monoclonal antibody, immobilization method and matrix have been chosen according to the criteria discussed above and methods previously described (7,8) the major factor in determining the cost of this purification method is the amount of antibody required. The amount of antibody required is determined by the capacity per cycle of the immunosorbent and the number of cycles that can be utilized in a given process. The capacity per cycle for the immunosorbent is given by Equation 1. 

As shown in Equation 1, the capacity per cycle is directly proportional to the amount of antibody immobilized, the immobilization yield, the M.W. of the protein and the column volume and an exponential function of the number of cycles. The amount of antibody immobilized will usually be less than 10 gL l. Higher activation of the matrix required for greater than 10 gL l loading results in a decrease in the immobilization yield. The maximum immobilization yield is 1.0 (100%) while 0.8 (80%) is not difficult to obtain. The M.W. of the protein to be isolated is fixed. The only way to increase the capacity per cycle significantly is to increase the volume of the immunosorbent or increase the number of cycles prior to reaching 50% of initial capacity (cycle half-life). Increasing the volume of immunosorbent increases the amount of monoclonal antibody required. 

Newly developed purification methodologies such as purification steps with immobilized monoclonal antibodies and other affinity chromatographic techniques not only increased the purity and specific activity of factor VIII concentrates, but also showed at the same time the substantial removal of potentially present pathogens... 

Larger analytes are readily detected by direct interaction with antibodies immobilized on the sensor surface (Fig. 9.5.6). Sensitivities for this type of assay are typically in the high nanomolar to high picomolar range. The sensitivity and specificity can be increased simultaneously with the use of secondary antibodies that are specific for an epitope other than the one bound by the surface immobilized antibodies. If very high sensitivity is required, several stages of amplification can be carried out, allowing for detection of femtomolar levels of protein. For example, mouse monoclonal capture antibodies can be immobilized on the sensor surface. 

Electron Paramagnetic Resonance Spectroscopy Studies of Immobilized Monoclonal Antibody Structure and Function... 

In order to maximize the utility of immobilized monoclonal antibodies and minimize costs when they are used, it is necessary to have a molecular-level understanding of the effects of immobilization on antibodies used in the preparation of immunosorbents. 

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