Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification (original) (raw)
Related papers
Journal of Immunological Methods, 1983
The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-c-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; J25I-HOP-DNPlysine was used in equilibrium dialysis and polyethylene glycol precipitation; and J25I-DNPs-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.
A method for titrating anti-hapten antibody by passive haemagglutination
Journal of Immunological Methods, 1973
A general method for measuring anti-hapten antibody by passive haemagglutination is described. It employs as indicator cells erythrocytes coated with haptenated Fab where the Fab is derived from antibody directed against the erythrocytes. 0.2 jag/ml anti-DNP antibody could be detected. * Abbreviations used: DNP: dinitrophenyl; DNP-BGG: dinitrophenylated bovine gamma globulin; Fab, Fc: fragments of IgG derived by papain digestion, respectively antigen-binding and crystallisable; FCS: foetal calf serum; PBS: Dulbecco 'A' phosphate buffered saline.
An ELISA-based approach to optimize elution conditions for obtaining hapten-specific antibodies
Analytical and Bioanalytical Chemistry, 2003
The correct choice of the elution conditions to break an affinity interaction is important for the successful purification of biomolecules. The optimal elution buffer liberates the bound substance in a minimum volume and maintains the activity of the purified material. The present study demonstrates an enzyme-linked immunosorbent assay (ELISA)-based approach for selection of specific elution conditions for eluting antibodies against a small molecule (atrazine) from pooled sera. Six different elution conditions were tried for the removal of antibodies from the complex. Large-scale purification of anti-atrazine antibodies from the sera was done with a hapten-specific column using an amino-terminal crosslinked agarose gel. Efficacy in terms of total amount of recovery and binding affinities of eluted antibodies from the column were further investigated by ELISA. Results indicate that the ELISA-based elution approach is ideal for the selection of suitable elution buffer that can subsequently be utilized for affinity purification applications.
Physical Properties of Antibody to Bovine Serum Albumin as Demonstrated by Hemagglutination
2000
The heterogeneity of antibody as determined by a variety of physical, chemical~ and biological methods has been well established. Since the demonstration of high molecular weight horse antipneumococcal antibody (1), numerous studies have shown electrophoretic, ultracentrifugal, and chromatographic differences in human and various animal antibodies (2, 3). Generally rabbit and human anfi-erythrocyte antibodies are characterized as electrophoreti-caUy slow migrating w-globulin of low molecular weight (7S) and low anionic binding, and fast migrating ~,-globulin of high molecular weight (19S) and high anionic binding (2, 3). Although most studies have been restricted to anticellular antibodies, Wassermann antibodies (4), skin-sensitizing antibodies , rheumatoid factor , and anti-thyroid auto-antibodies , have been shown to belong to the high molecular weight class of antibodies. Certain of these antibodies have been associated with the duration of immunization. For example, 19S horse anti-pneumococcal antibody formed early in immunization is followed by the production of low molecular weight antibody on prolonged antigenic stimulation (8). More recently Stelos (9) has reported early rabbit hemolysin to be mostly a ~-1 with a sedimentation rate of 19S and that a 3,-2 hemolysin associated with 6S protein to appear later in immunization. Employing partition chromatography, Porter (10) and Humphrey and Porter (11) found in the early stages of production that anti-protein rabbit antibodies were in the slow moving fractions, and that on further immunization antibody appeared in faster moving fractions. There is, however, a dearth of information of this kind with regard to antibodies to soluble protein antigens. The present study describes the electrophoretic, ultracentrifugal, and chromatographic separation of early and hyperimmune rabbit anti-bovine serum albumin antisera employing the passive hemagglutination (HA) method. Evidence is presented that rabbit anti-protein antibodies are similar to anti-cellular antibodies with respect to their sequential synthesis. tw ILl J-z O Iz t -.J ¢D CD bJ "1-
Influence of Antibody affinity on the performance of different antibody assays
Journal of Immunological Methods, 1984
The effect of antibody affinity on the performance of 5 commonly used assays was studied. The assays used were measurement of antigen binding capacity in a Farr type assay, haemagglutination, solid-phase radioimmunoas~ay (SP-RIA), solid-phase ELISA and precipitation. The first 4 assays were all more sensitive for high affinity antibodies. Precipitation was not related to affinity, suggesting that factors secondary to antigen-antibody binding may be more important in determining the level of precipitate formation.
Biochemical characterization of antibodies submitted as reactive with glycophorin species
Revue française de transfusion et immuno-hématologie, 1988
Thirty-one antibodies submitted for testing were examined in order to determine with which glycophorin species they were reactive and what region of the target molecule(s) were involved in antigen recognition. Antibodies were assayed by direct and indirect aggmutination techniques, using cells of various MNSs phenotypes before and after exposure to various enzymes. In addition, antibodies were tested by immunoblotting and, in a small number of instances, by radioimmunoprecipitafion, in order to determine or confirm the molecular species reactive with each antibody. MATERIALS AND METHODS Erythrocyte agglutination assays All antibodies were screened for reactivity with red cells (RBC) of various MN and SsU phenotypes by agglutination assay using routine blood bank serologic techniques [3]. Initial screening was done using tissue culture supernates undiluted and purified antibody or ascites fluids diluted 1 : 100 in phosphate buffered saline supplemented with 1% (w/v) bovine serum albumin. Indirect agglutination was accomplished using rabbit anti-mouse IgG (heavy and light chain) antibody diluted 1:500 (Jackson Laboratories). After reactive cells were identified, all antibodies were titered and subsequently used at the greatest dilution at which maximal binding occurred as measured by agglutination reactions. The effects of various enzymes on target antigens were also determined using agglutination assays, as previously described [3, 5]. Trypsin and chymotrypsin were used at final concentrations of 2 mg/ml, and neuraminidase was used at a ratio of 25 units per 0.5 ml packed RBC. Cells of various MN phenotypes, as well as U
Antibody engineering for the analysis of affinity maturation of an anti-hapten response
The EMBO journal, 1988
The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-...