Morten Bagge Hansen

Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill

The tetrazolium salt (MTT) method involving conversion of MTT to coloured formazan by cells serving as indirect measurements of cell growth/cell kill has been reported by several groups, although technical problems have been encountered. The present investigation was undertaken in order to delineate what laboratory variables have direct influence on the sensitivity and reproducibility of the method. The pH of the extraction buffer was of the utmost importance, since it was demonstrated that a pH greater than 5 would give rise to false signals. Furthermore, modifying the composition of the extraction buffer, all formazan dye grains were solubilised, totally. A direct comparison with published methods demonstrated that only the modified method would yield 100% higher signals without increasing the background. In contrast to previous reports, it was shown that phenol red does not interfere with the measurements and no washing steps are required since all ingredients can be added subsequently. Serum proteins at concentrations up to 25% have no influence on the result. All samples can be measured in an ELISA scanner at 570 nm with little intra-assay variation.

High-avidity autoantibodies to cytokines

Abstract The increased use of recombinant cytokines and cytokine analogues in various disease therapies emphasizes the importance of antibodies to cytokines. Here, Klaus Bendtzen and colleagues discuss how immunological tolerance to cytokines may be broken, and the clinical relevance of cytokine autoantibodies. They also comment on the implications for refined passive immunization and cytokine vaccination strategies.

Binding of cytokines to pharmaceutically prepared human immunoglobulin

Pharmaceutically prepared IgG, pooled from sera of over 2,000 normal individuals, contained both monomeric and dimeric IgG. Each type of IgG bound 125I-labeled interleukin (IL)-1 alpha, IL-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha. Increased binding to IgG was observed if 125I-IL-1 beta was denatured by heating to 39 degrees C. However, the binding of both nondenatured and denatured 125I-IL-1 beta was not inhibited by unlabeled IL-1 beta. In contrast, binding of 125I-IL-1 alpha, 125I-IL-6, and 125I-TNF alpha was inhibited by the corresponding unlabeled cytokine. Papain-digestion of IgG abolished binding of 125I-TNF alpha but failed to influence the displaceable binding of 125I-IL-1 alpha and 125I-IL-6. 125I-TNF alpha was a mixture of trimeric and monomeric forms, the latter being the predominant form at lower concentrations. The apparent saturability of 125I-TNF alpha was explained by a higher nonspecific binding of monomeric than of trimeric 125I-TNF alpha to IgG. The amounts of cytokine antibodies in IgG preparations would contribute approximately 2 micrograms anti-IL-1 alpha IgG and 1 microgram anti-IL-6 IgG per kg body wt during high dose immune globulin therapy. In conclusion, pharmaceutical preparations of human IgG contain specific and neutralizing, high affinity antibodies against IL-1 alpha and IL-6, but not against TNF alpha or IL-1 beta. There are significant methodological pitfalls that hamper detection of IgG autoantibodies against cytokines.

Anti-interleukin-6 antibodies in normal human serum.

High‐avidity IgG antibodies to the cytokine inlerleukin‐6 (IL‐6) were found in sera of apparently healthy adult individuals. These antibodies specifically interfered with an FLISA (enzyme‐linked immunosorbent assay) for IL‐6 in which specific polyclonal rabbit antibodies to human recombinant IL‐6 (rlL‐6) were used. Furthermore. using precipitation of 125‐I‐rIL‐6 with rabbit antibodies to human immunoglobulins (Ig). the sera of 7 out of 6S Danish blood donors were found to contain specific antibodies in substantial amounts. Judged by ELISA interference, gel filtration of sera incubated with 125I‐rIL‐6 and second antibody precipitation of 125I‐rIL‐6. IgG seemed to be the dominant IL‐6 binding protein in these normal sera. Using specific antibodies to human in light chains, it was found that the anti‐lL‐6 antibodies were of polyclonal origin. Moreover, there are at least two epitopes on the IL‐6 molecule, because more than one IgG bound lo some IL‐6 molecules at the same time, The anti‐IL‐6 antibodies did not cross‐react with a number of other human recombinant‐derived and native cytokines. The antibodies recognized native as well as rlL‐6. but preferentially monomeric lL‐6.

A new sensitive bioassay for precise quantification of interferon activity as measured via the mitochondrial dehydrogenase function in cells (MTT-method)

A biological method for precise quantification of interferons has been developed. The method is based upon the dehydrogenase system of the intact target cells, which will normally convert an artificial substrate, MTT, into formazan (blue), which, in turn, can be measured spectrophotometrically. This conversion is greatly reduced by cytocidal viruses in a dose‐dependent manner. The protection of target cells by interferon against challenge virus is reflected in a diminished reduction in the production of formazan, thus giving a very precise method for quantification of interferon. The lowest level of detection is around 0.10 international units. The intra‐ and inter‐assay variability appear to be below 10%. The assay, which makes no use of expensive ingredients, is performed in 96‐well micro‐trays and read in an inexpensive ELISA‐scanner.

High avidity IFN-neutralizing antibodies in pharmaceutically prepared human IgG.

This paper demonstrates and characterizes naturally occurring antibodies to interferon (IFN) in human IgG preparations. In vitro neutralization of the antiviral effect of IFN alpha and IFN beta, but not IFN gamma, was observed in 12 of 15 normal IgG preparations. The neutralizing capacity was higher against rIFN alpha 2A and rIFN alpha 2C than against lymphoblastoid IFN alpha and IFN beta. Frühsommer meningoencephalitis hyperimmune IgG and hepatitis-B hyperimmune IgG showed potent neutralization, whereas anti-rhesus D-, anti-rabies-, and anti-tetanus IgG showed weak neutralization. Saturable binding of 125I-rIFN alpha 2A was demonstrated only in those IgG preparations found to neutralize the antiviral effect of IFN. Significant correlation between IFN binding and neutralization capacity was observed. The antibodies bound with Fab to rIFN alpha 2A with an avidity of approximately 30 pM; the majority was of the IgG1 subclass. Maximum binding capacity was 490 pg rIFN alpha 2A/mg IgG. Cross-binding of rIFN alpha 2C, lyIFN alpha N1 and IFN beta occurred with 10 and 100-200 times lower activities than that of rIFN alpha 2A. There was no cross-binding with rIFN gamma or rIL-6. IgG preparations containing anti-IFN antibodies blocked the binding of 125I-rIFN alpha 2A to A549 cells. In conclusion, pharmaceutically prepared human IgG preparations contain variable but significant levels of high-avidity IFN alpha and IFN beta neutralizing antibodies.

Autoantibodies to crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN‐alpha 2b and human IFN‐gamma in healthy blood donors

Recently, naturally occurring antibodies to IFN‐α were discovered in a few systemic lupus erythematosus (SLE) and cancer patients; however, in most patients monitored for anti‐IFN antibodies before treatment, no antibodies were found. In an attempt to explain the ‘IFN‐blocking effect’ that we observed in all serum samples we investigated 200 sera from healthy blood donors. We isolated the globulin fraction, and used rabbit anti‐human IgG and IgM columns, protein A columns and T‐gel affinity chromatography to isolate human IgG and IgM. All sample fractions were tested in a biological IFN neutralization assay by means of a sensitive MTT‐assay. We found that normal human serum contained autoantibodies to crude human leucocyte IFN, native human fibroblast IFN, recombinant human leucocyte IFN‐α2b and recombinant human IFN‐γ, and that these naturally occurring antibodies were biologically active immunoglobulins of IgG and IgM type. These anti‐IFN antibodies were also present in purified human normal immunoglobulin pools. We conclude that all humans have naturally occurring anti‐interferon antibodies in their serum, and it is a tempting theory that human cytokines and lymphokines are, at least partly, regulated by immunoglobulins.

Human anti-interleukin 1α antibodies

Abstract Autoantibodies to the cytokine interleukin (IL)-1α are frequently found in sera of apparently healthy humans. We have developed a sensitive radioimmunoassay (RIA) and an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of human serum antibodies to IL-1α at concentrations below 10 pmol/l. The RIA is based on co-precipitation of 125 I-labelled human recombinant IL-1α (rIL-1α) by rabbit antibodies to human immunoglobulins. The ELISA is based on recovery of added rIL-1α to serum samples and takes advantage of the fact that free human autoantibodies to IL-1α in a dose dependent manner reduce recovery of added rIL-1α. The assays correlate exceedingly well ( r = 0.99, P

Specific binding of interleukin 1 (IL-1)β and IL-1 receptor antagonist (IL-1ra) to human serum. High-affinity binding of IL-1ra to soluble IL-1 receptor type I

Molecules that bind recombinant interleukin 1 (rIL-1) beta and rIL-1 receptor antagonist (rIL-1ra) with high affinity were detected in sera of healthy individuals. rIL-1 beta bound with dissociation constants in the nanomolar range, and the serum binding capacity was 40-50 ng/ml. rIL-1ra bound with 30 times higher affinity, and the serum binding capacity was 0.7-1 ng/ml. Rabbit antibodies against the recombinant-derived extracellular part of human IL-1 receptor type I (rsIL-1RI) selectively inhibited the binding of 125I-rIL-1ra to the serum factor(s). Almost 70% of the high-affinity IL-1ra-binding capacity was recovered after immunosorption with these antibodies. Binding of 125I-rIL-1ra to rsIL-1RI was blocked by rIL-1 alpha and by rIL-1 beta. In contrast, the purified rIL-1ra-binding factor (IL-1raBF) failed to bind rIL-1 alpha and rIL-1 beta. Gel filtration chromatography indicated a 1:1 binding of rIL-1 beta and rIL-1ra to their respective serum factors. The apparent molecular size of both serum factors was 70-80 kDa. Using SDS-PAGE and autoradiography, IL-1raBF had a molecular size of 60 kDa. We conclude that IL-1raBF, a serum factor which selectively and with high affinity binds IL-1ra (Kd = 70 pM), is related to or identical with a soluble form of IL-1RI. If upregulated during disease, IL-1raBF may constitute yet another level of natural regulation of IL-1 bioactivities.

High-affinity IgG autoantibodies to IL-6 in sera of normal individuals are competitive inhibitors of IL-6 in vitro.

Nano- to picomolar concentrations of high affinity IgG antibody to interleukin 6 (IL-6ab) were detected in sera of 71 of 467 normal Danish blood donors (15%). IL-6 bound to the Fab fragments of IL-6ab, and the antibody specifically and competitively interfered with enzyme-linked immunosorbent assays for human IL-6. Only IL-6ab-positive sera interfered with these ELISAs. A statistically positive correlation was found between total IL-6 binding and specific IL-6 binding to serum (P < 0.0001), suggesting that IL-6ab dominates as IL-6 binding factors in normal human serum. The IL-6ab also inhibited binding of IL-6 to receptors on the human U-937 macrophage-like cell line, the human U-266 myeloma cell line and the mouse hybridoma cell line B13.29, clone B9 (B9 cells). IL-6-induced proliferation of the B9 cells was competitively inhibited by IL-6ab. We conclude that sera of normal individuals may contain appreciable levels of autoantibodies to IL-6. These IgG autoantibodies may be physiologically and pathophysiologically important because they are potent inhibitors of IL-6 in vitro.