CD66 identifies a neutrophil-specific epitope within the hematopoietic system that is expressed by members of the carcinoembryonic antigen family of adhesion molecules.

Preliminary results from the IVth Leucocyte Culture Conference have classified the monoclonal antibody (MoAb), YTH 71.3.2, as CD66. Two other MoAbs, YPC 2/12.1 and CE6/2D3.1, share a common cellular specificity, reacting with cells of the neutrophil series and colonic epithelium. The YTH 71.3.2 and CE6/2D3.1 MoAbs both recognize a similar CD66 defined epitope that is distinct from that identified by YPC 2/12.1. By Western blotting, these antibodies react with different molecular species from cells of different lineages. The antibodies identify 50- to 55-Kd, 80- to 100-Kd, and 130- to 200-Kd components present in a semi-purified carcinoembryonic antigen (CEA) preparation from colonic adenocarcinomas and a 90- to 130-Kd molecule from HL-60 cells. With the colonic cell line, LS174T, YPC2/12.1 stains diffuse bands of 160 to 200 Kd and 90 to 130 Kd with equal intensity, whereas the binding of CE6/2D3.1 and YTH 71.3.2 is biased toward the lower molecular weight set of molecules. Remarkably, all three antibodies recognize CEA-related molecules. Defined analyses using HeLa cells transfected with CEA, NCA(NCA-50/90), and CGM6(NCA-95) cDNAs show that the three MoAbs identify CEA to varying degrees. While YTH 71.3.2 and CE6/2D3.1 also bind to NCA-50/90, YPC 2/12.1 recognizes an epitope expressed by both the NCA-50/90 and NCA-95 molecular species.

Thyroid blood group isoantigen expression: a parallel with ABH isoantigen expression in the distal colon.

An interesting and not previously reported parallel has been observed between the known pattern of ABO (H) blood group isoantigen expression in normal and neoplastic colonic epithelium and that in the thyroid. Epithelial expression of blood group isoantigens was not observed in 16 specimens of normal or non-neoplastic thyroid tissue. This contrasts with the progressive re-expression of these antigens in neoplastic thyroid tissue. Blood group isoantigens were detected in two of eight papillary adenomas and 13 of 17 papillary carcinomas. Antigen expression was in part related to differentiation, and stained cells were less readily detected in follicular tumours, only one of five adenomas and two of seven carcinomas displaying blood group antigens while three medullary and two anaplastic carcinomas were antigen-deficient.

Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor.

Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.

The expression of ABH and Y blood group antigens in benign and malignant breast tissue: the preservation of the H and Y antigens in malignant epithelium.

The ABO(H) and Y antigen status of epithelial cells from 45 breast carcinomas, 14 benign breast lesions and 7 normal breasts have been assessed using an indirect immunoperoxidase histochemical assay and a series of blood group specific monoclonal antibodies. All 20 A, AB and B group tumours had lost the A and B isoantigens, 13 of these tumours were however found to express H and Y antigens. Of 25 group O tumours 17 expressed the expected H and Y antigens. These findings were not dependent on the histological nature or the invasive characteristics of the tumour. Similar results were obtained when 28 metastases from breast carcinomas were examined, the H and Y antigens being identified in the tumour elements in 24 lymph nodes while we failed to identify either the A or B antigens. The development of breast malignancy appeared therefore to correlate best with the deletion of A and B glycosyl transferases. Normal breast tissue consistently expressed the expected blood group isoantigens. Areas of benign breast disease showed a more varied pattern of antigen expression. Seven of 14 lesions lacked ABH antigens, the loss of blood group structures could not however be correlated with any specific histological features and was not limited to the loss of A and B substances.

Are blood group isoantigens lost from malignant prostatic epithelium? Immunohistochemical support for the preservation of the H isoantigen.

Previous studies while demonstrating the presence of blood group isoantigens on normal prostatic epithelium have failed to identify such antigens on malignant prostatic tissue. Using a series of blood group specific monoclonal antibodies directed towards the A, B, H and Y antigens we have reinvestigated blood group isoantigen expression in both benign prostatic hypertrophy and prostatic adenocarcinoma. Results obtained from areas of benign prostatic hypertrophy are in broad agreement with those published however though we were unable to detect either A or B blood group isoantigens Type 2H and Y isoantigens were identified in 10 of the 12 tumours. These findings, while differing from previously reported results, lend support to the suggested connection between ontogenesis, oncogenesis and blood group isoantigen expression and also support the proposed link between Type 2 structures and malignant transformation.

Monoclonal antibodies for purification and assay of IL-2.

Mouse monoclonal antibodies (Mac 002 and Mac 003) raised against recombinant human Interleukin-2 (rec IL-2), were developed for use as assay and purification reagents. In the Immunoradiometric assay, (IRMA), 125I-Mac 002 is used as tracer, with sheep polyclonal anti-rec IL-2 on the solid phase. This reliably measures rec IL-2 in the range 3-1000 ng/ml. The assay measures natural IL-2 with a lower sensitivity. For some samples of IL-2, the amount detected by IRMA is greater than expected from the biological assays, presumably because there are IL-2 molecules with antigenic, but not biological activity. This is a possible source of variation in the specific activities observed in different preparations of of IL-2. In the purification reagent, Mac 003 is immobilised on sepharose CL-4B to purify recombinant IL-2 from less than 1% in an E. Coli extract, to greater than 90% purity, in a single step with greater than 80% yield.

A human-human monoclonal anti-D by direct fusion with a lymphoblastoid line.

The human lymphoblastoid cell line W1-L2-729-HF2 has been fused with B cells from a plasmaphoresed anti-D donor immunized with D+ cells. A stable monoclonal antibody-producing cell line has been produced which yields culture supernatant of good titre without the need for concentration. The production and use of this reagent as an alternative Rh D typing reagent for use by saline and enzyme manual tests and automated tests in a Technicon 16C machine is discussed. Du red cells are detected in enzyme enhanced tests.

Differing reactions of monoclonal anti-A antibodies with oligosaccharides related to blood group A.

Inhibition radioimmunoassays with blood group A-related oligosaccharides have been used to investigate the specificities of six monoclonal anti-A antibodies, three of which had been intentionally generated by immunization of mice with blood group A erythrocytes and A-active blood group substance, and three were incidentally produced following immunization of mice with human tonsil cell membranes or a human colon cancer cell line. By hemagglutination, these antibodies are highly specific for human blood group A erythrocytes. However, they differ from one another in their reaction patterns with mono- and difucosyl A antigen structures and the corresponding afucosyl sequences on Type 1 and Type 2 backbone structures. The six antibodies, together with four previously characterized anti-A monoclonal antibodies (originally raised against the receptor for epidermal growth factor) have been classified into five groups. The first two groups consist of antibodies with broad specificities for A-related structures. There are five antibodies in the first group (TL5, 29.1, A17/3D1, MH2/6D4, and MH1/5D1) reacting to varying degrees with the mono- and difucosyl A antigen structures on either type of backbone sequence. In the second group are two antibodies (A15/3D4 and A15/3D3) which are difficult to inhibit with the oligosaccharides tested, but they reacted best with monofucosyl A structure on either type of backbone. Each of the remaining three antibodies had a distinct and more restricted reaction pattern, with a specificity for the difucosyl A antigen on both types of backbone (antibody EGR/G49) or the Type 1-based mono- and difucosyl A antigen structures (antibody MAS 016c) or the Type 2-based monofucosyl A antigen structure (antibody 455). The reactions of four of the antibodies with N-acetylgalactosamine or with oligosaccharides containing the afucosyl sequence GalNAc alpha 1-3Gal suggest that they may react with certain glycoconjugates with alpha-N-acetylgalactosaminyl termini ("A-like" structures) that are unrelated to the products of the blood group A gene-specified alpha-N-acetylgalactosaminyl-transferase. Knowledge of the differing reactions of these monoclonal antibodies is important for interpreting their reactions with glycoproteins and glycolipids of diverse origins.

A radioimmunoassay for wheat gliadin to assess the suitability of gluten free foods for patients with coeliac disease.

Coeliac disease is a clinical condition characterised by malabsorption secondary to abnormalities of the small intestine. The condition is known to be exacerbated by wheat gliadin, rye, barley and possibly oats. The only assays that are available for testing for the presence of wheat gluten in foods are double diffusion against rabbit anti-gliadin antiserum and measurement of Kjeldahl nitrogen in products derived from wheat flour. We have developed a radioimmunoassay for wheat gliadin with a detection limit of 1 ng. Nominally gluten free foods based on wheat starch have been shown to contain up to 1.9 X 10(-2)% wheat gliadin. Bread made from Nutregen wheat starch which has now been withdrawn contains 6.4 mg gliadin per standard 30 g slice. A radioimmunoassay for wheat gliadin could be used to define standards for the suitability of gluten free products based on wheat starch for patients with coeliac disease.

Relation of antigenic structure of cereal proteins to their toxicity in coeliac patients.

Unfractionated gliadin and its alpha-, beta-, gamma- and omega-gliadin subfractions were used as rabbit immunogens. The antisera were characterized by (1) Ouchterlony double diffusion, (2) binding of 125I-labelled gliadin subfractions, (3) inhibition by several gliadin subfractions of binding between gamma-gliadin antiserum and 125I-labelled gamma-gliadin. Double diffusion showed identical cross-reactivity between the antisera and the gliadin subfractions with the exception of omega-gliadin. Precipitin lines of partial identity with gliadin were observed against rye secalins and barley hordeins but not oat avenins or maize zeins. Binding was observed between unfractionated 125I-labelled alpha-, beta-, gamma- and omega-gliadins and all the antisera. There was binding of 125I-labelled omega-gliadin to the omega-gliadin antiserum but poor binding of 125I-labelled omega-gliadin to unfractionated alpha-, beta- and gamma-gliadin antisera. Competitive inhibition of binding between 125I-labelled gamma-gliadin and gamma-gliadin antiserum diluted 1:250 (v/v) demonstrated similar competition between alpha-, beta- and gamma-gliadins and this antiserum but poor competition between omega-gliadin, wheat glutenins, albumins and globulins, rye secalins, barley hordeins and oat avenins. These findings suggest that there is a good correlation between the antigenic structure of gliadin proteins and their toxicity to patients with coeliac disease.

The control of transferrin receptor synthesis in mitogen-stimulated human lymphocytes.

Human lymphocytes cultured in the presence of the plant mitogenic lectin phytohemagglutin in (PHA) become activated and leave the G0 phase of the cell cycle. In the presence of PHA and lymphokines produced in situ the cells will enter S phase and undergo cell division. We have determined the time course of appearance for the receptor for transferrin as an initial attempt to understand the molecular mechanisms regulating the onset of lymphocyte differentiation and proliferation in the 48 h following PHA addition. Using three different assay methods we have shown that the increase in the number of surface receptor molecules is due to the accumulation of newly synthesized receptor and not to the redistribution of a previously existing pool of receptor molecules. The total amount of transferrin receptor increased at least four-fold. In vitro translation of RNA from activated lymphocytes indicates that the new receptor synthesis is due, at least in part, to increased availability of mRNA encoding the transferrin receptor.

Clinical testing of gliadin fractions in coeliac patients.

Since the toxic fraction of cereal flour which damages the small bowel mucosa of patients with coeliac disease has not been fully defined in vivo, we studied the effect of intraduodenal infusions of different doses of unfractionated gliadin and of alpha-, beta-, gamma- and omega-gliadin subfractions on the morphology of multiple jejunal biopsies taken from two patients with treated coeliac disease. A dose-response study with increasing quantities of unfractionated gliadin in one coeliac patient showed that 1000 mg produced marked damaged in serial jejunal biopsies taken 2-3 h after commencing the infusion and that the changes had almost completely disappeared 72 h later. alpha-, beta-, gamma- and omega-gliadin were prepared, checked for purity and investigated for toxicity in two coeliac patients. After an intraduodenal challenge with 1000 mg of the four gliadin subfractions these were shown to have induced damage in the mucosa of jejunal biopsies taken 6 h later. These observations confirm the results of studies in vitro, which suggest that not only alpha-but beta-, gamma- and omega-gliadin are enterotoxic in coeliac disease.

A new monoclonal anti-A. Culture supernatants with the performance of hyperimmune human reagents.

By proper selection for good growth and high avidity, we have prepared a new anti-A monoclonal antibody producing cell line that gives culture supernatants as potent as US-licensed commercial hyperimmune human reagents and which meet USA FDA standards without the need for concentration. The production and use of this reagent is cost effective and makes it a candidate to replace conventional anti-A typing reagents.

A radioimmunoassay for alpha- and beta-gliadins.

1. A rapid, sensitive specific radioimmunoassay for alpha- and beta-gliadin has been developed using an antiserum raised in rabbits to A-gliadin, a component of alpha-gliadin. 2. The antigen used in the assay was alpha-gliadin labelled with 125I; antigen-antibody complexes were collected after adsorption to Staphylococcus aureus in suspension. 3. The sensitivity of the assay, as judged by competitive binding with unlabelled antigen, was 1 ng of alpha- or beta-gliadin, which show complete cross-reaction with this antiserum. 4. Cross-reactivity to other wheat proteins was less than 1% and no cross-reactivity to extracts of rye, barley or oats was observed. 5. This radioimmunoassay for alpha- and beta-gliadin has been used to measure their amount in different varieties of wheat flour, several foods prepared from flour, e.g. bread, biscuits and products prepared as 'gluten free'. The possibility of assaying for alpha-gliadin in prepared foods is of special value since alpha-, beta-, gamma- and omega-gliadin have been shown to exacerbate coeliac disease.

Human blood group isoantigen expression on normal and malignant gastric epithelium studied with anti-A and anti-B monoclonal antibodies.

Variation in human blood group isoantigen expression on normal and malignant gastric epithelium was demonstrated with monoclonal antibodies to blood groups A and B in an indirect immunoperoxidase technique. The expected isoantigen expression was demonstrated on endoscopic biopsy specimens of normal gastric mucosa from 11 patients. Of 17 patients with gastric carcinoma (blood group A, 15; blood group AB, 2), complete loss of isoantigen expression was noted in 6 (35%). In these 6 patients, blood group isoantigen remained both in the adjacent uninvolved mucosa and at the margin of resection. The loss of isoantigen did not appear to be related to the degree of differentiation within the tumor, to the secretor status of the patient, or to the blood subgroup. Lymph node metastases reflected the isoantigen status of the primary tumor, being positive in 5 of 6 expression in all 17 patients or in an additional 15 patients studied with blood group O. These findings were discussed in the light of previously reported work on the localization of blood group isoantigens on malignant and nonmalignant gastric epithelium with the use of conventional antisera and a variety of immunohistologic techniques.

A monoclonal anti-HLA antibody recognizes a mouse tumor-associated antigen.

The monoclonal antibody W6/32.1 recognizes a public determinant on the HLA-A, B and C antigens of all tested human haplotypes. Though the antibody does not bind to normal mouse cells of any H-2 haplotype, it does show an unexpected specificity for the T cell leukemia line MBL-2 from a C57BL/6 mouse. It is shown that the murine antigen recognized by W6/32.1 is on an H-2-like molecule which also carries the determinant recognized by the monoclonal antibody B22-249 R1, specific for the H-2Db antigen. Unlike B22-249 R1, however, W6/32.1 does not bind to normal H-2b lymphocytes, nor to a variety of tumor cell lines of the H-2b haplotype. This cross-reaction is specific to W6/32.1, and is not shared by other monoclonal antibodies of similar anti-HLA specificities. Moreover, the affinity of W6/32.1 for its human antigen is substantially higher than for its mouse antigen. We conclude that W6/32.1 fortuitously recognizes a novel determinant on the H-2Db antigen of MBL-2, rather than an extensive region of structural homology shared between HLA and H-2. Thus for cells of the H-2b allotype this determinant is detected only on MBL-2, and by definition is thus an example of a tumor-associated antigen.

Localisation of metastatic carcinoma by a radiolabelled monoclonal antibody.

Rat monoclonal antibodies were prepared by immunising rats with human colorectal carcinoma cell membranes and fusing splenic lymphocytes with a rat myeloma. Hybridoma supernatants were screened by binding assays on membranes prepared from colorectal carcinoma tissue. One hybridoma supernatant, containing a monoclonal antibody with high binding activity on malignant compared to normal colon sections, was grown in large quantities in serum-free medium. After ammonium sulphate precipitation the antibody was purified by ion-exchange chromatography and labelled with 131I. Radiolabelled antibody was administered i.v. to 27 patients with colonic and other tumours. Scintigrams were obtained at 48 h. Computerised subtraction of the blood pool image revealed localised areas of uptake corresponding with areas of known disease in 13/16 patients with colorectal carcinoma and 3/4 patients with breast cancer.

How are tumour antigens related to normal antigens?

Tumour cells share with normal cells antigens characteristic of defined states of differentiation. Is there anything else? An example of tumour antigens not expressed anywhere in normal tissue is the set of tumour-specific transplantation antigens (TSTA) of murine chemically induced sarcomas. There is evidence that at least one TSTA specificity is retrovirus-derived, is carried on the envelope protein gp70, and probably arises by the recombination events that yield the diverse gp70s of the MCF strains of murine leukaemia viruses. Whether a similar mechanism can generate human tumour antigens depends on the yet unanswered question of whether human cells have retroviruses in their genomes capable of recombination. Aside from this, the only other mechanism known for antigen expression on tumours is via their oncogenes, which seem to make normal cell products. Such products, or secondary consequences of their production, were they normally expressed only at an early stage of development, would be candidates for 'fetal antigens'. While only the two mechanisms mentioned above seem the ready sources of 'tumour-associated antigens', it would be too early--in the face of ever more startling information about gene mobility and rearrangements--to think we have exhausted possible mechanisms for generating tumour antigens.