Yoshimi Kawade

The neutralization of interferons by antibody. II. Neutralizing antibody unitage and its relationship to bioassay sensitivity: the tenfold reduction unit.

The importance of establishing a common method of reporting neutralizing antibody levels is emphasized by the fact that patients injected repeatedly with a human interferon (HuIFN) may develop such antibodies that can abrogate the beneficial effects of the treatment. The earlier experimental and theoretical constructs of Kawade led to certain recommendations by the World Health Organization (WHO) concerning the methodology of neutralization tests and how to report the resultant data. A WHO international collaborative study on two human sera with antibodies against HuIFN-alpha and HuIFN-beta provided the opportunity not only to test the theoretical concepts concerning the neutralization reaction with data obtained in different bioassay systems in different laboratories but also to obtain enough data points for statistical evaluation with bioassays having a great range of sensitivity to IFN. The analyses substantiate and extend the original conclusions of Kawade that the neutralization follows the reaction mode of low-affinity antibody, in accord with the constant proportion hypothesis by which antibody reduces IFN activity in a set ratio of added/residual biologically active IFN, a consequence of the low molar concentration of free IFN at the neutralization end point. The present results support the recommendation that the preferred way to state the index of neutralization of antibodies is a titer (t), calculated by the formula t = f(n - 1)/9, where f is the reciprocal of the antibody dilution achieving the end point, and n is the IFN concentration measured in that days titration. The tenfold reduction unit (TRU) of neutralization is proposed for use in expressing the quantity, or unitage, of IFN neutralizing antibody. The utility of its application is explained. The use of the index of neutralization described and the proposed derivative term of antibody unitage, TRU, should help make the results from different laboratories employing different bioassay systems more readily comparable and interpretable, provided the bioassays are sufficiently sensitive to IFN.

Induction of interferon-γ in mouse spleen cells by OK-432, a preparation of Streptococcus pyogenes

Abstract A bacterial antitumor and immunopotentiating agent, OK-432, induced Interferon in the spleen cell cultures but not in the thymus cell cultures of various inbred strains of mice. When 1 × 10 7 spleen cells were cultured in the presence of 5 μg/ml of OK-432, interferon activity was detected as early as 4 hr later and reached a maximum level of about 160 to 500 units/ ml 24 hr later. OK-432-induced interferon was mainly an IFN-γ of molecular weight approximately 40,000, but also contained IFN-α and IFN-β.

Characterization of rapidly labeled ribonucleic acid of animal cells in culture

Abstract The rapidly labeled RNA of growing FL cells in culture was shown to consist of two classes of molecules, q 1 and q 2 , with sedimentation constants of about 40 S and 50 S, respectively. The time course of 32 P-labeling and the chase experiment indicated that q 2 is metabolically much more active than q 1 . The base composition of q 1 was similar to ribosomal RNA, while that of q 2 closely resembled DNA. As a working hypothesis, q 1 may be assumed to be the precursor of ribosomal RNA and q 2 the messenger RNA. When the total RNA was fractionated on a methylated albumin column, and the fractions comprising ribosomal RNA were examined by analytical ultracentrifugation using ultraviolet optics, a species of RNA with a sedimentation constant of 42 S was found in addition to the ribosomal RNA. This component could be greatly concentrated relative to other RNAs by chromatographic and centrifugal fractionations, and was identified as one of the rapidly labeled RNAs, q 1 .

The Neutralization of Interferons by Antibody. I. Quantitative and Theoretical Analyses of the Neutralization Reaction in Different Bioassay Systems

The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of expression of neutralizing potency and substantiate the earlier operational 10/1 LU/ml approach recommended by the World Health Organization. The accompanying paper relates neutralization results to the sensitivity of the bioassay to IFN and describes the rationale for a recommended unit of antibody neutralization.

Antigenicity of mouse interferons: Distinct antigenicity of the two L cell interferon species

Abstract Antibodies were raised in rabbits against Newcastle disease virus-induced L cell interferon of high purity, and against each of its two major species, F(24K) and S(36K) interferons. The two interferons were found antigenically distinct. Thus, the anti-F and anti-S sera failed to neutralize appreciably the antiviral, as well as the cell growth-inhibiting, activity of the heterologous interferon. Heterologous reactions were also undetectable in a modified neutralization test, in which secondary antibody against rabbit γ-globulin was used to remove, before the assay for the residual interferon, any interferon-antibody complexes that might remain biologically active. Affinity chromatography of interferons on immobilized antibodies also showed antigenic distinctness of F and S interferons. Poly (I) ·poly (C)-induced L cell interferon and Newcastle disease virus-induced C243 cell interferon also consisted of two distinct species which are antigenically similar to F and S.

Neutralization of the biological activity of cytokines and other protein effectors by antibody: theoretical formulation of antibody titration curves in relation to antibody affinity

Patients treated with interferons, other cytokines, or various biologically active proteins may form neutralizing antibodies, which can adversely affect clinical outcome. It is therefore important to understand how antibodies neutralize such soluble protein antigens and how best to quantitate such antibodies. By applying the mass action law to antigen-antibody reactions, we previously developed a mathematical model applicable in two situations: first, for antibodies having low affinity for the antigen concerned (the Constant Proportion (CP) case), and, second, for antibodies having high affinity (the Fixed Amount (FA) case). The results allowed calculation of neutralization titers which were independent of the particular assay method used. Neutralization by antibodies of intermediate affinity, however, requires different mathematical treatment because the mode of neutralization does not fit the two cases mentioned above. In this paper, theoretical neutralization curves were derived, based on the same mathematical model, for antibodies of intermediate affinity. We show that the slope of the neutralization curve relating residual active antigen to the concentration of antibodies is determined by the antibody association constant and the molar concentration of the effector antigen. It is therefore possible to infer the magnitude of the association constant from the observed neutralization curve. We show that values obtained for the neutralization titer of antibodies of intermediate affinity by the use of the formula previously described for the Fixed Amount and Constant Proportion cases may deviate from the theoretically sound values; the magnitude of the deviation can be estimated by applying the formulas described herein. These relationships should apply generally to antibody neutralization reactions with all biologically active soluble protein effector molecules that have a single and nonrepetitive epitope.

The expression of potency of neutralizing antibodies for interferons and other cytokines.

The occurrence of antibody formation in patients administered biologically active human proteins as biotherapy for different diseases emphasizes the importance of establishing a common method of reporting neutralizing antibody levels for such cytokines. For quantitative neutralization bioassays, the preferred expression of the neutralizing potency of an antiserum is a titer, that is, the dilution of serum that reduces 10 Laboratory Units (LU)/ml of the cytokine to 1 Laboratory Unit/ml, the endpoint of most bioassays. This 10-to-1 LU/ml expression, which has been recommended by the World Health Organization for recording the results of interferon neutralization by the constant interferon method (with varying dilutions of serum) can also be used with the constant antibody method (with varying concentrations of interferon). For various reasons, interferon doses in International Units (IU)/ml should not be used for the neutralization test. Should the interferon concentration vary, intentionally or otherwise, from the intended dose of 10 LU/ml, a simple calculation allows expression of the neutralizing potency as the recommended reduction of 10-to-1LU/ml as follows: the titer to be reported is the reciprocal of the antibody dilution (achieving the endpoint), multiplied by the interferon concentration (measured in that day’s titration) minus one, divided by 9. This index of neutralization is the preferred method to represent the neutralizing potency of polyclonal and monoclonal antibodies and should make the results from different laboratories more readily interpretable and enable comparison.

Formation of nuclear polyhedral bodies and nuclear polyhedrosis virus of silkworm in mammalian cells infected with viral DNA

Abstract The infectious DNA of nuclear polyhedrosis virus of silkworm ( Bombyx mori Linne) caused production of characteristic nuclear polyhedral bodies in cultured FL cells of human amniotic origin, which did not support virus growth. The activity of the viral DNA in producing polyhedral bodies in FL cells was lost by pretreatment of the inoculum with DNase and by heating, but RNase was inactive. The polyhedral bodies formed in the cells were identical with those formed in cells of the silkworm with respect to their morphological and serological character. The polyhedral bodies isolated from FL cells and dissolved in alkali caused nuclear polyhedrosis when injected into silkworm pupae.

Purification of Two Components of Mouse L Cell Interferon: Electrophoretic Demonstration of Interferon Proteins

Mouse L cell interferon induced by Newcastle disease virus was purified by the procedure described previously (Yamamoto et al. 1974) followed by gel filtration. The two fractions obtained containing interferon species S (36000 daltons) and F (24000 daltons), respectively, were analysed electrophoretically at pH 4-3, or in the presence of sodium dodecyl sulphate (SDS) at pH 7-2. In both fractions, interferon activity was invariably associated with distinct protein bands. In the F-containing fraction there were essentially no other proteins, and in the S-containing fraction, impurity proteins were well separated from the interferon activity. The apparent mol. wt. determined by SDS-gel electrophoresis showed little or no dependence on gel concentration, suggesting that the interferons had low carbohydrate contents, and did not change after reduction with thiol reagents in SDS and urea.

Purification and characterization of recombinant murine immune interferon

The recombinant murine immune interferon (rMu‐IFN‐γ) was purified to homogeneity from Escherichia coli harboring the expression vector of murine IFN‐γ. The purified rMu‐IFN‐γ showed an M r of 15000 in SDS‐polyacrylamide gel electrophoresis. Results of amino acid analysis, amino‐ and carboxyl‐terminal analyses and peptide mapping of rMu‐IFN‐γ suggest that it has the complete protein sequence predicted on the basis of cDNA except for lack of four amino acid residues from the mature carboxyl‐terminus.