Single chain antibodies that recognize the N-glycosylation site

We aimed to identify antibodies that can recognize the Asn-Xaa-Ser/Thr(NXS/T) N-glycosylation site that guides oligosaccharyltransferase (OT) activity. We used synthetic Asn-Cys-Ser/Thr(NCS/T) tripeptides conjugated to bovine serum albumin to isolate single chain antibody fragments of a variable region (scFv) from the Griffin 1 phage antibody library. Although Ser and Thr have different side chains, the scFv proteins thus isolated bound to both NCS and NCT with Kd values of the order of 10(-6) M and accepted the substitution of the Cys residue with various amino acids, including Ala, Gly, and Val. However, these proteins recognized neither Asn-Pro-Ser/Thr nor non-NXS/T tripeptides. The scFv proteins recognized NCS/T and N-glycosylation site of mutant yeast protein disulfide isomerase when they were in their native but not denatured state. These results indicate that antibody recognition of the NXS/T motif is conformation dependent and suggest that NXS/T spontaneously adopts a specific conformation that is necessary for antibody recognition. These features are likely to correlate with the known binding specificity of OT.     

Development of monoclonal antibodies that recognize antigens associated with human cervical carcinoma

Six monoclonal antibodies, generated by immunization of mice with human cervical carcinoma cells maintained in tissue culture or with cells from fresh tumor tissue, reacted specifically with the malignant cells in 71% to 90% of the tumor tissue imprints and cervical smears containing neoplastic cells but not with normal cervical epithelial cells in smears from 21 to 23 healthy donors. Antibody CE 402 bound to epithelial cells associated with regeneration in 2 of the 23 normal smears tested. Considerable heterogeneity of antibody binding by malignant cells was observed. Antibody CE 400 was the most reactive, binding to more than 50% of the tumor cells in all reactive specimens. Five of these monoclonal antibodies detected protein antigens in the 80 K to 110 K molecular weight range. Our studies demonstrate the feasibility of producing monoclonal antibodies with selected specificity for cervical carcinoma. These antibodies may be of considerable diagnostic value.     

Monoclonal anti-idiotypic antibodies that recognize the binding site for nicotine on rat brain receptor

Anti-idiotypic monoclonal antibodies have been prepared that represent the internal image of nicotine and are specific for the nicotine binding site on rat brain receptor. Specificity of these antibodies for the combining site on anti-nicotine was demonstrated by their ability to inhibit binding of monoclonal anti-nicotine to immobilized nicotine-polylysine. Furthermore, purified rat brain nicotine receptor but not acetylcholine receptor from fish electric organ effectively competed with anti-nicotine for immobilized nicotine and for immobilized anti-idiotype. Only 9 pmoles of naturally occurring (-)-nicotine inhibited idiotype-anti-idiotype binding by 50% whereas 11 times more (+)-nicotine was required. Acetylcholine, several cholinergic agonists and antagonists, nicotine metabolites, and other structurally related compounds were poor inhibitors.     

Cross-reactivity of antigens and antibodies belonging to different pathogenic types of human papillomaviruses

The analysis of the properties of a quadrivalent peroral vaccine against the cervical cancer, which was created in a plant expression system on the base of transgenic tomato fruits, by immunoassay and Western blot hybridization showed that the antibodies against human papilloma virus 16 L1 (HPV16 L1) actively interacted not only with the antigenic proteins HPV18 L1, HPV31 L1, and HPV45 L1, but also with the antigenic protein HPV6 L1, which belongs to another HPV family. Thus, new data on the possibility of crossreactivity between antibodies and antigens belonging to remote HPV families were obtained.     

Toxoplasma gondii: characterization of monoclonal antibodies that recognize rhoptries

We have previously reported on a series of monoclonal antibodies that recognize the rhoptries of Toxoplasma gondii and that interfere with the action of penetration enhancing factor. The antibodies immunoprecipitate several related antigens from [35S]methionine-labeled parasites that range in size from 60 to 43 kDa. By immunoblot, one of the antibodies reacts with the 60 kDa protein in the presence of protease inhibitors. Trypsin digestion of the antigen destroyed antigenic reactivity indicating that the 60 kDa antigen is a protein. The antigen was stable to periodate oxidation and failed to react with Schiff's reagent, indicating that the antigen contains little or no carbohydrate. Two-dimensional gel electrophoresis followed by immunoblot showed that the antigen recognized by Tg 49 was an acidic protein with an approximate pI of 5.8.     

Antileishmanial IgG and IgE antibodies recognize predominantly carbohydrate epitopes of glycosylated antigens in visceral leishmaniasis

The specificity of human antileishmanial IgG and IgE antibodies to glycosylated antigens of Leishmania chagasi was evaluated. An ELISA was performed with soluble leishmanial antigen (SLA) and a panel of 95 sera including samples from patients with subclinical infection (SC) and visceral leishmaniasis (VL), subjects cured of visceral leishmaniasis (CVL), and from healthy individuals from endemic areas (HIEA). Antileishmanial IgG were verified for 18 (40%) of 45 SC subjects (mean absorbance of 0.49 +/- 0.17). All nine sera from VL patients had such antibody (0.99 +/- 0.21), while 11 (65%) of 17 CVL individuals were seropositive (0.46 +/- 0.05). Only three (12%) of 24 HIEA controls reacted in IgG-ELISA. Antileishmanial IgE was detected in 26 (58%) of 45 SC patients (0.35 +/- 0.14), and in all VL patients (0.65 +/- 0.29). These antibodies were also detected in 13(76%) of 17 CVL subjects (0.42 +/- 0.14) while all HIEA controls were seronegative. There was no correlation between antileishmanial IgG and IgE antibody absorbances. Mild periodate oxidation at acid pH of SLA carbohydrates drastically diminished its antigenicity in both IgG and IgE-ELISA, affecting mainly the antigens of 125, 102, 94, and 63 kDa as demonstrated by western immunoblotting.     

High incidence of antinuclear antibodies that recognize the matrix attachment region.

The matrix attachment region (MAR) is a distinctive genomic DNA involved in a variety of nuclear processes through association with the nuclear matrix. Recent studies suggest that nuclear matrix is altered in the process of apoptosis and presented to the immune system, leading to the production of autoantibodies against its protein components. To see whether MARs are also recognized by autoantibodies, a collection of human sera containing antinuclear antibodies was screened for the presence of binding activities against cloned MARs. We found that MAR-binding activities are quite common in these sera. There was a positive correlation among the MAR-binding titers for three different MAR probes. As expected, the MAR-binding activity was copurified with serum IgG, and subclass analysis with affinity-purified IgG on MAR-Sepharose showed a predominance of IgG2 isotype. Several lines of evidence implied that the anti-MAR antibodies detected here is distinct from the ordinary anti-DNA antibodies that are reactive to bulk DNA.     

Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten

Two mouse mAb specific for the hapten p-azophenylarsonate and encoded by the same combination of germ-line V, D, and J genes differ 200-fold in affinity for hapten. We determined the amino acid sequences of the V regions of the high affinity antibody and compared them to the published sequences of the low affinity antibody which is not somatically mutated. Of 19 amino acid substitutions, two, Ile57 and Thr58 in the H chain, also occur, either alone or together, in other somatically mutated antibodies specific for p-azophenylarsonate; these antibodies have been independently isolated. Introduction of either one of these mutations alone into the low affinity antibody by oligonucleotide-directed mutagenesis increased the antibody affinity for hapten three- to fourfold, whereas introduction of both mutations together conferred an eightfold increase in affinity. These results support the hypothesis that somatic mutations are selected on the basis of the affinity for antigen that they confer, and suggest that even relatively small increases in affinity may be selected, probably in a sequential manner.     

Generation of cloned calves from different types of somatic cells

Six types of bovine somatic cell lines, including a granulosa cell line of Chinese red-breed yellow cattle (YGR), a granulosa cell line of Holstein cow (HGR), two skin fibroblast cell lines of two adult Holstein cows respectively (AFB1 and AFB2), a skin fibroblast cell line (FFB) and an oviduct epithelial cell line (FOV) of a Holstein fetus, were established. Somatic cell nuclear transfer (SCNT) was carried out using these cells as nuclei donor, and a total of 12 healthy calves were cloned. The effects of different types of donor cells on developmental potential of bovine SCNT embryos were investigated, (i) There was no significant difference in development rates to the blastocyst stage for SCNT embryos from YGR and HGR (33.2% and 35.1%, respectively). Pregnancy rates of them were 33.3% and 30.2%, respectively; and birth rates were 16.7% and 11.6%, respectively, (ii) Development rates to the blastocyst stage for SCNT embryos from diffetent individuals (AFB1 and AFB2) differed significantly (27.9% and 39.4%, respectively, P < 0.05). Pregnancy rates of them were 36.2% and 36.4%, respectively; and birth rates were 14.9 % and 27.3%, respectively, (iii) There was significant difference in development rates to the blastocyst stage for SCNT embryos from FFB and FOV of the same fetus (37.9% and 41.5%, respectively,P < 0.05). Pregnancy rates of them were 45.7% and 24.1%, respectively; and birth rates were 22.9 % and 10.3%, respectively. Finally, developmental potential of bovine SCNT embryos from all four types of somatic cells from Holstein cows (HGR, AFB, FFB and FOV) were compared. Forin vitro development stage, development rates to the blastocyst stage for SCNT embryos from HGR, AFB, FFB and FOV were 35.1%^A, 29.4%^B, 37.9%^A and 41.5%^C, respectively (P ^ABC < 0.05); forin vivo development stage, pregnancy rates of them were 30.2%, 36.2%, 45.7% and 24.1%, respectively; and birth rates of them were 11.6%, 17.2%, 22.9% and 10.3% respetively.     

Bi-specific monoclonal antibodies: selective binding and complement fixation to cells that express two different surface antigens

A new dimension of immunotherapeutic selectivity might be achieved if antibodies could distinguish cells that co-express two different surface antigens. Bi-specific monoclonal antibodies (BSMAB) with two different antigen combining sites that share a common Fc region theoretically might have such a potential. Two such BSMAB were produced by hybrid-hybridoma clones prepared by fusion of pre-existing hybridomas and were purified by isoelectric focusing. CD3,4 (IgG2a, IgG2b) recognizes the T cell surface antigens CD3 and CD4, and CD3,8 (IgG2a, IgG2a) recognizes CD3 and CD8. These BSMAB promote complement-mediated lysis of target cells that bear both surface antigens 25 to 3125 times more efficiently than those that express only one of the antigens. This selectivity results from the increased avidity of these antibodies for cells with both antigens, as reflected by the increased surface immunoglobulin concentration detected by flow cytometry. It was also demonstrated that there exists a threshold surface immunoglobulin density necessary for antibody-dependent complement-mediated cytotoxicity microtiter assays for the various IgG antibodies tested in both bivalent and monovalent binding. These results support the associative model of IgG-mediated complement fixation.     

Characterization of monoclonal antibodies that recognize the Eimeria tenella microneme protein MIC2

The apicomplexan pathogens of Eimeria cause coccidiosis, an intestinal disease of chickens, which has a major economic impact on the poultry industry. Members of the Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes and dense granules) that mediate invasion of host cells and formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from Eimeria tenella(EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced against recombinant EtMIC2 proteins is described. All mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina and E. maxima in an immunofluorescence assay. By Western blot analysis, the mabs identified a developmentally regulated protein of 42 kDa corresponding to EtMIC 2 and cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2 related proteins in Eimeria species.     

Identification of somatic hybrids in plant protoplast fusions with monoclonal antibodies to plasma-membrane antigens

Monoclonal antibodies generated by immunization with a plasma-membrane preparation from suspension-cultured cells of Nicotiana glutinosa L. were used in combination with fluoresceinor rhodamine-labeled goat anti-mouse immunoglobulins to identify heterokaryons in protoplast fusion procedures. Antibody labeling did not inhibit callus formation nor plantlet regeneration. The antibodies are non-invasive and surface labeling provides clear optical discrimination of true heterokaryons from unfused aggregates as well as from parental protoplasts and homokaryons. Labeling is stable throughout fusion and hence by pre-labeling parental protoplast populations the strategy is both versatile and of general applicability.     

Antitubulin antibodies. II. Natural autoantibodies and induced antibodies recognize different epitopes on the tubulin molecule

Natural and induced antitubulin antibodies were compared for their epitope recognition on alpha- and beta-tubulin subunits by immunoenzymatic assays and Western blot techniques on partially digested tubulin molecules. Our results indicated that natural autoantibodies recognized different epitopes from those recognized by induced antibodies, because: 1) all polyspecific natural autoantibodies tested so far recognized the same or very overlapping epitopes in the central part of both alpha- and beta-subunits (between positions 100 and 300 on the tubulin amino acid sequence) and that this epitope differed from the various epitopes recognized by induced antitubulin antibodies on the amino-terminal or carboxy-terminal parts of the tubulin subunits; 2) one human myeloma protein (monoclonal (m)IgA, kappa) with a monospecific antitubulin activity bound to an epitope around position 310 on both alpha- and beta-subunits and a second human mIg (mIgM, kappa) with a monospecific anti-beta activity bound to an epitope on the carboxy-terminal part of the subunit around amino acid position 350. Both epitopes differed from epitopes recognized by induced antitubulin antibodies. These results thus confirmed our previous findings indicating that natural and induced antitubulin antibodies do not share cross-reactive idiotopes.     

Monoclonal antibodies recognize localized antigens in the eye and central nervous system of the marine snail Bulla gouldiana

The eyes of the marine snail Bulla gouldiana act as circadian pacemakers. The eyes exhibit a circadian variation in spontaneous optic nerve compound action potential frequency in constant darkness, and are involved in controlling circadian rhythms in behavioral activity expressed by the animal. To initiate an investigation of the molecular aspects of circadian rhythmicity in the Bulla eye and to identify specific molecular markers in the nervous system, we raised monoclonal antibodies (MAb) to the eye and screened them for specific patterns of staining in the eye and brain. Several MAb recognize antigens specific to groups of neurons in the brain, whereas others stain antigens found only in the eye. In addition, some antigens are shared by the eye and the brain. The antigens described here include molecules that mark the lens, retina, neural pathways between the eye and the brain, specific groups of neurons within the central ganglia, and an antigen that is shared by basal retinal neurons (putative ocular circadian pacemaker cells) and glia. These molecular markers may have utility in identifying functionally related groups of neurons, elucidating molecular specializations of the retina, and highlighting pathways used in transmission of information between the retina and the brain.     

High yields of lethal mutations in somatic mammalian cells that survive ionizing radiation

When mammalian cells are irradiated in vitro, the component cells of a normal-appearing survivor colony or clone are commonly thought to have proliferative capacity equivalent to that of the unirradiated cells. We have found, however, that cells appearing in survivor colonies may carry heritable lethal defects which come to light, perhaps only after numerous successful divisions, in the form of plating efficiencies that are reduced below those of unirradiated cells in a dose-dependent manner. We regard these heritable defects as signs of the induction of lethal mutations, which, like non-lethal mutations, may require many generations before they are expressed. This effect has been noted in two very dissimilar mammalian cell lines, one a primary culture from adult tissue, the other an immortal cell line. We suggest that induction of lethal mutations may occur also in somatic cells in vivo; this would account for the well-known observation that previously irradiated but apparently healed tissue is subsequently proved to be extraordinarily sensitive to subsequent exposure to irradiation or cytotoxic drugs. The results of our experiments in vitro suggest that current methods of estimating mutation or transformation yields may yield underestimates. If lethal mutations are induced also in vivo, interpretations of the results of fractionation experiments on normal tissues may have to be reconsidered.     

Ordering of antibodies, that react with cardiac muscle fiber and interstitial connective tissue antigens, in different immunoglobulin classes

Sera of patients suffering from rheumatic diseases and myocarditis were examined on the sections of human and bovine myocardial tissue by indirect immunofluorescence with the use of pure IgG antibodies or monospecific sera against IgG, IgA and IgM. It was shown that antibodies reacting with different myofibers and interstitial connective tissue of the heart belong to the main immunoglobulin classes (IgG, IgA and IgM). There was a significant predominance of IgG antibodies as shown by the frequency of their detection and by the titer height. The predominance of antibodies to certain classes of immunoglobulins did not correlate with a specific disease entity. The frequency of detecting antibodies to a certain immunoglobulin class was in good agreement with the time of the disease onset. Moreover, the frequency of positive reactions due to IgG, IgA, and IgM antibodies correlated with the level of the appropriate immunoglobulins in the test sera.     

Monoclonal antibodies that recognize different membrane proteins that are deficient in Rhnull human erythrocytes. One group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.

1. Rhnull human erythrocytes lack all of the antigens of the Rh and LW blood group systems and have abnormal shape and an increased osmotic fragility. In this paper two murine monoclonal antibodies raised against intact human erythrocytes were used to investigate further the abnormalities in these cells. BRIC 125 reacts weakly with Rhnull erythrocytes and BRIC 69 does not react at all. The results showed that BRIC 125 reacts with a component of Mr 47,000-52,000 which has a substantial content of N-glycans. In contrast, BRIC 69 reacted with a band of Mr 31,000 together with a very diffuse band of Mr 35,000-52,000. Treatment of BRIC 69 immunoprecipitates with endoglycosidase F/peptidyl-N-glycosidase F resulted in the loss of both BRIC 69 reactive components and the appearance of a new band of Mr similar to that of the Rh(D) polypeptide. 2. BRIC 125 had a broad reactivity with cells in peripheral blood, whereas the reactivity of BRIC 69 was confined to erythrocytes. BRIC 125, but not BRIC 69, reacted with human kidney tissue and bound to endothelium in peritubular capillaries, arteries and veins as well as the epithelial tissue of distal tubules. BRIC 125 stained haemopoietic cells, foetal hepatocytes and megakaryocytes in foetal liver and sinusoidal cells, hepatocytes and portal tracts in adult liver. In contrast, BRIC 69 reactivity was confined to haemopoietic cells in foetal liver. The BRIC 125 epitope has a wide tissue distribution, suggesting the occurrence of a related group of polypeptides which have a general functional role on cell surfaces. 3. Rhnull erythrocytes are deficient in at least four different membrane polypeptides.