Aflatoxin B; specific antibodies and their use in radioimmunoassay.

New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxy-methyloxime derivatives of either aflatoxin B1 (AFB1) or an analogue, 5,7-dimethoxycyclopentenon (2,3-c) coumarin, produced antibodies that bind 3H-AFB1. The specificities of the antisera with respect to aflatoxins BI, B2a, G1, G2, Q1, P1, and some other structually related compounds were determined. Radioimmunoassays that can detect levels as low as 0.27 pmoles (0.06 ng) of AFB1-were used to analyze serum, urine, and crude extracts of corn and peanut supplemented with aflatoxin. In the foodstuffs, as little as 1 mug AFB1/kg was measured. The immunoassay was at least as sensitive and specific as other available analytic methods, but did not require the purification of samples by chromatography before analysis. The technique may be particularly useful in epidemiologic studies designed to study the possible relationship between chronic aflatoxin ingestion and cancer.

Complement-dependent cytotoxic antitumor antibody. II. Quantitative determination of cell-bound immunoglobulin M1.

A method for the quantitative determination of rabbit IgM bound to cell surfaces has been developed. This method was based on the ability of goat IgG specific for rabbit IgM heavy chains to bind 125I-labeled protein A when bound to the antigen. With the use of this technique the production of specific IgM antitumor antibodies in New Zealand White rabbits after immunization with guinea pig hepatoma cells line-1 and line-10 was followed. Differences in the production of IgM were observed between the different bleedings from rabbits immunized with line-1. No significant IgM antibody was produced following immunization of rabbits with line-10 tumor cells. This indirect method for determining IgM on the cell surfaces was objective, easy to perform, and detected complement-fixing and noncomplement-fixing antibodies. In addition, this technique could be applied to quantify other components on the cell surface for which a suitable specific antibody was available.

Correlation between antitumor activity of protein A and in vivo formation of defined high molecular weight complexes with immunoglobulin G in BALB/c mice.

The purpose of this study was to test the hypothesis that antitumor activity of staphylococcal protein A (SpA) is related to the composition of complexes formed in vivo with IgG. BALB/c mice were inoculated intradermally with 10(6) Meth A fibrosarcoma cells on day 0 and treated i.v. on days 3 and 7 with between 1 and 405 micrograms of SpA. The 45- and 15-micrograms doses significantly inhibited tumor growth and enhanced survival time compared to saline controls in four of four and two of four experiments, respectively. Sucrose gradient ultracentrifugation was used to show that serum from tumor-bearing or normal mice given 45 or 15 micrograms of 125I-labeled SpA contained only high molecular weight [(IgG)2SpA]2 complexes for up to 24 h after injection, whereas serum from mice given higher ineffective doses (135 and 405 micrograms) contained low molecular weight (IgG)(SpA) complexes over the first 1-4 h. Serum from mice undergoing successful therapy with 45 micrograms of unlabeled SpA also contained only [(IgG)2SpA]2 complexes. In contrast, when mice with large tumors (120-150 mm2) were treated on days 16 and 20, only the 135- and 405-micrograms doses significantly inhibited further tumor growth. Serum from mice with 16-day tumors contained only [(IgG)2SpA]2 complexes even after 5 min and when 135 or 405 micrograms of 125I-SpA was given. This result is consistent with significantly higher (2.7-3.4-fold, P less than 0.0001) levels of total and SpA-reactive IgG in serum from these mice compared to normal mice or mice with 3-day tumors. Our results demonstrate a correlation between antitumor activity of SpA and in vivo formation of [(IgG)2SpA]2 complexes in an established animal model, and help to define the mechanism of SpA action at the molecular level.

Tumoricidal response following perfusion over immobilized protein A: identification of immunoglobulin oligomers in serum after perfusion and their partial characterization.

Previously, we showed that perfusion of plasma from hosts bearing breast adenocarcinoma over immobilized staphylococcal protein A resulted in objective tumor regressions. In the present study, sera perfused in vitro over immobilized staphylococcal protein A were analyzed by physicochemical and immunochemical methods to characterize newly formed products. Sera from normal and breast adenocarcinoma-bearing dogs showed increased levels of C1q-binding IgG after perfusion over a strain of staphylococcus that is protein A rich (Cowan I), but not protein A deficient (Woods 46). C1q binding levels were also increased in normal and tumor-bearing canine or human sera which were perfused over purified protein A immobilized in collodion charcoal (PACC), and this increase was localized in sucrose density gradient fractions ranging from 7S to 19S. Polyacrylamide gel electrophoresis analysis of the high-molecular-weight fraction in postperfusion canine sera, isolated by G-200 fractionation and immunoaffinity chromatography, showed predominantly heavy and light immunoglobulin chains of canine IgG. Furthermore, protein A was released from PACC after perfusion with serum or solutions containing IgG or albumin from humans, dogs, and chickens. After serum perfusion over PACC, protein A was identified in the effluent by additional studies as follows: (a) polyacrylamide gel electrophoresis analysis showed that eluted 125I-protein A comigrated with the protein A marker; (b) postperfusion C1q-binding complexes, isolated by gel filtration under dissociating conditions and affinity chromatography on IgG-Sepharose showed a single precipitin band with normal human (protein A reactive) but not chicken (protein A unreactive) serum. Protein A released from PACC which appeared in postperfusion sera was associated with immunoglobulins in macromolecular complexes since (a) eluted 125I-protein A was largely (NH4)2SO4 and polyethylene glycol precipitable, whereas free protein A was not, and it sedimented in sucrose density gradient fractions distributed beyond the 7S marker, compared to free protein A which localized below 7S; (b) radiolabeled protein A eluting from PACC after serum perfusion showed 8-fold greater binding to C1q-coated tubes compared to free protein A; and (c) increased C1q-binding IgG in postperfusion sucrose density gradient fractions corresponded to the appearance of protein A in parallel gradient fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma adriamycin and daunorubicin levels by fluorescence and radioimmunoassay.

In 38 adriamycin experiments and 4 daunorubicin experiments, radioimmunoassay readily and reproducibly detects and estimates these drugs and immunologically similar metabolites in patients' plasma and urine to at least 120 hr after dosing without interference by concurrent medication. The plasma drug decay follows first-order kinetics in a triphasic pattern. Radioimmunoassay and fluorescence assay show similar decay up to 4 hr but diverge at that point with the fluorescence assay yielding higher values. Pharmocokinetic differences are amplified in patients with liver dysfunction and may be caused by fluorescent drug metabolites not sensitive to radioimmunoassay or nonspecific fluorescent materials. The radioimmunoassay offers the capability to measure adriamycin and daunorubicin in clinical settings in which fluorescence assay is not available.

125I-labeled protein A as a general tracer in immunoassay: suitability of goat and sheep antibodies.

The immunoassay method in which 125I-labeled staphylococcal Protein A ([125I]PA) serves as a general tracer has been extended to include goat and sheep IgG antibodies. Goat and sheep IgG normally do not react significantly with PA. However, once IgG antibody is bound to immobilized antigen or hapten, binding of [125I]PA is enhanced markedly. Binding efficiencies of [125I]PA to immune complexed goat anti-human IgM, human IgE, methotrexate and sheep anti-IgE were determined and compared quantitatively to rabbit IgG with the corresponding specificity. Immunoassays were developed based on the inhibition of [125I]PA binding as a measure of antibody inhibition by fluid-phase homologous ligand. In terms of sensitivity and specificity, assays using goat and sheep antibodies were comparable to assays developed using rabbit IgG. Goat antibody to the monovalent hapten methotrexate behaved anomalously: for each concentration of IgG tested, there was an optimal amount of methotrexate beads that gave maximum binding of [125I]PA. In the other immune systems, for each antibody concentration maximum binding of tracer was a function only of the amount of immobilized antigen added. In contrast to the results obtained with solid-phase antigen, solutions containing antibody and amounts of antigen ranging from large antigen excess to antibody excess to antibody excess failed to react significantly with PA or [125I]PA.

A simple procedure to use whole serum as a source of either IgG- or IgM-specific antibody.

Absorption of rabbit antiserum to Forssman antigen with immobilized staphylococcal Protein A or concanavalin A selectively removed IgG or IgM antibodies, respectively. This absorption procedure was more rapid than ion exchange chromatography on DEAE cellulose or molecular sieving on Sephadex G-200 and gave a better yield of functionally purer antibody. This absorption method gave antiserum suitable for the preparation of either IgM or IgG sensitized sheep erythrocytes and should be of value for the large-scale preparation of indicator cells required for the study of complement action and for detection of specific receptors on cell surfaces.

Monoclonal antibodies to the chemotherapeutic agent methotrexate: production, properties and comparison with polyclonal antibodies.

Hybridomas that secrete monoclonal antibodies to the chemotherapeutic agent methotrexate (MTX) were produced by fusion of FO myeloma cells with splenocytes obtained from BALB/c mice immunized with L-MTX covalently bound to keyhole limpet hemocyanin (KLH). When the MTX/KLH molar ratio was 430/1 or 530/1 (high MTX series), 238/412 (58%) or 157/405 (39%), respectively, of antibody producing hybridomas secreted only antibodies to MTX. In contrast, only 15/412 (4%) or 54/405 (13%) hybridomas secreted only antibodies to KLH. When the molar ratio was 50/1 (low MTX series), only 14/343 (4%) of the wells contained anti-MTX and 93/343 (27%) contained antibodies to KLH. The predominance of anti-MTX over anti-KLH secretors in the 2 high MTX series, and the reverse predominance of anti-KLH secretors over anti-MTX hybridomas in the low MTX series was highly significant (P less than 0.001). The anti-MTX antibodies were mainly IgG1 with fewer IgG2a and IgG2b subclasses. All were kappa chain isotypes. No IgM or IgA antibodies were detected. Titers and binding constants of monoclonal anti-MTX antibodies in ascites fluid obtained from mice injected with selected cell lines were higher than those of conventional rabbit, mouse, or goat antibodies. Based on results of competitive inhibition radioimmunoassays, the ascites monoclonal antibodies were at least as specific as the conventional antibodies for MTX compared to structurally related compounds. IgG2a and IgG2b antibodies had high titers in complement mediated hemolysis of sheep erythrocytes that contained MTX on the cell surface (E-MTX) and also bound 125I-labeled protein A when complexed to MTX on these cells. IgG1 antibodies were relatively weak in the hemolysis assay and failed to bind 125I-labeled protein A directly. Hemolytic activity and 125I-labeled protein A binding were markedly enhanced when E-MTX cells were treated sequentially with IgG1 anti-MTX then rabbit IgG antibodies to mouse IgG1.

Immunoassay of antigens and haptens by inhibition of passive immune hemolysis.

Improved methods are presented for the detection of antigens and haptens by the use of the passive hemolysis inhibition test. The test is capable of detecting nanogram quantities of proteins (e.g., ferritin, IgE) and haptens (e.g., folinic acid, methotrexate). The method is also useful for studying quantitative and qualitative aspects of antibody-antigen interaction.

Comparison of monoclonal and polyclonal antibodies to cotinine in nonisotopic and isotopic immunoassays.

Monoclonal antibodies (McAb) were used to develop nonisotopic and radioimmunoassays (RIA) for quantitative determination of the major nicotine metabolite, cotinine, in physiological fluids. ELISAs and fluorescence immunoassays were carried out in microtiter plate wells coated with a conjugate of cotinine 4'-carboxylic acid bound covalently to poly-L-lysine. The detection systems were horseradish peroxidase (HRP)-labeled staphylococcal protein A, HRP-streptavidin-biotin, and biotinylated alkaline phosphatase-4-methylumbelliferyl phosphate. With the three McAb tested, I50 values ranged between 0.024-0.063 ng cotinine and as little as 0.005-0.015 ng gave 15% inhibition. These assays were 5-20 times more sensitive than similar assays using six rabbit antisera. With McAb the standard inhibition curves were steeper and complete inhibition of immune binding was achieved with approximately 1 ng cotinine. In contrast, 100-500 ng cotinine failed to give greater than 80-90% inhibition with rabbit antibodies either in the plate assays or in RIA using a 125I-labeled tyramine derivative of cotinine as the tracer. In this RIA, the sensitivity with McAb (mean I50 of 0.55 ng cotinine) was over three-fold greater than with rabbit antisera (mean I50 of 1.84 ng). The presence of antibodies directed to the amide linkage group common to the polylysine conjugate. 125I-tyramine derivative and the immunogen likely accounts for the inferior quality of assays using rabbit antisera. Consistent with this conclusion, superimposable inhibition curves were obtained in the RIA when monoclonal or rabbit antibodies were used with [3H]cotinine. Cotinine levels in saliva, serum and plasma from smokers and non-smokers determined with McAb-based assays showed a strong correlation with values obtained by RIA using rabbit antisera or by gas chromatography. Properly selected McAb offer distinct advantages over conventional antisera in nonisotopic immunoassays and RIAs for cotinine as a biochemical marker of active or passive smoking.

125I protein A: applications to the quantitative determination of fluid phase and cell-bound IgG.

An improved method for the preparation of 125I-labelled Protein A (125I PA) of high specific and functional activity is described. 125I PA has been used in combination with purified rabbit IgG bound to a solid support to develop a competitive binding assay capable of detecting Protein A or human, rabbit and guinea pig IgG at the nanogram level. An optimal set of assay conditions was established and levels of IgG measured in normal human, rabbit and strain-2 guinea pig serum. 125I PA has also been used to detect IgG anti-Forssman antibody bound to sheep erythrocytes and to line-1 and line-10 tumor cells and as an indirect assay for tumor associated antigen in the ascitic fluid of tumor-bearing guinea pigs.

Monoclonal antibody ELISA for cotinine in saliva and urine of active and passive smokers.

The value of a monoclonal antibody-based ELISA for measuring cotinine in saliva and urine of active and passive smokers was assessed. Cotinine (mean +/- SEM) was detected in all 26 saliva (392 +/- 74 ng/ml) and 27 urine (4264 +/- 508 ng/mg creatinine; 2566 +/- 364 ng/ml) samples from smoking parents, but in only two of 36 salivas and one of 37 urines from nonsmokers (P less than 0.001). Similarly, mean cotinine levels in 30 salivas (4.67 +/- 1.10 ng/ml) and 33 urines (35.5 +/- 8.8 ng/mg creatinine; 25.3 +/- 8.1 ng/ml) from passively exposed children were significantly higher (P less than 0.001) than in fluids of 36 unexposed children. Children's levels showed a strong correlation (P less than 0.001) with the number of cigarettes smoked in the home, but only when data from nonsmoking households were included in the analysis. In adult smokers there was a positive correlation between salivary and urinary cotinine (P = 0.002) and a close relationship between urinary cotinine and cigarettes smoked per day (P = 0.066). The ELISA gives a reliable quantitative measure of cotinine as an indicator of active and passive exposure to tobacco smoke. However, correlations with cotinine can be overestimated if large numbers of nonsmokers are included in the comparison.

Radioimmunoassays for protein A of Staphylococcus aureus.

Radioimmunoassays have been developed that can detect nanogram amounts of protein A (SpA), a product generated by Staphylococcus aureus that binds selectively to the Fc region of IgG from most mammalian species. Competition assays for fluid phase SpA utilize antibodies produced in chickens, 125I-labeled SpA as the tracer molecule, and either F(ab')2 fragments of rabbit IgG anti-chicken IgG or 40% ammonium sulfate as the precipitating agent to separate antigen-antibody complexes from free antigen. The double antibody assay could be carried out in serum from species that form only soluble complexes with SpA (e.g., rabbit), that react poorly with SpA (e.g., rat), or under appropriate conditions in serum from species (e.g., dog) that show high reactivity with SpA and form precipitating complexes. Chicken antibodies prepared by affinity chromatography on SpA-Sepharose and labeled with 125I were used in a direct binding assay for SpA present either on the cell wall of Cowan strain I or Wood 46 bacteria, in insoluble complexes prepared from SpA and whole serum or purified IgG, or in Clq binding complexes that were formed by passage of serum from normal or tumor bearing humans or dogs over SpA-collodion charcoal. Since both types of assays could detect SpA even in the presence of serum or IgG, they offer advantages over other techniques in which the SpA-Fc interaction may interfere.

Stereospecific monoclonal antibodies to nicotine and cotinine and their use in enzyme-linked immunosorbent assays.

Stereospecific monoclonal antibodies (McAb) have been prepared against the tobacco alkaloid (S)-(-)-nicotine and its major metabolite (S)-(-)-cotinine. Nine anti-nicotine and 4 anti-cotinine hybridomas, selected by a screening procedure that utilized immunoprecipitation of the 3H-labeled natural isomers of nicotine or continine, were grown in the ascites fluid of pristane-primed syngeneic BALB/c mice. Antibodies in concentrations up to 7.5 mg/ml ascites and with binding affinities that generally exceeded 10(8) M-1 were obtained. Enzyme-linked immunosorbent assays (ELISAs) were developed in which nicotine or cotinine derivatives bound covalently to poly-L-lysine were coated onto wells of polyvinyl chloride microtiter plates. Coated wells were incubated sequentially with McAb in the presence or absence of inhibitor, rabbit anti-mouse immunoglobulin, then horseradish peroxidase-labeled protein A (HRP-SpA) before addition of substrate. The antibodies are highly specific and show minimal cross-reactivity with several nicotine metabolites and other structurally related compounds. In the respective assays, only 0.25 ng (S)-(-)-nicotine and 0.12 ng (S)-(-)-cotinine are required to give 50% inhibition of antibody binding, and as little as 0.05 ng nicotine and 0.02 ng cotinine give 15% inhibition. These assays are 5-10 times more sensitive than analogous ELISAs developed with rabbit antisera and HRP-SpA or conventional radioimmunoassays (RIAs) that utilize the rabbit antisera and 3H-labeled ligands. There was good correlation between the levels of nicotine (r = 0.967) and cotinine (r = 0.981) found in saliva samples from smokers and non-smokers assayed by McAb-based ELISAs and conventional RIAs.

Peptide nucleic acid probe detection of mutations in Mycobacterium tuberculosis genes associated with drug resistance.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a serious public health problem. Many of the specific gene mutations that cause drug resistance in M. tuberculosis are point mutations. We are developing a PCR-peptide nucleic acid (PNA)-based ELISA as a diagnostic method to recognize point mutations in genes associated with isoniazid and rifampin resistance in M. tuberculosis. Specific point mutation-containing sequences and wild-type sequences of cloned mycobacterial genes were PCR-amplified, denatured, and hybridized with PNA probes bound to microplate wells. Using 15-base PNA probes, we established the hybridization temperatures (50 degrees C-55 degrees C) and other experimental conditions suitable for detecting clinically relevant point mutations in the katG and rpoB genes. Hybridization of PCR-amplified sequences that contained these point mutations with complementary mutation-specific PNAs resulted in significant increases in ELISA response compared with hybridization using wild-type-specific PNAs. Conversely, PCR-amplified wild-type sequences hybridized much more efficiently with wild-type PNAs than with the mutation-specific PNAs. Using the M. tuberculosis cloned genes and PCR-PNA-ELISA format developed here, M. tuberculosis sequences containing point mutations associated with drug resistance can be identified in less than 24 h.

Cotinine analytical workshop report: consideration of analytical methods for determining cotinine in human body fluids as a measure of passive exposure to tobacco smoke.

A two-day technical workshop was convened November 10-11, 1986, to discuss analytical approaches for determining trace amounts of cotinine in human body fluids resulting from passive exposure to environmental tobacco smoke (ETS). The workshop, jointly sponsored by the U.S. Environmental Protection Agency and Centers for Disease Control, was attended by scientists with expertise in cotinine analytical methodology and/or conduct of human monitoring studies related to ETS. The workshop format included technical presentations, separate panel discussions on chromatography and immunoassay analytical approaches, and group discussions related to the quality assurance/quality control aspects of future monitoring programs. This report presents a consensus of opinion on general issues before the workshop panel participants and also a detailed comparison of several analytical approaches being used by the various represented laboratories. The salient features of the chromatography and immunoassay analytical methods are discussed separately.

Issues and perspectives on the biocompatibility and immunotoxicity evaluation of implanted controlled release systems.

Over the past decade, significant advances and discoveries in cell and molecular biology and biomaterials have provided a foundation for the research and development of new, complex controlled release systems. Many of these new controlled release systems utilize active biological components, i.e., proteins and cells, to achieve their intended therapeutic goal. Utilization of bioactive biological materials in implantable controlled release systems has prompted a broad as well as an in-depth interest in the safety and efficacy of these systems. This short review is intended to provide individuals with a perspective on standards and guidance documents which specifically address biological and immunotoxicity response evaluation for safety of controlled release systems from a regulatory point of view.