Determination of antibody affinity using ELISA

Some new approaches for the determination of antibody affinity were proposed. It was pointed out that the proposed methods are more simple, convenient, precise, and informative than that of Friguet et al. (1985). The approach that allows determination of two-valence antibodies affinity was also proposed. The example of two monovalent antibodies presented in the examined mixture was considered. It allows to estimate the affinity of both kinds of antibodies as well as to determine their concentration relations in the mixture.     

Determination of antibody affinity by ELISA. Theory

New approaches for the measurement of antibody affinity by ELISA are suggested and considered theoretically. It was shown that not only more precise and more convenient in comparison to that suggested earlier, but also more informative graphical representation of the experimental data in the appropriate coordinate could be used for evaluation of antibody affinity. The following cases were considered: (i) determination of antibody affinity for one kind of univalent antibodies, (ii) determination of antibody affinity for one kind of bivalent antibodies, (iii) determination of antibody affinity for two kinds of univalent antibodies, which are in a mixture, and (iv) determination of antibody affinity for two kinds of bivalent antibodies, which are in a mixture. Advantages and disadvantages of the different approaches are discussed.     

Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.     

Alterations in monoclonal antibody affinity and antigenic receptor site expression on mycoplasma-infected human colorectal cancer cells

The affinity of MoAb CO 17-1A and expression of its antigenic target were studied on uninfected and mycoplasma-infected colorectal cancer cell lines SW 1116 and SW 948. Binding of 125I-labeled CO 17-1A to SW 1116 cells was quantified at 37 degrees C by determination of the affinity constant (Ka) and the number of antigenic receptor sites (r) per cell using Scatchard plots. When mycoplasma-free SW 1116 cells were used as targets, Ka was 0.92 +/- 0.06 x 10(8) M-1 and r = 1.32 +/- 0.14 x 10(6) at 37 degrees C. One batch of unspeciated, mycoplasma-infected SW 116 cells had reduced affinity and a decreased number of antigenic receptor sites per cell for 125I-labeled 17-1A, while another batch of infected SW 1116 cells had a 4- to 5-fold increase in r and diminished Ka for the antibody compared with uninfected cells. When unspeciated, mycoplasma-infected SW 948 cells were exposed to 125I-labeled 17-1A and the data subjected to Scatchard analysis, the affinity of the antibody deviated markedly from linearity and rendered analysis for Ka and r meaningless. These data indicate that mycoplasma infection can produce variable effects on the cellular expression of antigenic receptor sites and the affinity of antibody for its target, and emphasize the importance of using mycoplasma-free cell lines in studies of these parameters.     

Purification of urokinase by monoclonal antibody affinity chromatography

Matrix-bound monoclonal antibodies against urokinase have been used to purify this enzyme by affinity chromatography. In a single-step procedure, urokinase can be isolated from crude preparations with high yield and high purity, and without loss of enzymatic activity.     

Isolation of chromosaponin I-specific antibody by affinity chromatography

Chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea and other leguminous plants, modulates several developmental processes of plant roots and activates the sugar taste receptor cells in blowflies. CSI is a unique saponin for its reducing power and biological activities in both plants and insects. In the present paper, we described the method of preparation for CSI-specific antibody using CSI-affinity and soyasaponin I-affinity columns. The antibody's-specific binding activity to CSI was confirmed by a bioassay using Arabidopsis roots and a ligand-molecule interaction analysis using BIAcore 3000. Because of the lability of CSI, the CSI-affinity column was made only by a moderate reaction condition in which CSI was coupled to EAH Sepharose 4B in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). The special control of the reaction temperature was essential to complete the coupling reaction; the reaction with EDC at 0 degrees C followed by a gradual increase in temperature.     

Efficient bispecific monoclonal antibody purification using gradient thiophilic affinity chromatography

Bispecific monoclonal antibodies (bsMAbs), due their unique design, have a wide range of potential applications in immunodiagnostics and immunotherapy. One of the major limitations for the use of bsMAbs produced by hybrid–hybridomas is the concomitant production of parental monospecific antibodies. The relative amount of bsMAb secreted may vary between different hybrid–hybridomas. Hence, the purification of the desired bispecific molecule from other forms is crucial. Current purification methods include anion-exchange, HPLC on different matrices, and dual affinity methods. Most of those methods include multiple steps and have limitations on the purity or yield of the desired species. We report here a simple single-step purification method, using inexpensive thiophilic chromatography. This new method can potentially be scaled up, for industrial proposes. Finally, based on the amino acid sequences and assembly of the two heavy chains we attempt to explain the possible mechanism by which thiophilic chromatography was able to resolve the bsMAbs from the monospecific species.     

Purification of individual tRNAs using a monoclonal anti-AMP antibody affinity column

A murine monoclonal anti-AMP antibody affinity matrix was used for isolation of individual species of amino acid transfer nucleic acids (tRNAs). The antibodies had been prepared using 5'-AMP covalently attached to bovine serum albumin as antigen and exhibited high affinity for 5'-AMP but greatly reduced affinity for 3'-AMP. Native uncharged tRNAs that terminate in a 5'-AMP group on the amino acid acceptor arm of the molecule bind tightly to the anti-AMP affinity matrix, whereas aminoacylated tRNAs are not retained. This allows separation of a particular tRNA species as its aminoacyl derivative from a complex mixture of uncharged tRNAs under very mild conditions.     

Modulation of antibody affinity by a non-contact residue.

Antibody LB4, produced by a spontaneous variant of the murine anti-digoxin monoclonal antibody 26-10, has an affinity for digoxin two orders of magnitude lower than that of the parent antibody due to replacement of serine with phenylalanine at position 52 of the heavy chain variable region (Schildbach, J.F., Panka, D.J., Parks, D.R., et al., 1991, J. Biol. Chem. 266, 4640-4647). To examine the basis for the decreased affinity, a panel of engineered antibodies with substitutions at position 52 was created, and their affinities for digoxin were measured. The antibody affinities decreased concomitantly with increasing size of the substituted side chains, although the shape of the side chains also influenced affinity. The crystal structure of the 26-10 Fab complexed with digoxin (P.D.J., R.K. Strong, L.C. Sieker, C. Chang, R.L. Campbell, G.A. Petsko, E.H., M.N.M., & S.S., submitted for publication) shows that the serine at heavy chain position 52 is not in contact with hapten, but is adjacent to a tyrosine at heavy chain position 33 that is a contact residue. The mutant antibodies were modeled by applying a conformational search procedure to position side chains, using the 26-10 Fab crystal structure as a starting point. The results suggest that each of the substituted side chains may be accommodated within the antibody without substantial structural rearrangement, and that none of these substituted side chains are able to contact hapten. These modeling results are consistent with the substituents at position 52 having only an indirect influence upon antibody affinity.(ABSTRACT TRUNCATED AT 250 WORDS)     

单克隆抗体亲和层析法纯化重组溶葡萄球菌酶

溶葡萄球菌酶能够特异性杀灭金黄色葡萄球菌且不易产生耐药性, 有望成为治疗葡萄球菌属细菌引发感染的特效药物。为获得高纯度的重组溶葡萄球菌酶以达到药用标准, 本研究构建了一种以重组溶葡萄球菌酶单克隆抗体为配体的亲和层析纯化方法。纯化后的重组溶葡萄球菌酶纯度大于95%, 得率大于90%, 即使重复使用30多次, 纯化效率不变。且经比色法鉴定纯化后的重组溶葡萄球菌酶仍具有良好的活性。该方法步骤简单, 纯化效果好, 为生产高纯度重组溶葡萄球菌酶奠定了基础。     

Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis

Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.     

Heterogeneity of monoclonal antibody affinity

In experiments with Pasteurella pestis monovalent capsular antigen and hybridoma monoclonal antibodies obtained after cloning and recloning the heterogeneity of the active centers of antibodies with respect to their affinity was revealed. In ascitic fluids obtained from the animals inoculated with different hybridoma clones 5 groups of antibodies, differing in their affinity, were determined in each fluid sample.     

The human multicatalytic proteinase: affinity purification using a monoclonal antibody

A monoclonal antibody, coupled to Sepharose CL-4B, was used for the rapid purification of the human multicatalytic proteinase in a single chromatographic step under mild conditions. The enzyme was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. Electrophoresis under dissociating and reducing conditions revealed at least ten components with molecular masses in the range 22-34 kDa. Affinity-purified enzyme was identical to conventionally purified enzyme with respect to enzymatic properties, molecular mass and subunit composition.     

Western blot antibody determination in sera from patients diagnosed with Anisakis sensitization with different antigenic fractions of Anisakis simplex purified by affinity chromatography

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.     

Separation of antigens by immunological specificity. Studies on the desorption of homologous antigen from disulphide-linked antibody immunosorbents

1. Glycine-hydrochloric acid buffer, pH2.2, desorbed (131)I-labelled human serum albumin (100%), lysozyme (100%), ovalbumin (90%), fluorescent ovalbumin (50-60%) and fluorescent human gamma-globulin (20%) from their respective homologous disulphide-linked antibody immunosorbents; reasons are suggested for the low recoveries of the fluorescently labelled proteins. 2. Approx. 40% of the recovered (131)I-labelled human serum albumin and fluorescent ovalbumin was desorbed above pH6.0, but lysozyme was not eluted until the pH was 3.0 or below. 3. In all cases where high recoveries of antigen were obtained, the immunosorbents could be regenerated and recycled at least four times with full retention of specificity and minimal diminution of capacity. 4. The desorbed antigens were unchanged when compared with the original antigens by quantitative precipitin, specificradioactivity, fluorescent and enzymic analyses and by cellulose acetate electrophoresis. 5. Desorption of antigen with a variety of reagents was investigated. These reagents were less satisfactory than glycine-hydrochloric acid.