Retention Of Antigen-binding Activity Research Articles

Targeted molecular imaging and cell homing in cardiovascular disease via antibody-sortagging

Aim: Targeting of contrast agents to unstable atherosclerotic plaques offers the potential to identify such plaques before rupture, allowing suitable interventions and thus avoiding myocardial infarction and death. Similarly, homing of stem cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen binding activity and functionality of the targeted biological component (e.g. stem cells). Here we report a novel, gentle, robust, highly reproducible, and site-specific coupling method utilizing the Staphylococcus aureus sortase A enzyme to conjugate a single-chain antibody (scFv), anti-GPIIb/IIIa-scFv, to nanoparticles and cells for molecular imaging and stem cell homing in cardiovascular disease.

Kinetics of Whole Serum and Prepurified IgG Digestion by Pepsin for F(ab')2 Manufacture

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.

Development and validation of an antigen-binding capture ELISA for native and putrescine-modified anti-tetanus F(ab′)2 fragments for the assessment of the cellular uptake and plasma kinetics of the antibodies

Cationization is a strategy to enhance the permeability of antibodies to physiological membranes for potential therapeutic and diagnostic applications of these proteins, with one of its crucial points being the retention of antigen binding activity. Here, we describe the cationization of horse polyclonal anti-tetanus F(ab′) 2 fragments and the development and validation of an ELISA for quantitative measurements of the binding activity of the native and cationized F(ab′) 2 in cell lysates and rat plasma samples, assessing the cellular uptake and plasma kinetics of these antibodies, respectively. The method used tetanus anatoxin coated on microtitre plates as capture antigen to bind sample or standard F(ab′) 2 , the amount of antibody binding being quantified using, first, a secondary biotinylated anti-horse antibody/streptavidin-alkaline phosphatase complex in situ and then a measurement of the substrate product. Cationization of the F(ab′) 2 was performed with putrescine at pH 4.5 using soluble carbodiimide as carboxyl activator. The average substitution ratio was determined at 3 putrescine molecules per F(ab′) 2 molecule. The cationized F(ab′) 2 retained roughly 80% of the initial antigen binding activity and was stable over a 1 year period of storage at − 20°C. The ELISA validation data showed that the method was linear for both the native and cationized F(ab′) 2 using Hanks' balanced saline solution with 0.2% bovine serum albumin as assay diluent for the cell lysate samples. The useful F(ab′) 2 concentration range was 2.5–25 ng/ml and the limit of quantification was 2.5 ng/ml. With rat blank plasma used as assay diluent for the rat plasma samples the useful F(ab′) 2 concentration range was 3.5–25 ng/ml and the limit of quantification was 3.5 ng/ml. Specific requirements for the limits of quantification were fulfilled: precision ≤ 20% CV and accuracy within ± 20% of the nominal values. Intra- and inter-assay coefficients of variation were within 9% and accuracies within ± 10% of the nominal values. The validated method was applied to the study of the cellular uptake of native and cationized anti-tetanus F(ab′) 2 in an HL 60 cell model, and of plasma kinetics after i.v. administration to rats.

Facile F(ab′) 2 Manufacturing: Strategies for the Production of Snake Antivenoms

Asimple process for the manufacture of polyclonal F(ab′)2 fragments that might be adopted for the facile preparation of antivenoms is described. The production of polyclonal F(ab′)2 fragments from serum commonly involves the initial purification of IgG's prior to their proteolytic cleavage and further purification. To reduce the number of processing steps the authors have compared the digestion of whole serum by free and immobilized pepsin with that of pure IgG. It was observed that with equal units of pepsin activity, caprylic acid pre-purifi ed IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was signifi cantly greater in the latter case. The effi ciencies of whole serum digestion by the same total units of free and immobilized pepsin was also compared. IgG digestion was complete after 12 hours, as was serum digestion. About 80% of the antigen binding activity was retained in either case after 24 hours though the immobilized pepsin lost about 40% of its proteolytic activity after first use. The purifi cation of F(ab′)2 on two ion exchange adsorbents (StreamlineTMSP and paramagnetic CM beads) on paramagnetic thiophilic beads and on a hydrophobic custom assembled guanidine agarose was studied. The binding capacity on CM beads was too low to be useful for F(ab′)2 preparation. Most of the F(ab′)2 bound to the Streamline SP at low pH (pH 4.0 and 3.0), as did significant amounts of low molecular weight digestion products. The F(ab′)2 fragments were eluted completely with 0.3M NaCl whilst the low molecular weight digestion products required 1M NaCl to elute them. F(ab′)2 also bound to the thiophilic beads, free from the low molecular weight digestion products, and could be eluted with low ionic strength solutions. The guanidine agarose beads showed a lower capacity than the thiophilic beads but their selectivity was comparable. Overall the thiophilic and hydrophobic matrices showed better selectivity than the cation exchange matrices.

A novel covalent modification of antibodies at their amino groups with retention of antigen-binding activity

A novel method of covalent modification of antibodies at their amino groups with retention of antigen-binding activity is described. The procedure is as follows: (a) blockade of those amino groups of antibodies whose integrity is essential to their antigen-binding activity with 2,3-dimethylmaleic anhydride, a reversible amino group-blocking reagent; (b) modification of residual amino groups with reagents reactive with the amino groups; and (c) removal of dimethylmaleyl groups by hydrolysis. This procedure was used for covalent conjugation of methotrexate (MTX) with two monoclonal antibodies against human melanoma-associated antigens using MTX N-succinimidyl ester. MTX attached to the antibodies at sites other than the amino groups via less stable bond(s) was removed by treatment with hydroxylamine.