Screening for okadaic acid by immunoassay.

Increasing incidences of phytoplankton blooms with the potential danger of toxin release into the food chain have necessitated the search for new diagnostic methods that can detect toxins quickly and reliably. A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantitate okadaic acid in shellfish and phytoplankton extracts. To determine the specificity of the assay, a number of toxins, such as calyculin A, brevetoxin-1, and dinophysistoxins-1, -2, and -3 were analyzed. Both dinophysistoxins-2 and -1 could be detected by the assay but in concentration ranges 10- and 20-fold higher than that for okadaic acid, respectively. Dinophysistoxin-3, calyculin A, or brevetoxin-1 could not be detected with this assay. To validate the accuracy of the method, 18 mussel and 7 phytoplankton extracts were analyzed in parallel for okadaic acid content by ELISA and liquid chromatography combined with either fluorescence or mass spectrometric detection. Very high correlation between the results was found.

Evaluation of a 99mTc-antimyosin kit for myocardial infarct imaging.

The Fab fragment of a mouse monoclonal antibody AM(3-48) that recognizes alpha and beta-heavy chains of human atrial and ventricular myosin and beta-heavy chain of human slow skeletal muscle myosin [CardioVisionTM] was labeled with 99mTc using stannous reductant in a simple, instant kit method. The infarcted heart uptake in dogs of 99mTc-AM(3-48)Fab' was compared with that of established radiopharmaceuticals routinely used for cardiac imaging in humans. The dog infarct was induced by bringing a catheter from the femoral artery to the coronary artery where an artificial blood clot was generated. The 99mTc-AM(3-48)Fab' preparation was selectively taken up by infarcted myocardium, resulting in diagnostic quality images of the infarcted area as early as 6 hour post-injection, rendering CardioVisionTM particularly useful for SPECT imaging. Good agreement was found between the images obtained with 99mTc-Pyrophosphate and those obtained with 99mTc-AM(3-48)Fab', while the infarcted area was clearly delineated as a cold spot with 99mTc-MIBI or 201 Tl-thallous chloride. The biodistribution of 99mTc-AM(3-48)Fab' was also studied in healthy and isoproterenol-infarcted rats, from which dosimetry values in man were extrapolated. The data indicate that the kidneys will receive the highest radiation dose and that they will be the main contributors to the total radiation burden, which was estimated at 0.005 rad/mCi.

An anti-okadaic acid-anti-idiotypic antibody bearing an internal image of okadaic acid inhibits protein phosphatase PP1 and PP2A catalytic activity.

Okadaic acid (OA), produced by marine phytoplankton, is the parent compound of a family of marine toxins responsible for diarrheic shellfish poisoning (DSP). A monoclonal antibody to OA (6/50) (Ab1) has been raised and in turn used for immunization of syngeneic animals. Mice inoculated with the 6/50 idiotype produced both anti-idiotypic antibodies (Ab2) and OA binding antibodies (Ab3). The selected anti-idiotypic antibody 1/59 bound to the immunizing 6/50 idiotype but not to F(ab')2 fragments of pooled normal mouse Ig. It inhibited the binding of OA to solid-phase attached F(ab')2 of 6/50 IgG as well as the binding of 6/50 IgG to a solid-phase bound OA. Like OA, 1/59 anti-idiotypic antibody inhibited protein phosphatase 1 and 2A catalytic subunits in a 32P-phosphorylase a phosphatase radioassay. Thus, 1/59 IgG is a novel internal image anti-idiotypic antibody (Ab2 beta) and can serve as a surrogate of OA in biological assays.

An idiotypic-anti-idiotypic competitive immunoassay for quantitation of okadaic acid.

A competitive indirect enzyme-linked immunosorbent assay for the measurement of okadaic acid, a marine toxin, was developed. The assay uses a murine monoclonal anti-idiotypic antibody bearing an internal image of okadiac acid epitope to capture an anti-okadaic acid monoclonal antibody in the presence of free okadaic acid. Bound anti-okadaic acid antibody is detected with peroxidase-conjugated anti-mouse immunoglobulin antiserum. If present, free toxin will lessen the amount of anti-okadaic acid antibody binding to its corresponding anti-idiotypic antibody in a dose-dependent manner that can be quantified from the standard curve. The assay permits reliable measurement of okadaic acid in the 9-81 ng/ml range. The intra- and interassay coefficients of variation in the measurement of OA in the toxin spiked mussel samples averaged 9% and 12%, respectively. The assay is rapid, accurate, reproducible and relatively simple to perform. It may be of potential use to laboratories involved in monitoring the toxin levels in plankton, seafood or sponges.

Immunohistochemical characterization of a new anti-carcinoembryonic antigen monoclonal antibody.

A novel anti-CEA monoclonal antibody C234 with no cross-reactivity to other members of CEA gene family was produced by immunization with heat-treated CEA. The immunoreactivity of C234 was tested on formalin fixed normal and neoplastic tissues by peroxidase/anti-peroxidase technique. None of normal tissues from healthy or diseased individuals, except for normal colonic mucosa were stained. On the other hand, all 26 colorectal adenocarcinomas, 8 gastric adenocarcinomas, 3 of 7 pancreatic adenocarcinomas, and one small bowel duodenum carcinoma were immunoreactive. None of primary hepatocellular carcinomas expressed CEA. However, only 40 of 87 neoplasms of non-gastrointestinal origin were stained. Thus, the antibody can recognize gastrointestinal tumors and differentiate them from neoplasms of other histological origin.

Infarcted heart uptake and biodistribution of radiolabelled anti-myosin monoclonal antibody in rat and dog myocardial infarct models.

A new mouse monoclonal antibody that recognizes alpha- and beta-heavy chains of human atrial and ventricular myosin and beta-heavy chain of human slow skeletal muscle myosin was obtained. The 125I- and 111In-labelled antibody, and its F(ab')2 and Fab fragments localize in isoproterenol induced infarcted rat heart, with the F(ab')2 fragment showing the highest uptake. Comparison with 99Tc-pyrophosphate uptake in infarcted dog heart, induced by selective obstruction of a coronary artery, suggest that the 111In-labelled F(ab')2 localizes specifically in infarcted myocardium only.

Experimental antitumor activity of new azathioprine analogues.

Two newly synthesized azathioprine (AZA) analogues, 6-(1,2-dimethyl-4-nitro-5-imidazolyl)thiopurine (Met-AZA) and 6-(2-methyl-5-nitro-4-imidazolyl)thiopurine (IZO-AZA), were investigated against KB human tumor cells. In 5 transplantable murine tumor models, including sc Sa180, sc Ca755, ip LL and ip leukemias; L1210 and P388 both drugs were found to be antitumor active in all the experiments carried out regardless of dosing regimen or the route of administration. Similar good activity was shown in the KB, ip Sa180, and Ca755 systems and partly against LL as compared to AZA. However, Met-AZA against ip P388 demonstrated therapeutic advantage following qd 1-9 daily dosing, 0.33 log10 tumor cell kill; therapeutic index (TI = ILSmax/ILS 25) = 2, and ILS = 69% in comparison to AZA and IZO-AZA, TI = 1.3, 1.2, and ILS = 31%, 40%, respectively. Met-AZA is comparable to AZA, while seeming to display greater antileukemic activity than AZA.

Tumor-associated antigens as immunotherapy targets.

The potential of liposomes to act as immunoadjuvant carriers of tumor-associated antigens (TAA) has been investigated. The incorporation of B16 melanoma TAA within liposomes resulted in immunological recognition by non-tumor-bearing mice, and subsequent inhibition of tumor growth upon tumor challenge. The immunogenicity and protective activity were enhanced by the concomitant incorporation of a lipophilic immunoadjuvant, MDP-GDP, in the liposome preparation. The ability of liposomal preparations to augment the immunogenicity of a human oncofetal antigen, CEA, was also studied. The incorporation of CEA within liposomal carriers resulted in immunological recognition in mice at doses (0.1 micrograms) significantly less than required in Freund's complete adjuvant (25 micrograms), maximal responsiveness being found with liposomal-CEA-MDP-GDP preparations. Liposomal TAA vaccines may therefore require the presence of immuno-adjuvant-active agents for the induction of effective immunological responses in individuals at risk from recurrent disease.

Clinical applications of carcinoembryonic antigen.

Although carcinoembryonic antigen (CEA) has been the subject of interest for many investigators for over 20 years, many questions still remain unanswered concerning the CEA molecule. These include the ultimate clinical potential of CEA as a tumor marker (specificity, sensitivity, distribution), the biological role of CEA, and the genetic control of CEA synthesis. Initially, much of the work with CEA concerned its physicochemical and immunochemical properties, as well as of cross-reacting molecules, and methods of serologic detection of CEA. More recent studies have focused on cloning of the CEA gene and genetic control of CEA production. An extensive literature exists concerning the role of serum CEA assays and their potential value in determining the prognosis and monitoring of patients affected with cancers of various organs. Despite extensive research into the biology of CEA, few papers deal with the application of CEA immunohistochemistry and immunocytochemistry as concerns normal cellular development, the degree of tumor anaplasia, and the various diagnostic problems of surgical pathology. There has also been a great deal of interest in the utility of both polyclonal and monoclonal antibodies to CEA both in the radioimmunolocalization and potential therapy of CEA-producing tumors. This review summarizes the past and current findings of the clinical applicability of the serum measurement of CEA and examines the status of radioimmunolocalization of tumors as a basis for effective antibody targeted immunotherapy in the future.

Liposomal incorporation and immunogenicity of carcinoembryonic antigen.

The ability of liposomal carriers to act as immuno-adjuvants for carcinoembryonic antigen (CEA) has been evaluated. Liposomal incorporation was consistent with association with the aqueous phase of the liposome, little if any of the protein being associated with the phospholipid bilayer. The incorporation of CEA within liposomal carriers resulted in immunological recognition in mice at doses (0.1 microgram) significantly less than required in Freund's complete adjuvant (25 micrograms), maximal responsiveness being found with liposomal-CEA preparations containing a lipophilic immunoadjuvant, N-acetyl-muramyl-L-alanyl-D-isoglutamyl-glyceryl-dipalmitate. Such liposomal formulations may have utility as immunoadjuvants for cancer immunotherapy.

Evaluation of an enzyme-linked immunosorbent assay for the measurement of autoantibodies against eye muscle membrane antigens in Graves' ophthalmopathy.

We have tested for antibodies against human and pig eye muscle membrane antigens in the serum of patients with Graves' ophthalmopathy using an enzyme-linked immunosorbent assay (ELISA). Several different membrane preparations were used as source of putative antigen including a 100,000 X g pellet, a pellet depleted of the 100,000 X g (microsome) fraction, and solubilized membranes. With eye muscle membrane pellets there were no significant differences for either serum or immunoglobulins between patients with ophthalmopathy, those with autoimmune thyroid disorders without eye disease, and normal subjects for either human or pig membranes, although tests were positive determined from the upper limit of normal in a few patients with or without eye disease. This was the case regardless of the enzyme-antibody conjugate used, the membrane protein concentration or serum or immunoglobulin dilution. Pre-absorption of tissue fractions, serum, or immunoglobulins, with red blood cells or liver powder, eye muscle membranes or skeletal muscle membranes did not significantly reduce back-ground binding which was often very high, or enhance the difference between patients with ophthalmopathy and normal subjects. It was found that non-specific binding to the plastic surface of the microplates and/or tissue proteins, the presence, in human tissues, of blood-derived immunoglobulins which gave strong reactions in the ELISA, and variable fixation of membrane pellets to the plates were factors which made ELISA unsatisfactory when crude membrane pellets were used as antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-thyroglobulin anti-idiotypic antibodies in sera of patients with Hashimoto's thyroiditis and Graves' disease.

The objective of this study was to find naturally occurring anti-idiotypic (anti-Id) antibodies to anti-human thyroglobulin (anti-hTg) idiotype in sera of patients with autoimmune thyroid disease. Sera from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE) and sera from normal subjects were tested for the presence of anti-Id antibodies against mouse anti-hTg monoclonal antibodies (McAb) in indirect ELISA and in indirect solid-phase RIA. Microtitration plates were coated with six McAb, five of them directed against different epitopes on hTg molecule, and then incubated with patients' sera. The bound antibody was detected with either peroxidase or 125I-labeled anti-human IgG. The specific positive reaction was observed in four of 40 patients with HT, in two of 26 patients with GD, in seven of 58 patients with RA, and in none of 20 normal subjects. The detected binding was due to the presence of anti-hTg anti-Id antibodies and not to Tg-anti-Tg circulating immune complexes, as the positive sera did not contain hTg when resolved on SDS-PAGE, nor did they bind to all anti-hTg McAb tested. The binding was dose dependent, and titers of anti-Id antibodies varied from 1:243 to 1:2187. The binding could be inhibited up to 50% by hTg, but not by the thyroid microsomal antigen, indicating that some of those anti-Id might represent the internal image of the antigen. Serum from the patient 3403, showing the strongest reactivity against McAb A-3, was chosen for IgG purification and F(ab')2 fragment isolation. The 3403 F(ab')2 fragment, but not the Fc fragment, was found to react specifically with four mouse anti-hTg McAb but not with the control mouse IgG. Thus, the obtained results permit the conclusion that anti-hTg anti-Id antibodies could occur naturally during the course of thyroid autoimmune disorders.

Use of monoclonal antibodies to investigate a possible role of thyroglobulin in the pathogenesis of Graves' ophthalmopathy.

One possible mechanism for Graves' ophthalmopathy is that the progressive orbital inflammation is initiated by formation of thyroglobulin (Tg)-anti-Tg immune complexes at sites of Tg binding to extraocular muscle membranes. In this study monoclonal antibodies (MCAB) against human Tg were used as probes (1) to identify Tg in eye muscle membranes prepared from normal subjects and (2) to measure binding of human Tg and Tg-anti-Tg immune complexes to eye muscle membranes. Reactivity of anti-Tg MCAB with Tg, thyroid, and eye muscle membranes was determined by binding of [125I]anti-Tg monoclonal antibody, an enzyme-linked immunosorbent assay (ELISA), and the indirect immunofluorescence technique. Seven membrane fractions, prepared by differential sucrose gradient centrifugation, were used. Whereas [125I]anti-Tg MCAB bound to all thyroid membrane fractions tested, no [125I]anti-Tg bound to eye muscle membranes. Similarly, reactivity of anti-Tg MCAB with eye muscle membranes was not demonstrated in ELISA or immunofluorescence tests. Although Tg-anti-Tg immune complexes bound to thyroid membranes, such complexes did not bind to eye muscle membranes. Significant binding of [125I]human Tg to eye muscle or thyroid membranes was not demonstrated for any membrane preparation. On the other hand moderate, but significant, binding to skeletal muscle was shown. Similar results were found using an ELISA. Binding of [125I]anti-Tg-Tg complexes of [125I]Tg to thyroid and eye muscle membranes was not affected by the presence of normal human serum, phosphate ions, pH, or incubation temperature, conditions claimed by others to be critical for Tg and Tg-anti-Tg immune complex binding. Since Tg is not present in normal human eye muscle a major role of Tg, or Tg-anti-Tg immune complexes, in the pathogenesis of Graves' ophthalmopathy appears to have been excluded by these findings.

Demonstration of a circulating autoantibody against a soluble eye-muscle antigen in Graves' ophthalmopathy.

Mouse monoclonal antibodies against antigens in human eye muscle, orbital connective tissue, and lacrimal tissue and guineapig harderian gland have been produced by means of the hybridisation technique. From 14 fusions, over 30 antibodies have been produced, of which 24 have been maintained and characterised for the present studies. All were IgG1. Most were organ-specific, but eye-muscle antibodies tended to cross-react with skeletal muscle, one eye-muscle antibody cross-reacted with thyroidal microsomes, and both lacrimal monoclonal antibodies reacted strongly with mucus-secreting cells in human small intestine. Only one of the antibodies fixed complement. Orbital monoclonal antibodies were used as probes to identify orbital antigens. Most antigens were in the cytosol fraction, but a few were demonstrated in cytoplasmic membranes. Circulating autoantibodies against a human-eye-muscle soluble antigen were detected in 17 of 23 patients with Graves' ophthalmopathy but in only 1 of 14 patients with Hashimoto's thyroiditis, in 2 of 11 patients with subacute thyroiditis, in no patient with Graves' hyperthyroidism without eye disease, and in none with multinodular goitre.

Selective induction of T-cell subsets capable of accelerating and retarding Lewis lung carcinoma.

The influence of three T-cell subsets on tumor growth was studied. The first, induced by a thymic microenvironmental fraction, accelerated the appearance of Lewis lung carcinoma in Winn assay. The second subset, derived from the first by incubation with a thymic hormone preparation, had a marked retarding effect on tumor appearance. The third subset, also induced by thymic hormone, but directly from bone marrow precursors, does not appear to influence tumor cells.

Studies on the influence of adrenaline, acetylcholine and cysteine on suppressive activity of lymphocytes engaged in GvH reaction in rats.

Thirty-minute preincubation of lymphocytes deriving from popliteal lymph nodes of hybrid W X A F1 rats experiencing GvH reaction in 10(-6)M solution of acetylcholine resulted in an increased [3H]-thymidine incorporation both in cultures stimulated and nonstimulated with PHA. When the cells were preincubated for 30 min in 10(-5)M solution of adrenaline the level of [3H] TdR incorporation into non-stimulated cells was increased. The cells pretreated in such a way and added to cultures containing normal peripheral lymph node lymphocytes responding to PHA exhibited diminished or even abrogated suppressive effect which they normally exert if they are not treated with these two drugs. The PLN-cells preincubated in 2 mM solution of L-cysteine for 30 min, when added to the culture system showed an enhanced intensity of their suppressive effect.

Levamisole and graft-versus-host reaction (GvHR) in rats.

The influence of levamisole on local GvHR was studied. The drug injected at doses of 10 mg/kg or at 2.5 mg/kg of body weight into rats was found to stimulate GvHR. The intensity of the reaction was measured by the popliteal lymph node (PLN) enlargement, the number of cells contained in the node and by [3H]-thymidine incorporation into those cells. The values of all of these parameters were enhanced when the PLN was analyzed between 5-7 days of GvHR. Levamisole influenced the kinetics of GvHR by increasing its intensity, causing its earlier initiation and prolonging its duration. The drug incubated for 30 min with donor lymphocytes, which then elicited the reaction, had little but reproducible effect on the development of the reaction. The optimal schedule of levamisole administration has been determined. This drug was found to be the most effective when given after the GvHR initiation. Levamisole caused the induction of GvHR when suboptimal doses of donor lymphocytes were injected.