Percy Minden

The primary interaction of antibody to components of aspergilli: II. Antibodies in sera from normal persons and from patients with aspergillosis

Abstract The capacity of antibodies in 102 human sera to bind components of aspergilli was studied. Sera were from patients with pulmonary aspergillosis or a variety of other clinical disorders and from healthy individuals. The antigens employed were anionic mucopolysaccharide components derived from A. fumigatus and A. nidulans and were not precipitated by antibodies in either human or rabbit sera. The antigens were soluble in 10 per cent trichloroacetic add and 50 per cent saturated ammonium sulfate, and antigen binding was measured by radioimmunodiffusion and the ammonium sulfate test. Sera from patients with aspergillosis, particularly the mycetomal form, bound significantly greater amounts of the antigens than did the other sera. Binding was associated with IgM and IgG, was to the F(ab)′2 portion of immunoglobulin, and was immunologically specific. Inhibition studies demonstrated the presence of shared antigens between aspergilli and a variety of other fungi, mycobacteria, non-acid-fast bacteria, and commercial house dust concentrates. The minimal but significant binding observed with sera from normal individuals is probably the result of antigenic stimulation by aspergilli as well as other fungi and organisms in the environment.

The primary interaction of antibody to components of aspergilli: I. Immunologic and chemical characteristics of a nonprecipitating antigen☆

Abstract The experiments presented in this report describe the preparation of a radiolabeled antigen which can be used in the ammonium sulfate test to measure antibodies against aspergilli. The antigenic components were in the trichloroacetic acid (TCA)-soluble fraction from sonically disrupted aspergilli. The major radiolabeled ligand appeared to be an immunologically nonprecipitating anionic mucopolysaccharide which was insensitive to DNase or RNase, slightly sensitive to pepsin, and very sensitive to periodate oxidation and borohydride reduction. Results of quantitative studies indicate that the radiolabeled antigens had a relatively low affinity for antibody populations produced in hyperimmunized rabbits. Quantitative inhibition experiments as well as immunodiffusion studies demonstrated similarities and differences among species of the genus Aspergillus as well as other fungi. Commercial house dust extracts appear to contain significant amounts of the TCA-soluble aspergilli antigens.

Immune complexes in children with leukemia relationship to‐disease characteristics and to antibody response to mycobacterium bovis (BCG) in patients receiving BCG immunotherapy

The incidence and possible clinical relevance of immune complexes in sera from children with acute leukemias was investigated. Determinations were made in sera obtained at three‐month intervals from individual patients over a two‐year period. Two tests, the C1q binding and the polyethylene glycol precipitation assays, were employed. At time of diagnosis, immune complexes were found in sera from 6 of 41 patients (15%) with acute lymphoblastic leukemia (ALL). Three of 6 patients with T‐cell leukemia had immune complexes at diagnosis as compared to 3 of 35 with null‐cell leukemia. Only 1 of the 6 patients with T‐cell leukemia had a white blood cell count of less than 50,000/μl3. Of the patients followed for six months or more, 34% had immune complexes during therapy and the incidence of relapse was unrelated to the presence or absence of immune complexes. Fifty‐two percent of patients that remained in remission during the period of study and 5 of 8 patients that relapsed (63%) had immune complexes at one time or another suggesting that the presence of immune complexes by themselves did not portend either a favorable or an unfavorable prognosis. Sera taken at the time of diagnosis from children with acute non‐lymphoblastic leukemia (ANLL) had a higher incidence (36%) of immune complexes than similar sera from ALL patients (15%). Antibodies in sera from leukemia patients at diagnosis had a lower capacity to bind a BCG antigen than did sera from control subjects. Eleven of 17 patients that had BCG immunotherapy had a humoral antibody response to BCG. There was no relation between an antibody response to BCG and a favorable clinical response.

A solid-phase radioimmunoassay to detect antibodies produced by hybridomas to antigens derived from human melanoma cells

SummaryA solid-phase radioimmunoassay to detect antibodies that react with antigens derived from human melanoma cells is described. A soluble preparation derived from Nonidet P-40 lysates of tissue-cultured melanoma cells was dried on the surfaces of wells of polyvinyl chloride microtiter plates and fixed with 0.02% glutaraldehyde. Antibody preparations were added and incubated for 18 h at 4° C. The wells were washed and bound antibodies were detected using radioactive Staphyloccoccal protein A (125I-SpA). Optimal conditions are described for all the steps employed. Concentrations of antigen selected, the amount of 125I-SpA employed and the duration of incubation of antibodies with antigen were found to be critical.The assay was sensitive and reproducible, and lent itself to the simultaneous evaluation of many individual antibody samples in a short period of time. The assay was particularly valuable for rapid screening of hybridoma supernatants for antibodies to antigens derived from melanoma cells and from a panel of other tumor and normal cells.

Primary interaction between antibody and components of Alternaria: I. Immunological and chemical characteristics of labeled antigens

Components were isolated from Alternaria tenuis and its culture filtrate, and were radiolabeled with 123-I. The labeled antigenic components had a high polysaccharide content as determined by staining patterns following electrophoresis in polyacrylamide gels and by inactivation with sodium metaperiodate. A primary binding test was employed to detect and measure serum antibodies to the components from A. tennuis. This procedure was more sensitive in detecting antibodies that bound to antigens than were comparable tests dependent upon precipitin types of reactions. The labeled components of A. tenuis cross-reacted or shared antigens with 3 other species of molds: Stemphylium sp., Curvularia sp., Aspergillus fumigatus, but not with a variety of other fungal and nonfungal materials.

Some variable controls for accurate heterologous anti-immunoglobulin (HAI) tests.

Abstract Optimum conditions for carrying out the heterologous anti-immunoglobulin test (HAI) are described, using a bovine serum albumin (BSA): anti-BSA system. The anti-immunoglobulin employed should always be assayed to determine the proper amount for each experiment and dilutions of test antisera should be made in protein solution. Under such conditions, the HAI test is a reliable and sensitive procedure to measure the capacity of antisera to bind specifically with antigens.

Primary interaction between antibody and components of Alternaria: II. Antibodies in sera from normal, allergic, and immunoglobulin-deficient children☆

Antibodies in sera from normal, allergic, and immunoglobulin-deficient children were studied for binding to radiolabeled components of Alternaria tenuis. Significant binding levels were found in 103 of 105 sera from normal children. The levels were age-dependent, rising from a low point in the 7- to 12-month age group to adult levels by the age of 8 years. Levels of binding to two antigens, a culture filtrate derivative (125I-CLF) and a mycelial derivative (125-I-HS), were similar. Sera from asthmatic children with strong immediate skin test reactions to Alternaria extracts bound significantly higher levels of 125-I-CLF than did sera from allergic children with negative skin tests or from control children. Binding levels in sera from children with hypogammaglobulinemia were significantly less than binding levels in sera from normal children in any age group. Sera from children with selective IgA deficiency bound 125-I-CLF at normal levels. The almost universal occurrence of anti-Alternaria antibodies in children was partly explained by the finding of partial cross-reactivity and/or shared antigens among several fungal species, including A. tenuis, Stemphylium sp., Curvularia sp., and Aspergillus fumigatus. The biological significance of these antibodies is not clear, but the procedures described lend themselves to further investigations.

The use of cellular immunoadsorbents to prepare polyclonal antibodies that distinguish between antigens derived from human melanoma cells and autologous lymphocytes

SummaryCellular immunoadsorbents were employed to isolate xenogeneic antibodies that reacted with a restricted group of antigens on human melanoma cells. Melanoma cells and autologous lymphoid cells were grown in tissue culture. Cellular immunoadsorbents were prepared by coupling formalin-treated melanoma and lymphoid cells to diethylaminoethyl cellulose. Rabbits were immunized with melanoma cells and antisera were passed sequentially through immunoadsorbents made of fetal bovine serum, and lymphoid cells. Unbound effluents were then passed through an immunoadsorbent prepared with melanoma cells. Antibodies binding to melanoma cells were eluted and their reactivity to melanoma-derived antigens was tested using a solid-phase radioimmunoassay. Antigens for this assay were NP-40 lysates of melanoma and lymphoid cells and fetal bovine serum. Radioactive Staphylococcal protein A was used to detect binding by the antibodies to the test antigens.The effects of formalin-fixation and storage of melanoma and lymphoid cells were studied. Storage of fixed melanoma cells for periods up to 4 months did not affect their capacity to bind antibodies. A single exposure of formalin-fixed cells to a low-pH elution buffer which was followed by neutralization did not affect binding by these cells. Antibodies isolated in this manner were of the IgG class and reacted with antigens derived from melanoma cells but not from autologous lymphocytes or fetal bovine serum. This study demonstrated the feasibility of using cellular immunoadsorbents to prepare xenogeneic polyclonal antibodies with a high degree of reactivity to antigens derived from human melanoma cells.

Purification of antibodies to bacterial antigens by an immunoadsorbent and a method to quantify their reaction with insoluble bacterial targets

A combination of procedures was employed to develop a radioimmunoassay which quantified the binding of antibodies to antigens of either intact Propionibacterium acnes or to antigens of insoluble extracts derived from the bacteria. Reactive antibody populations were purified by use of bacterial immunoadsorbents which were prepared by coupling P. acnes to diethylaminoethyl cellulose. Binding of antibodies was detected with [125I]staphylococcal protein A ([125I]SpA) and optimal conditions for the assay defined by varying the amounts of antibodies, bacterial antigenic targets and [125I]SpA. In antibody excess, 100% of available [125I]SpA was bound by the target-antibody complexes. However, when antibody concentration was limiting, a linear relationship was demonstrated between per cent specific binding of [125I]SpA and antibodies bound to bacterial targets. These results were achieved only with immunoadsorbent-purified antibody populations and not with hyperimmune sera or IgG. The radioimmunoassay detected subtle antigenic differences and similarities between P. acnes, P. acnes extracts and a variety of unrelated microorganisms.

The Association between Antigens of Human Malignant-Melanoma Cells and Mycobacterium bovis (BCG)

Antigenic components shared between Mycobacterium bovis (BCG) and certain experimental and human tumors have been demonstrated by a variety of methods (Borsos and Rapp, 1973; Minden et al., 1974; Bucana and Hanna, 1974; Minden et al., 1976a; McCoy et al., 1978; Lewis et al., 1976; Holan et al., 1979). There are also data suggesting that other microorganisms have determinants in common with some tumors (Minden et al., 1976b; Minden, 1976; James et al., 1976; Kwapinski et al., 1978). With respect to human tumors, there is evidence that antigens from human malignant melanoma (Bucana and Hanna, 1974; Minden et al., 1976a) and acute myeloid leukemia (Minden et al., 1976b) are shared with or are similar to antigens in BCG.