(Auto)antibodies in human breast cancer sera against antigens associated with breast cancer cells, detected by immunoblotting.

Sera of patients with breast cancer (as well as control normal sera and sera of patients with ovarian cancer or melanoma) were screened for the presence of antibodies against antigens expressed by the MDA breast cancer cell line. The techniques employed were radioimmunoassay with radioiodinated protein A and immunodotting with peroxidase-conjugated anti-human immunoglobulin antibodies. Sera reacting strongly by immunodotting were subsequently tested against antigens of the MDA and T47D cell lines in immunoblotting experiments. Both the breast cancer and the control sera yielded highly complex band patterns, which varied from serum to serum. The cancer sera differed from the normal sera, however, as they produced in most cases one or several bands that were distinctly stronger than the others. One of the strong bands, in fact a doublet of approximately 50 kilodaltons (kd), was produced preferentially (although not exclusively) when breast cancer sera were reacted with T47D cell membrane antigens. Absorption of selected sera with normal tissue or MDA antigens abolished or greatly reduced the intensity of some of the bands. It is concluded that, with the possible exception of the 50-kd band, most (probably all) of the bands seen in immunoblots resulted from the binding of autoantibodies to normal antigens expressed by the breast cancer cell lines. The main difference between cancer and normal sera would seem to be an increased content of autoantibodies in cancer, the specificity of these autoantibodies varying, however, from serum to serum.

Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein-Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population.

A DNA fragment containing Epstein-Barr virus (EBV) terminal fragment sequence was obtained from a genomic library of nasopharyngeal carcinoma (NPC). One of the clones (clone 1510) contained the gene encoding latent membrane protein (LMP). Sequence analysis revealed that this gene had 95% homology with the LMP sequence of the B95-8 strain. Among the sequence variations, there was a change from G to T at nucleotide position 169,426, resulting in the loss of an XhoI site in exon 1 of the LMP gene. A pair of primers bracketing the XhoI site were designed to synthesize the EBV DNA fragment from nucleotides 169,081-169,577 by using the polymerase chain reaction (PCR) method. The PCR products were then subject to XhoI digestion and to DNA sequencing analysis. This restriction enzyme site polymorphism along with the sequence variations were also observed in 50 biopsy tissues as well as in the throat washings of 6 out of 20 healthy individuals that we examined, indicating that the EBV strain predominantly existing in these biopsy tissues was different from strains of B95-8, Jijoye or nude mouse passaged cells (C15) with an African origin, but closely resembled other nude mouse passaged CAO cells which were originally derived from China. Balb/c 3T3 cells carrying this NPC-LMP gene showed a transformed cell morphology and were tumorigenic in nude mice. The relationship between this unique type of EBV and NPC has yet to be established.

A radioactive antibody binding-inhibition assay, for the detection of cell-membrane related antigens in body fluids.

Antisera raised in rabbits against glutaraldehyde-fixed human breast cancer cells contain antibodies to human cell membrane components, as determined by immunofluorescence. Adsorption of such antisera onto polymerized human serum, followed by acid elution, yields purified antibodies reacting with human cell surface antigens, indicating that membrane related antigens are present in the serum. The purified antibodies were radioiodinated and shown to bind to an immunoadsorbent prepared by entrapping in a polyacrylamide gel pleural exudate of breast cancer patients. The specificity of the binding was confirmed by inhibition experiments. Data are presented demonstrating that at least some of the antibodies reacting in this radioimmunoassay are directed against antigens related to cell surface components.

A technique for the purification of immune complexes using rheumatoid factor.

A method for the purification of immune complexes (IC) from human serum is described, using as a model complexes of tetanus toxoid with human antitoxoid antibodies (TAT). The technique is based on the ability of IC to bind to tubes coated with rheumatoid factor (RF). Small amounts of TAT IC were added to human serum and the mixtures were rotated overnight in tubes coated with RF. The tubes were then washed and the bound material was eluted with sodium dodecyl sulphate and iodinated with 125I. Analysis of the labeled preparation by electrophoresis in polyacrylamide gels revealed the presence of the toxoid component. The technique should be useful in isolating small amounts of IC for analytical purposes.

Detection of Plasmodium falciparum using a radioimmunoassay based on a crossreacting, monoclonal anti-P. berghei antibody-P. berghei antigen system.

An antibody binding-inhibition test is described, which allows the detection of P. falciparum in red blood cells (RBC) infected in vitro, using a crossreacting, monoclonal anti-P. berghei antibody and P. berghei coated microtiter plates. Experiments carried out to determine the coating efficiency of various P. berghei and P. falciparum derived antigen preparations showed that intact, saponin freed P. berghei parasites and sonicated, RBC parasitized with P. falciparum had the highest binding activity. Binding of the monoclonal antibody to the antigen coated plates was effectively inhibited by preincubation with sonicated, P. falciparum infected RBC. The minimal degree of infection detectable was about 0.008% parasitemia (400 parasitized RBC/microliters blood). The sensitivity of detection was not appreciably affected by the source of the coating antigen. We conclude that the difficulty and expense involved in the use of P. falciparum based immunodiagnostic tests for large scale screening for malaria can be obviated by making use of P. berghei based assays.

An improved technique for the isolation and analysis of immune complexes.

A technique for isolating and analyzing immune complexes (IC) using rheumatoid factor (RF) as immunoadsorbent is described. It contains several modifications to a previously published original method and has given greatly improved results: a small amount of BSA-anti-BSA (aBSA) model IC was added to human serum and the mixture was filtered through a column of Sephacryl S-300, the large molecular weight fraction was concentrated and added to tubes coated with RF. The bound IC were eluted, radioiodinated and freed to the major contaminants (HSA and immunoglobulins) by adding the corresponding antisera and removing the resulting complexes by coprecipitation or with the aid of protein A-containing Staphylococci. The purified preparations were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography. BSA was clearly identified in the autoradiograms. An example is given of the application of this technique to the isolation and analysis of IC from an abdominal effusion obtained from a patient with ovarian cancer.

A technique for the identification of glycoprotein antigens in immune complexes and its application to the detection of a common glycoprotein in sera of patients with Burkitt's lymphoma and nasopharyngeal carcinoma.

A new technique for the detection of glycoprotein antigens in immune complexes (IC) isolated from serum is described. The technique was developed with a model IC system consisting of ovalbumin (OVA)-rabbit anti-ovalbumin antibodies (aOVA), at 3 times antigen excess. OVA-aOVA IC added to normal human serum (NHS) were purified by absorption onto and elution from tubes coated with rheumatoid factor (RF) and were subjected to electrophoresis in polyacrylamide gels. Concanavalin A (Con A)-binding proteins were detected by treating the gels with radioiodinated Con A (125Con A), followed by autoradiography. IC isolated from sera of patients with Burkitt's lymphoma (BL) and Nasopharyngeal Carcinoma (NPC) were analyzed before and after reduction with dithiothreitol. Two closely spaced proteins of about 40 kdalton were identified in the reduced samples in 26 of 30 BL sera (86%) and in 24 of 30 NPC sera (80%) but were not seen in 30 sera of African patients with a variety of unrelated tumors nor in 12 sera of European blood bank donors.

A solid-phase antibody binding-inhibition test, for the assay of Plasmodium berghei antigen and antibodies, using radioiodinated protein A.

Sonicated red blood cells (RBC) of rats infected with Plasmodium berghei (Pb) were used to coat plastic tubes with Pb antigens. The antigen-coated tubes were employed to detect Pb antigens and antibodies, with high efficiency. Anti-Pb antibodies were estimated by treating the tubes with rabbit or rat anti-Pb sera and assaying the bound Ig with radiolabeled Staphylococcus PrA. Pb antigens were detected by their capacity to inhibit the binding of the anti-Pb antibodies. Using a rabbit-Pb serum, sonicated, infected RBC (50% parasitemia) gave detectable inhibition up to 1 : 106 dilution.

A highly sensitive solid-phase radioimmunoassay for the assay of Plasmodium falciparum antigens and antibodies.

A highly sensitive radioimmunoassay for detection of P. falciparum antibodies and antigens is described. A partially purified P. falciparum antigen preparation is obtained from in vitro cultured parasites enriched after gelatin sedimentation by sonicating the infected red blood cells and precipitating the proteins with 50% saturated ammonium sulfate. The precipitate is dissolved in buffer, ultracentrifuged and used to coat wells of microtiter plates. Anti-P. falciparum antibodies are detected by incubating antiserum dilutions in the coated wells and detecting the bound IgG with radioiodinated staphylococcal protein A. P. falciparum antigens are detected by their ability to inhibit binding of antibodies to the coated wells. Sera of individuals with a history of P. falciparum infection contain antibodies detectable at a dilution of 1:75,000. P. falciparum RBC infected in vitro can be detected at levels of parasitemia of the order of 1 parasite or less per 10(6) RBC.

A high molecular weight cytotoxic lymphokine in supernatants of lymphokine-activated killer cells cross-reacts with transforming growth factor beta.

Supernatants of lymphokine-activated killer (LAK) cells were highly cytotoxic for melanoma A375 cells. A high-molecular-weight fraction was isolated from such supernatants by gel filtration on an S-300 Sephacryl column (Fraction 1; Fr1). The cytotoxic activity in Fr1 was heat- and acid-resistant and was completely abolished by a rabbit antibody against TGF-beta. We conclude that Fr1 contains TGF-beta or a cross-reactive molecule, associated with a high-molecular-weight carrier.

Degenerative changes in the A375 melanoma line induced by transforming growth factor beta 1.

A375 human melanoma cell cultures grown in the presence of TGF beta contained greatly reduced cell numbers and exhibited drastic alterations in cell morphology compared to the control cultures. Preincubation of the cells with the cytokine for only 18 h was sufficient to induce these changes irreversibly. Examination of TGF beta-treated cells in the electron microscope revealed large numbers of lipid-filled vacuoles in the cytoplasm, greatly contracted nuclei and some loss of the otherwise abundant microvilli. Thus TGF beta may have a direct toxic effect on the A375 melanoma cells.

Use of a monoclonal, anti Plasmodium berghei antibody, cross reacting with P. falciparum, for the detection of P. falciparum in in vitro infected blood.

This report describes an immunoradiometric assay for Plasmodium falciparum in infected blood, based on a cross-reacting monoclonal antibody (mAb) raised against P. berghei. In this assay, binding of the mAb to intact P. berghei parasites coated on microtiter plates is inhibited by solubilized P. falciparum infected red blood cells. The use of P. berghei parasites in conjunction with monoclonal antibodies should facilitate the development of an inexpensive and reproducible test for the immunodiagnosis of malaria.

Culture supernatants of lymphokine-activated killer (LAK) cells contain a high-molecular-weight cytotoxic lymphokine.

Supernatants of human lymphokine-activated killer (LAK) cells grown in vitro were tested for cytotoxic activity against several mouse and human neoplastic cell lines. All LAK preparations tested (14/14) exhibited cytotoxic activity (40-90% killing of the target cells). Sephacryl S-300 Gel filtration experiments indicated that the biological activity of the LAK supernatant is associated with molecular moieties ranging from 800 kDa or more, to less than 10 kDa. The finding of strong cytotoxic activity in LAK supernatants against several tumor lines points to the possibility of employing soluble products of these cells, rather than the living cells themselves, for therapeutic purposes.

Antibodies in human sera against the Epstein-Barr virus encoded latent membrane protein (LMP).

Antibodies reactive with the Epstein-Barr (EBV)-encoded latent membrane protein, LMP, were detected in human sera. Membrane fractions of the EBV-carrying Raji cells and a fusion protein (LMFP), that represents the carboxy part of LMP, were used as antigens. These were assayed by the reduction of the leukocyte migration inhibition (LMI) reaction. Reactivity with LMFP was detected in 10 of 14 sera from patients with Burkitt's lymphoma (BL) and in 3 of 23 sera from healthy EBV-seropositive individuals. The antibody levels were higher in the BL sera. Since the tumor cells do not express LMP, this may be due to the high virus load in the patients.

Levels of fibrinogen/fibrin degradation fragment E and related substances in sera and effusions of patients with malignant disease.

A radioimmunoassay (RIA) was developed and used to determine the level of fragment E [a fibrinogen/fibrin degradation product (FDP)] and of fragment-E-containing substances (FES) in sera and effusion fluids of patients with malignant diseases. Sera of patients with other diseases and sera of healthy individuals served as controls. Results were expressed as units/ml (U/ml), one unit being equivalent to 40 ng pure fragment E. Effusion fluids of both malignant and nonmalignant origin contained relatively high levels of fragment-E-containing substances, up to 7,500 U/ml. Normal sera had less than 30 U/ml, while sera of patients with a variety of neoplastic or nonneoplastic conditions contained larger amounts, reaching to hundreds and, in rare cases (some patients with rheumatoid arthritis), even thousands of U/ml. Some of the highest levels in the malignant sera were found in samples from patients with Burkitt's lymphoma and stomach cancer. About 10%-20% of the reactive material in effusions and 20%-40% in the sera consisted of fragment E. These results confirm earlier findings of high FDP levels in neoplasia. Given the higher accuracy of the radioimmunoassay and its suitability for large scale testing, it would appear worthwhile to continue such studies to explore the clinical usefulness of the RIA for fragment E.

A radioimmunoassay for the diagnosis of malaria.

A newly developed radioimmunoassay for the diagnosis of malaria has been tested in South Africa. The radioimmunoassay is an antibody binding-inhibition assay, based on a monoclonal antibody (D5) cross-reacting with Plasmodium berghei and P. residual binding activity was tested on antigen-coated microtiter plates. A sample was considered positive if it inhibited binding of the antibodies to an extent exceeding that of the microscopically negative blood samples. Blood was collected on 3 separate occasions from a total of 530 individuals living in a malaria-endemic area and was examined by radioimmunoassay and microscopy. Group 1, consisting of 194 samples, yielded 12 samples positive by microscopy and 10 of these (83%) were also positive by radioimmunoassay. One sample in this group was "positive" in the radioimmunoassay but negative on microscopy (false positive). In the 320 samples of group 2, 13 were positive by microscopy and 6 (46%) by radioimmunoassay. Group 3, which included 16 samples preselected as positive by microscopic examination and 16 controls, was examined after 4 weeks storage at -20 degrees C. Twelve samples (75%) were positive by radioimmunoassay. Tests carried out to determine the effect of blood storage on the activity of the antigen indicated that activity was preserved with little loss over a 3-month period.

Preliminary field trial of a radioimmunoassay for the diagnosis of malaria.

A radioimmunoassay (RIA) has been developed for the detection of Plasmodium falciparum in infected blood. The assay is based on the ability of solubilized, infected red blood cells (RBC) (P. falciparum "antigen") to combine with anti-P. falciparum antibodies and thus prevent the subsequent interaction of the latter with "antigen"-coated microtiter plates. A preliminary trial was carried out in Thailand to determine the usefulness of the RIA for the immunodiagnosis of malaria. Blood samples from malarious and non-malarious patients were examined both by standard microscopy and by RIA. Efficient solubilization of the parasites proved to be a major requirement for the successful performance of the RIA. Sonication or freezing and thawing, which were perfectly satisfactory for the solubilization of cultured, infected RBC, were found to be totally inadequate when applied to RBC taken from patients. However, parasites in RBC from patients could be solubilized efficiently by treatment with detergents (e.g., NP40, Triton X-100, etc.). Of the 108 blood samples tested, 23 were found positive for falciparum parasitemia by microscopy and 39 by RIA. One sample from a patient with patent falciparum parasitemia and three with patent vivax parasitemia were negative by RIA. Ten of the samples positive only by RIA belonged to patients with recent malarial infection, as shown by microscopy. Thus, the RIA detected almost all of the patients with microscopic evidence of falciparum malaria. The proportion of false positives in the RIA test was low.

Plasmodium falciparum: assay of antigens and antibodies by means of a solid phase radioimmunoassay with radioiodinated staphylococcal protein A.

Human red blood cells (RBC) infected in vitro with Plasmodium falciparum were employed to prepare several types of antigens (sonicated, infected RBC and purified, sonicated merozoites and schizonts). These antigens, as well as control preparations derived from non-infected RBC, were used to coat plastic tubes, which were subsequently tested for capacity to bind anti-P. falciparum antibodies. Binding was detected by means of radio-iodinated staphylococcus protein A. Sera from patients with recent disease or patients who had a history of P. falciparum infection gave strong binding, while sera of normal individuals had only a low binding activity. Some of the antibodies in the positive sera were directed against RBC, since they could bind to tubes coated with normal RBC antigens and could be removed by absorption with RBC. The specificity of the P. falciparum antibodies was confirmed by inhibition tests: preparations derived from infected blood but not from normal blood inhibited the binding activity of the positive sera, to antigen coated tubes.

Relation of antigen-binding cells to immunological memory.

Priming of mice with a conjugate of HSA with sheep red cells induced a high level of memory to HSA, with very little antibody production ("pure priming"). HSA specific antigen binding cells in the spleens of the primed mice were assayed by means of a rosette technique, using HSA conjugated to donkey red cells. Rosette formation was almost completely inhibited by soluble HSA, thus confirming that the RFC were specific for this antigen. Spleens of primed mice contained up to 0.6% RFC, as compared to 0.08% HSA specific RFC in the spleens of non immunized animals. Suspensions enriched in rosettes (containing up to 16% RFC) were prepared by centrifugation on BSA density gradients. Adoptive transfer experiments showed that the rosette rich fraction contained all the memory cells. A marginal level of memory could be transferred to irradiated recipients with 3000 rosettes. A comparable degree of responsiveness to HSA could also be transferred with 70,000 RFC enriched from spleens of non immunized mice, but only when injected together with primed, RFC depleted spleen cells. Kinetic studies showed that the level of memory correlated well with the number of RFC up to two months after priming. The number of RFC decreased at later time intervals (though remaining higher than in controls at all times), without a corresponding decrease in the level of memory. A change in the quality of the memory cell with time is postulated.

Autoantibodies to red blood cells in rats infected with Plasmodium berghei.

A radio-iodinated Protein A (125SPA) binding assay was used to identify autoantibodies to red blood cells (RBC's) in sera of rats infected with Plasmodium berghei. Sera taken from rats at various times after infection were reacted with washed, normal RBC's, then the RBC's were washed and treated with 125SPA. The bound radioactivity was taken as a measure of the amount of Ig attached to the RBC's membranes. Using this test, anti-RBC autoantibodies were detected in rat sera as early as 5 days after infection. The level of the autoantibodies rose to a maximum at 12 to 14 days, at which time parasitemia was also maximal, then declined sharply. Autoantibodies were still detectable at 21 days after infection. Red blood cells from infected rats had readily detectable, membrane-bound Ig, as shown by their capacity to bind 125SPA directly. The amount of this Ig rose and fell in a fashion closely paralleling course of parasitemia.