H.F.J. Savelkoul

Semi-preparative purification and validation of monoclonal antibodies for immunotherapy in mice

A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS or 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy.

Terasaki-ELISA for murine IgE antibodies.II.Quantitation of absolute concentration of antigen-specific and total IgE

A Terasaki tray-based ELISA system was developed for the quantitative measurement of antigen-specific and total IgE antibodies in 5 microliter samples of mouse serum dilutions. The assay was based upon non-competitive binding of mouse IgE antibodies between the immobilized appropriate antigen or capture antibodies and the detecting rabbit antibodies. A conjugate of protein A-labelled beta-galactosidase and the fluorigenic substrate methylumbelliferyl-beta-D-galactoside were used as a detecting system. The resulted fluorescence could be measured rapidly and automatically using an inverted micro-fluorimeter. These measurements were automatically transformed into absolute concentrations by a microprocessor-based program using a four-parameter logistic function and an absolute IgE standard. The assay was shown to have a detection limit of 0.04 ng/ml and a range of linearity of 0.04-20 ng/ml, which is sufficient to measure IgE concentrations in mouse serum.

Rapid procedure for coupling protein antigens to red cells to be used in plaque assays, by prewashing in chromium chloride

A rapid and efficient procedure is described for the coupling of proteins (protein A, ovalbumin, albumin and chicken gamma globulin) to sheep red blood cells (SRBC) to be used in antigen-specific or protein A plaque assays. This modification of the original procedure has three distinct features: prewash of the red cells with a low concentration of essentially freshly prepared CrCl3, use of a relatively high concentration of CrCl3 in the reaction mixture and a coupling time of only 4 min. Protein A plaque assays performed with such target cells have the same sensitivity as those employing red cells coupled with protein A according to the original procedure. Studies with hybridomas secreting antibody specific for a protein antigen showed that antigen-specific plaque assays employing target red cells coupled with the protein antigen according to the modified procedure have the same sensitivity as the protein A plaque assay. The modified procedure greatly facilitates cellular studies on antibody formation after immunization with protein antigens.

Terasaki-ELISA for murine IgE-antibodies: I. Quality of the detecting antibody: Production and specificity testing of antisera specific for IgE

In order to develop an ELISA for the quantitative determination of murine IgE, antisera specific for murine IgE were prepared in the goat and rabbit. As immunogen, monoclonal IgE antibody mixtures of several allotypically different hybridomas were used. Before use, these antibodies were purified employing procedures that allow maximum recovery of binding activity. The goat and rabbit mouse epsilon chain-specific antisera were adsorbed on normal mouse serum. The purified antisera were found to be free of allotypic activity. However, immunoadsorption on NMS could not always remove contaminating anti-idiotypic antibodies. Repeated adsorptions with monoclonal antibodies of different isotypes carrying a similar idiotype were necessary to remove all detectable anti-idiotypic activity. Only after these precautions were the antisera suitable for detecting IgE molecules on nitrocellulose blots as well as for quantitating circulating IgE antibodies (ELISA) and IgE-secreting cells (plaque assay and reverse ELISA plaque assay). The purity and reactivity of several commercially available anti-IgE preparations were tested in similar types of specificity assays. Since the specificity of antibodies used in ELISA determines the monospecificity of the assay, retesting for contaminating cross-reactivities in commercial preparations was shown to be necessary.

Occurrence of damaged heavy chains during purification of murine IgE antibodies by fast protein liquid chromatography (FPLC) and their effect on the determination of concentration and affinity in ELISA

ABSTRACT A three-column automated FPLC system was employed to isolate mouse monoclonal-IgE antibodies (mAb) from ascitic fluids and culture supernatants with high yield and recovery. This system allowed large scale purification as well as analytical isolation of IgE antibodies. On the analytical level this purification method was used to study the different problems that are often encountered when dealing with mAb, especially when dealing with ascitic fluids. One problem is degradation caused by proteolytic and glycolytic enzymes. The activity of proteolytic enzymes in the starting material (culture supernatant of mouse IgE secreting hybridomas) results in loss of binding by monoclonal anti-IgE antibodies as determined in ELISA, leading to an underestimation of absolute IgE concentration and affinity constant. This proteolysis also leads to a loss of charged residues as established by a decreased elution strength from a cationic exchanger. A mixture of protease inhibitors can protect IgE antibodies against this proteolytic breakdown. Elution of bound IgE from an affinity column with acetic acid as compared to competitive hapten elution results in damage of the Fc portion leading to a decreased binding in ELISA and on an ion exchange column.

Terasaki-ELISA for murine IgE. III: Determination of concentration and functional affinity by sequential equilibrium binding analysis

A simple Terasaki tray-based ELISA technique with a fluorescent detecting system has been used to determine the affinity of murine IgE antibodies. The system was shown to be sensitive enough to measure affinities in the range of 10(-6)-10(-10) M as well as detect IgE antibodies down to a limit of 0.1 ng/ml. The results, expressed as arbitrary fluorescence units (AFU), were compared with those obtained using equilibrium dialysis for several DNP-specific IgE monoclonal antibodies of known affinities yielding KD values. The relationship between KAFU and KD established a conversion factor which could then be used to compute KD from KAFU, provided the detection system remained identical. Based on the equations proposed, an alternative method for the quantitation of murine IgE is described which is independent of the affinity of IgE for the coated antigen.

Quantitation of Murine IgE in an Automatic Elisa System

We describe a two-site micro ELISA system for rapid, automatic and quantitative measurement of total IgE in mouse serum, that is based upon a hybridoma IgE standard and heterologous anti-IgE antibodies. With this method we determined the absolute total IgE concentration in the serum of aging athymic nude mice and their heterozygous litter mates.

Isolation, Characterization and Quantitation of Murine Ige

Abstract A reproducible and effective large scale purification procedure is described to isolate and to purify murine IgE from ascitic fluid induced by a TNP-specific mouse IgE secreting hybridoma. By means of SDS-PAGE and HPLC the purity and identity of the isolated material can be evaluated. Using an ELISA method, the isolated IgE can be quantitated.