Sidney E. Grossberg

A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250

Abstract An assay for proteins in solution is described that depends on the conversion of Coomassie brilliant blue G250 in dilute acid from a brownish-orange to an intense blue color. As soon as the dye solution is mixed with a protein sample, the absorbance of the mixture can be measured. The absorbance of the solution is stable for 60–90 min at room temperature. The test can be used with a variety of proteins and polypeptides with molecular weights greater than 3000. The assay has high reproducibility and can detect less than 1.0 μg of albumin. The single reagent required is very stable, and free amino acids and several chemicals that interfere with the Lowry protein assay cause no interference. The test is adaptable to a micromethod, which is also described.

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.

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.

Serum Neopterin, an Immune Activation Marker, Independently Predicts Disease Progression in Advanced HIV-1 Infection

BACKGROUND CD4+ T lymphocyte (CD4) counts and plasma human immunodeficiency virus (HIV) type 1 RNA concentrations predict clinical outcome in HIV-1 infection. Our objective was to assess the independent prognostic value for disease progression of soluble markers of immune system activation. METHODS This retrospective marker-validation study utilized previously obtained clinical and laboratory data, including CD4+ cell counts, and made use of stored frozen serum samples to assay for levels of beta2-microglobulin, neopterin, endogenous interferon, triglycerides, interleukin-6, soluble tumor necrosis factor- alpha receptor II, and HIV-1 RNA, and to determine HIV genotypic reverse-transcriptase inhibitor resistance. The 152 patients who participated in this study represented a subsample of participants in AIDS Clinical Trials Group (ACTG) 116B/117, a randomized trial that demonstrated the clinical benefit of didanosine over zidovudine monotherapy in persons with advanced HIV-1 infection. Marker data were analyzed in relation to protocol-defined clinical disease progression, using Cox proportional hazards models. RESULTS The median duration of follow-up was 344 days. Elevated baseline values for neopterin (P=.0009), endogenous interferon (P=.00039) and interleukin-6 (P=.0007) were each associated with greater subsequent risk of clinical disease progression. In a head-to-head comparison that was adjusted for CD4+ cell count (P=.0165) and HIV-1 RNA level (P=.1220), we found that elevated values for neopterin (P=.0002) and, to a lesser extent, endogenous interferon (P=.0053) were the strongest predictors of increased risk of clinical disease progression 6 months later. CONCLUSIONS Soluble markers of immune activation add prognostic information to CD4 counts and viral load for risk of disease progression in advanced HIV-1 infection. The robust performance of neopterin, an inexpensive and reliably measured serum marker, supports its potential suitability for patient monitoring, particularly in resource-limited settings.

Quantification of neutralizing antibodies to human type I interferons using division-arrested frozen cells carrying an interferon-regulated reporter-gene.

Development of neutralizing antibodies (NAbs) to interferons (IFNs) can reduce the clinical response to IFN therapy. As current cell-based assays for quantifying NAbs have limitations, a highly sensitive and reproducible assay was developed, using division-arrested frozen human U937 cells transfected with the luciferase reportergene controlled by an IFN-responsive chimeric promoter, which allows IFN activity to be determined with precision within hours. Assay-ready PIL5 cells can be stored frozen for >3 years without loss of IFN sensitivity or the need for cell propagation. The assay is highly IFN sensitive (detecting <1.0 IU/mL), reproducible (SE +/- 15%) over concentrations from <1.0 to 100 IU/mL and able to measure different IFN subtypes and their pegylated variants. The use of this assay has shown that NAbs from patients treated with IFN-alpha2 exhibited markedly lower titers against 10 LU/mL of low specific activity IFNs, namely, IFN-alpha1, PEG-Intron(TM) (Schering-Plough, Levallois-Perret,France), or Pegasys(TM) (Hoffmann-La Roche, Neuilly-sur-Seine, France, than against 10 LU/mL IFN-alpha2. Similarly, NAbs from patients treated with IFN-beta1a exhibit lower titers against 10 LU/mL of low specific activity IFN-beta1b than against IFN-beta1a. The combination of the use of division-arrested, IFN-responsive human cells transfected with the luciferase reporter-gene makes the rapid PIL5 assay for NAbs highly advantageous.

Validating parameters of a luciferase reporter gene assay to measure neutralizing antibodies to IFNβ in multiple sclerosis patients

Neutralizing antibodies (NAbs) can occur in some multiple sclerosis (MS) patients receiving interferon beta (IFNbeta) therapy. NAbs reduce drug bioavailabity and high NAb titers reduce drug efficacy. We describe the validation of the R. Farrell and G. Giovannoni luciferase reporter gene assay to measure NAbs to INFbeta. We assayed 163 sera from IFNbeta treated MS patients with an optimized luciferase method and compared the results to those obtained with the reference cytopathic effect (CPE) method using A549 cells and an encephalomyocarditis virus (EMCV). Binding antibodies (BAbs) were measured using a capture ELISA as a screening test for NAbs in the CPE assay. NAb status measured by the luciferase and the ELISA/CPE method did not yield a significant difference. Log10 NAb titers obtained from the luciferase assay and the A549/EMCV CPE methods correlated very well. The inter-assay coefficient of variation for titers was between 17.8-29.3%, and the intra-assay coefficient of variation was between 6.3-15.2%. The luciferase assay is reliable, appropriately sensitive and requires less time than the currently available NAb methods.

A sensitive interferon assay for many species of cells: encephalomyocarditis virus hemagglutinin yield reduction.

Summary A new, highly sensitive assay for human interferon was developed in which the reduction in HA yield of a single-cycle infection with EMC virus is measured. The sensitivity and precision of the assay are comparable to or better than the other assays tested, depending most of all upon the cells used in the test. This simple method requires no sophisticated instrumentation, can be used on a microtiter scale, provides objective numerical results, and is readily applicable to testing large numbers of samples. The assay can be used in human lines arid diploid cell strains as well as in monkey, rabbit, cat, mouse, pig, cow, dog, and hamster-mouse hybrid cells. We are grateful to C. K. Schoenherr for her excellent technical assistance. We thank Dr. M. Essex for making it possible for one of the authors (P.J.) to work with the feline cell system in his laboratory. We appreciate the gifts of interferon from Dr. K. Cantell and Dr. J. Valenta, and of cells from Drs. G. G. Jackson, S. Baron, and E. Kilbourne.

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.

Molecular cloning, complete sequence, and biological characterization of a xenotropic murine leukemia virus constitutively released from the human B-lymphoblastoid cell line DG-75

A previously undetected retrovirus has been isolated from the human Epstein-Barr virus (EBV)-negative, B-lymphoblastoid DG-75 cell line, widely used for EBV gene transfection studies. The complete 8207-base genome of the DG-75 retrovirus was molecularly cloned from viral mRNA and sequenced (Accession No. AF221065). Northern blot analysis with probes specific for the putative RU-5, gag, pol, and env regions identified a full-length viral RNA and spliced env mRNA. DG-75 viral RNA was isolated from the DG-75 cell sublines UW and KAR, but not from the HAD subline. The DG-75 retrovirus was isolated with primer-binding sites that match tRNA(Thr) and tRNA(Gln2). Homology searches revealed homology to (i) xenotropic NZB-9-1 env mRNA, (ii) Moloney-MLV pol region, and (iii) a truncated Evi-2 endogenous proviral sequence gag and pol region. Viral interference and infectivity assays confirmed the xenotropic nature of the DG-75 retrovirus. The DG-75 retrovirus is the first isolate of an exogenous xenotropic MLV in which the full-length genomic sequence has been characterized.

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.