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Minor H (histocompatibility) loci govern immune responses causing slow skin-graft rejection and graft versus host disease in major histocompatibility complex (MHC)-compatible animals. It is generally assumed that minor H antigens represent allelic variants of normal proteins and that peptides bearing the allelic amino acid substitutions are presented by MHC molecules in the context of which they may be recognized by H-allotype-specific T cells. 24 refs., 1 fig., 2 tabs.

A genomic probe of the human RT6 gene detects a frequent biallelic BglII polymorphism. Allele A has a frequency of 63%, whereas that of allele B is 37%. This restriction fragment length polymorphism provides the first known genetic marker for this gene.

T cells expressing the RT6 surface alloantigen perform important immunoregulatory functions in the rat. Diabetes prone (DP) BB rats are deficient in circulating RT6+ T cells and develop spontaneous autoimmune diabetes mellitus. Transfusions leading to engraftment of RT6+ T cells prevent the disease. Coisogenic diabetes resistant (DR) BB rats do circulate RT6+ T cells and are free of disease. We investigated the basis for the deficiency of RT6+ T cells in the DP-BB rat and made the following observations. 1. Thymectomy causes the rapid loss of most peripheral T cells in the DP-BB rat. 2. Concomitant with the loss of T cells is the total loss of mRNA encoding RT6. 3. In contrast to the effects observed in peripheral lymphoid tissues, thymectomy does not lead to a detectable loss in RT6+ protein found in the small intestine. We conclude that the deficiency of RT6+ peripheral T cells in the DP-BB rat is due either to their short life span or to their reduced proliferative capacity following release from the thymus.

Abstract 5087 Background: Cancer-testis antigens (CTA) are attractive targets for cancer immunotherapy based on their tumor-restricted expression and immunogenicity. A number of CTA, including Melanoma-associated antigen 3 (MAGE-A3), are already under clinical investigation and CTA have been shown to induce strong T cell and humoral immunity in cancer patients receiving active immunotherapy. However, little is known about the fine specificity and the function of vaccine-induced humoral immune responses and it is unclear how they relate to spontaneous CTA-specific immune responses occurring in a minority of patients. Methods: We have performed a longitudinal analysis of spontaneously occurring antibody responses against the CT antigen MAGE-A3 in sera (N=1537), which were collected from patients with multiple myeloma (N=355) over a period of 6 years. Antibody titers were determined by ELISA technique and a B cell ELISPOT assay was applied to estimate the number of MAGEA3-specific memory B cells in peripheral blood of the patients. Fine specificity of the antibody responses was examined using overlapping 20mer peptides spanning the whole sequence of MAGE-A3. The given IgG subtype was determined, and the quality of MAGE-A3-specific antibodies was analyzed using western blot as well as affinity assays. Results were compared to those obtained with MAGE-A3-specific antibody responses induced by vaccination with full-length MAGE-A3 protein and adjuvants AS02B or AS15 in patients with non-small cell lung cancer (NSCLC; N=15). Results: Out of 355 myeloma patients 4 (1.1%) evidenced spontaneous antibody responses against MAGE-A3 at least at one point during the course of their disease. Spontaneously occurring anti-MAGE-A3 humoral responses were usually of low titer. In contrast, all of the vaccinated patients showed high-titered and persisting antibody responses which usually appeared around week 6 after the first application of the vaccine. Accordingly, we found high frequencies of vaccine-induced MAGE-A3-specific memory B cells in the peripheral blood of NSCLC patients while they remained undetectable in most myeloma patients. Vaccine-induced antibody responses underwent affinity maturation reaching affinity levels of spontaneous immune responses after repeated cycles of treatment. MAGE-A3-specific antibodies consisted of IgG1 and IgG3>IgG2>IgG4 subtypes in vaccinated patients whereas spontaneously occurring antibodies were mainly of the IgG2 subtype. Spontaneous as well as vaccine-induced IgG antibodies both recognized the natural full-length protein. Analysis of the fine specificity of the antibody responses revealed that vaccine-induced antibodies recognized a much larger number of MAGE-A3 epitopes than spontaneously occurring antibodies. However, both, spontaneous as well as vaccine-induced responses, most frequently and strongly recognized a specific region within the MAGE-A3 protein corresponding to amino acids 51–70. Conclusions This study demonstrates for the first time important qualitative differences between spontaneously occurring and vaccine-induced antibody responses against the MAGE-A3 antigen in cancer patients. While the potential of both types of antibody responses to promote antigen uptake and induction of T cell responses by antigen-presenting cells might differ, they both recognized the same restricted region within the MAGE-A3 protein. The latter finding might be of importance for the design of future immunotherapies targeting MAGE-A3. Disclosures: No relevant conflicts of interest to declare.

T-cells expressing the RT6 surface alloantigen appear to perform important immunoregulatory functions in the rat. Diabetes-prone BB rats lack circulating RT6 + T-cells and spontaneously develop autoimmune diabetes mellitus and thyroiditis. The coisogenic diabetes-resistant BB rat does circulate RT6 + T-cells and is free of disease. Transfusions leading to engraftment of RT6 + T-cells prevent both diabetes and thyroiditis in the diabetes-prone rat. To investigate the absence of this subset in the lymphopenic BB rat, we used both molecular and biochemical procedures and made the following observations: 1 ) an mRNA encoding RT6 protein is present in diabetes-prone spleen cells; 2 ) nucleotide sequencing of this transcript reveals an intact coding sequence for the RT6.1 alloantigen; 3 ) sensitive chemiluminescent assay of diabetes-prone lymph node cell detergent extracts shows that diabetes-prone RT6 mRNA is translated in vivo; 4 ) quantitatively, diabetes-prone lymph node cells express ≤ 10% of the RT6.1 protein found on similar numbers of diabetes-resistant BB cells; and 5 ) finally, we obtained evidence of an intact phosphatidylinositol linkage of the molecule to the cell surface and successfully immunoprecipitated the phosphatidylinositol-linked protein with DS4.23 monoclonal antibody, indicating that the RT6.1 antigen is correctly processed and folded in diabetes-prone lymph node cells. We conclude that the near total absence of RT6 + T-cells in the diabetes-prone BB rat is unlikely to be because of a defect in RT6 gene expression per se. Defects in RT6 gene regulation or other cellular defects leading to premature cell death in the T-cell lineage, alone or in combination, may instead be responsible.

Abstract 1900 Cancer-testis (CT) antigens show an expression restricted to malignancies and the human germline among healthy tissues and, therefore, represent attractive targets for cancer vaccines. CT antigen SSX-2 is expressed in about 20% of multiple myeloma patients (MM), however, little is known about the occurrence of spontaneous humoral immune responses against SSX-2 in these patients. Moreover, no information is available regarding the functionality of anti-SSX2 antibody responses and a possible impact on the course of the disease. Screening 1098 peripheral blood samples and 200 bone marrow samples of 194 MM patients, as well as 100 healthy donors serving as controls, we found six patients (3%) to present with SSX-2 specific antibodies. When we mapped the target epitopes using overlapping peptides for the whole SSX-2 sequence, we found the antibodies to be exclusively directed against amino acids 81–90. Remarkably, all antibodies were of the IgG3 subtype. In addition, SSX-2 antibodies were strictly antigen-specific and represented potent activators of the complement system. Correlating the antibody responses with clinical events, we found that the majority (5 out of 6) of seropositive patients had been antibody-negative before allogeneic stem cell transplantation (alloSCT) and had only developed anti-SSX2 antibodies after transplantation. Donor-derived antibody responses increased with depth of remission and, in case of recurrence of the disease, dropped to low or undetectable levels. Our data suggest that alloSCT is able to induce immune responses against myeloma specific SSX-2 antigen which is of low immunogenicity under normal conditions. Such antibody responses are of an effective phenotype and seem to correlate with protection from disease recurrence. To further characterize the biological role of transplantation-induced anti-SSX2 immune responses, we will analyze our patients for the presence of SSX-2-specific T cells which might be induced together with serological responses in the framework of an integrated immune response. Active immunotherapy might contribute to amplifying and shaping anti-SSX2 immune responses in myeloma patients, particularly after alloSCT. Disclosures: No relevant conflicts of interest to declare.

Seven to 55% of ischemic strokes amoung young adults remain etiologically unclaryfied. Lechat and coworkers (N Engl J Med 1988; 318:1148–52) recently had focussed attention on paradoxical embolism via the patent foramen ovale as a more important cause for stroke in the young than previously thought.

RT6 is a 25–30kd phosphatidylinositol-linked membrane protein of T cells. Recently, we have obtained cDNA clones encoding rat and mouse RT6 allelic variants. The RT6 antigens are encoded by a single gene locus in rats and by two closely linked gene loci in mice. RT6 allelic gene products of rats and mice are unusual in that they are highly polymorphic. Conspicuous features of the mutations suggest that they may be of functional significance.

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.

RT6 is an alloantigenic system expressed on the majority of mature peripheral T cells of the rat, encoded by two highly polymorphic alleles (RT6a and RT6b) of a single copy gene located on chromosome 1 in linkage with the genes for the β chain of hemoglobin (Hb) and tyrosinase (Tyr).1 We have determined the structure of the RT6 gene and identified two independent promoter regions (promoters 1 and 2) associated with exons 1 and 2, respectively, and shown that transcription of RT6 in lymphocytes of adult rats is mainly mediated by promoter 22 (see also Kuhlenbaumer et al, this issue). The RT6 alloantigens are asymmetrically expressed, ie, the percentage of RT6+ peripheral T cells is consistently lower in RT6a than in age-matched RT6b rats. F1 animals from (RT6a × RT6b) crosses show a subpopulation of RT6.2+/RT6.1− T cells. We have previously shown this asymmetric expression to be due to a pretranslational mechanism.3 This finding led us to look for allelic differences within the cis-regulatory elements of the RT6 gene.

The two alleles of the rat T-cell differentiation alloantigen RT6 are highly divergent and their expression is distinctively regulated. While the majority of T cells of RT6a/RT6b heterozygous laboratory rats (Rattus norvegicus) expresses both alleles, a subpopulation expresses only RT6b. To identify cis-regulatory elements that potentially control monoallelic expression, we compared the sequences of both alleles. A striking difference is the presence or absence of a rodent identifier (ID) sequence in intron 7. All investigated inbred RT6a rat strains (n=7) had this integration, while it was absent in all investigated RT6b rats (n=9). An ID element was also identified at precisely the same integration site in one RT6 allele of the closely related species Rattus rattus. The ID elements of both species showed nucleotide substitutions characteristic of different subfamilies, and their flanking repeats differed in length, indicating that two independent integration events had occurred into the same site adjacent to a mammalianwide interspersed repeat. Analysis of the surrounding sequences did not disclose any motifs to explain preferential integration into one allele. Our data indicate that the RT6 alleles diverged about 1 myr ago and that the ID element integrated into the RT6a locus soon after this. We have previously shown that DNA methylation plays an important role in regulating monoallelic RT6 expression. The possibility that the ID element in the RT6a allele interferes with the required demethylation process and thus accounts for monoallelic expression is discussed.

A hybridoma producing the monoclonal antibody HIS42 was isolated from a fusion between spleen cells from a BALB/c mouse immunized with rat thymocytes and the fusion partner SP2/0. This antibody recognizes a minor subset of T cells in every haplotype of rat tested so far. The subpopulation of HIS42 positive T cells contains both CD4+ and CD8+ cells, in the same ratio as found in the peripheral T cell population. When bound to Sepharose beads, HIS42 induces T cell proliferation in the presence of interleukin-2. In contrast to lymph node T cells, a number of thymocytes were found to express HIS42 only in the cytoplasm or together with membrane expression. Most bright HIS42 surface labelled thymocytes were also positive for MRC OX-44, a marker predominantly identifying mature thymocytes. SDS-PAGE analyses of the membrane molecules immunoprecipitated by HIS42 show two bands on unreduced gels. One of these bands (85 kd) runs as two separate bands at 35 and 48 kd on reduction. The other much weaker broad band (approximately 100 kd) is hardly affected by reduced conditions. Taken together these data suggest that HIS42 is directed against a determinant on the rat T cell receptor for antigen, which is common to a small number of T cells.

A single-step immunoaffinity purification procedure for the rat T cell marker RT6.2 is described which permits the isolation of microgram quantities of protein from the RT6.2 + T-T hybridoma EpD3. The amino terminus was sequenced directly from a polyvinylidine (PVDF) membrane blot prepared after SDS-PAGE. Further internal sequence data were obtained from peptides generated from purified RT6.2 digested with different endoproteases and separated by reverse-phase micro-HPLC. A computer search in data banks did not reveal any significant homology to other proteins.

Signal-peptide-peptidase-like 2A (SPPL2a), an aspartyl intramembrane protease, has been implicated in the proteolysis of TNF-alpha, Fas Ligand and Bri2. Here, we show that endogenous SPPL2a – in agreement with overexpression studies – is localised in membranes of lysosomes/late endosomes. Furthermore, we have analysed the molecular determinants for lysosomal sorting of SPPL2a by creating chimaeric constructs between SPPL2a and its plasma membrane localised homologue SPPL2b. Lysosomal transport of SPPL2a critically depends on its cytosolic carboxyterminal tail. A canonical tyrosine-based sorting motif of the YXXø type at position 498 is sufficient to direct SPPL2a to lysosomal/late endosomal compartments. This motif accounts for the differential localisation of the homologous proteases SPPL2a and SPPL2b and thereby influences the access to substrates and biological function of SPPL2a.Structured summary of protein interactionsLAMP2 and SPPL2acolocalize by fluorescence microscopy(view interaction)

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7.

Following their release from cells, ATP and NAD, the universal currencies of energy metabolism, function as extracellular signalling molecules. Mammalian cells express numerous purinoceptors, i.e., the nucleotide-gated P2X ion channels and the G-protein-coupled P2Y receptors. Signalling through purinoceptors is controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides. These enzymes include ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) and ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP, CD203 family). Investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize these proteins in their native conformation. This study reports the use of genetic immunization to generate such antibodies against P2X(1), P2X(4), P2X(7), ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6. Genetic immunization ensures expression of the native protein by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs). Such antibodies are especially useful for immunofluorescence and immunoprecipitation analyses, whereas antibodies against synthetic peptides usually function well only in Western-blot analyses. Here we illustrate the utility of the new antibodies to monitor the cell surface expression of and to purify some key players of purinergic signalling.

Arginine adenosine-5'-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD(+) to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field.

ADP-ribosylation is a post-translational modification regulating protein function in which amino acid-specific ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD onto specific target proteins. Attachment of the bulky ADP-ribose usually inactivates the target by sterically blocking its interaction with other proteins. P2X7, an ATP-gated ion channel with important roles in inflammation and cell death, in contrast, is activated by ADP-ribosylation. Here, we report the structural basis for this gating and present the first molecular model for the activation of a target protein by ADP-ribosylation. We demonstrate that the ecto-enzyme ART2.2 ADP-ribosylates P2X7 at arginine 125 in a prominent, cysteine-rich region at the interface of 2 receptor subunits. ADP-ribose shares an adenine-ribonucleotide moiety with ATP. Our results indicate that ADP-ribosylation of R125 positions this common chemical framework to fit into the nucleotide-binding site of P2X7 and thereby gates the channel.

Además de los anticuerpos convencionales, los tiburones desarrollaron anticuerpos compuestos por la cadena polipeptídica pesada denominados IgNAR, el fragmento variable de éstos se designa como vNAR y constituye los dominios de anticuerpo más pequeños capaces de reconocer a un antígeno. El largo inusual de su CDR3, les permite penetrar en las cavidades de los sitios activos de las enzimas e inhibirlas. Considerando esta característica, uno de los objetivos de este proyecto consistió en aislar anticuerpos de dominio sencillo del tiburón Heterodontus francisci, a partir de una biblioteca inmune específica, capaces de unirse a la enzima CD38 murina. La proteína CD38 actúa como receptor en la sinapsis inmunológica, y como enzima está involucrada en la degradación extracelular del NAD+ . Clínicamente esta proteína se utiliza como marcador de prognosis en la Leucemia Linfocítica Crónica. En este proyecto se generaron y aislaron vNARs que específicamente se unen a CD38 murino. A partir de un tiburón inmunizado se obtuvo una biblioteca de vNARs altamente diversa, después de varias rondas de selección se aislaron 16 familias. Las proteínas recombinantes así como los fago-anticuerpos fueron utilizados para probar su actividad de reaccionar específicamente contra CD38 en un ensayo de ELISA, los resultados revelaron que todas las familias con excepción de una eran capaces de unirse a CD38. A pesar de esto, ninguno de los vNARs aislados fue capaz de inhibir la actividad hidrolítica de la enzima. Se realizaron ensayos de FACS sobre células que expresaban CD38 en su membrana, como células de bazo de ratón y células de linfoma. De esta forma se identificó que los vNAR tenían una capacidad baja de unirse a células de bazo de ratón, pero se unían de manera favorable a las células de linfoma EL4-R (CD38pos). Hasta el momento se sabe que la familia SAGTK es la que mejor reacciona contra la enzima CD38 de ratón. El segundo objetivo del proyecto, fue generar anticuerpos capaces de reconocer moléculas de tipo vNAR, para ello se inmunizaron tres ratones con un vNAR purificado. Los ratones inmunizados desarrollaron una respuesta inmune específica, por lo que se utilizó el bazo y los nodos linfñaticos de uno de ellos para generar anticuerpos monoclonales. De la fusión celular se aislaron clonas capaces de reconocer una variedad de vNARs, después de subclonar las células fusionadas, se aislaron los anticuerpos monoclonales 370 y 533 ambos con un isotipo IgG1k. Usando los anticuerpos monoclonales (MAb) #379 y #533 en ensayos de ELISA y dot blot, se detectó específicamente diferentes vNARs y suero de tiburón. Además se evaluó si ambos MAb eran capaces de detectar alguna molécula presente en el suero del tiburón H. francisci, demostrándose que una molécula de aproximadamente 80 kDa fue inmuno-precipitada y detectada por Western blot utilizando el MAb 370. Otras dos proteínas del suero de tiburón fueron precipitadas, pero no detectadas por western blot, la primera con un peso molecular de 160 kDa y la segunda con un peso entre los 160 y 260 kDa. Todas estas moléculas están actualmente siendo analizadas por espectometría de masas para identificar la naturaleza de cada una de ellas, hasta la fecha se tiene la secuencia parcial de la proteína de 80 kDa que se confirmó como un IgNAR de H. francisci. Por otro lado, los MAbs 370 y 533 se utilizaron para determinar si en el curso de una inmunización de un tiburón H. francisci existían cambios en la especificidad del suero con el antígeno utilizado. Los resultados obtenidos utilizando el MAb 370 como método de detección en un ensayo de ELISA, muestran que después de 10 días posteriores a la inmunización, existe un reconocimiento específico al antígeno, dicha señal alcanza su punto más alto entre los días 26 y 40. Se demostró también que ante la falta de la administración de antígeno, la respuesta específica disminuye e incrementa de forma rápida ante una nueva administración. Los resultados obtenidos hasta ahora representan el primer acercamiento para investigar si existe una respuesta antígeno específica durante los procesos de inmunización del tiburón H. francisci y para determinar si existe maduración de la afinidad de los anticuerpos mediante un proceso dirigido por el antígeno. Besides conventional antibodies, sharks have evolved unusual antibodies compose only of the heavy chain, its variable domains are designated as vNAR, and are the smallest known antigen-binding antibody fragments exhibiting a high degree of chemical and thermal stability. Its unusual long CDR3 allows them to penetrate into the active site of enzymes and inhibit its activity, which makes them excellent enzymatic inhibitors. Therefore, one of the goals of this project was to isolate single domain antibodies from shark immune library specific for murine CD38 with binding and inhibitory activity. The CD38 protein has a dual function, as an adhesion molecule plays important roles in the immunological synapse and as enzyme, acts as an ecto-NADase, involved in extracellular NAD+ degradation. Clinically CD38 is a prognostic marker in Chronic Lymphocytic Leukemia. In this work we present the generation and isolation of vNARs that specifically bind to murine CD38. High diversity phage display library was obtained from immunized shark, with it several rounds of selection were performed and 16 vNAR families were isolated. Purified vNAR proteins and vNAR-phages were used to test their reactivity to CD38; revealing that all the families but one, specifically reacts with the enzyme. Although vNAR families bound to soluble CD38 protein, they were unable to inhibit its NADase enzymatic activity. However, to corroborate the binding capacity of isolated vNARs, FACS analysis were further accomplished on cells that express CD38 as cell-surface protein, a low reactivity was shown by vNAR-phages on mouse spleenocytes, whereas on CD38pos mouse lymphoma cell line EL4-R the reactivity was higher and specific. At the very end, the family SAGTK had a better binding reactivity to CD38. The second major goal of this project was to generate second reagents able to recognize vNAR molecules. Thee mice were immunized with a purified vNAR protein (SAALAK), a specific immune response against it was developed by the mice. To make monoclonal antibodies, the lymph nodes and spleen from one of the mice were taken and fused together with a myeloma cell line. From fused cells, positive clones reacting against a wide variety of VNAR molecules were isolated, after subcloning; two hybridoma cells #370 and #533 retained the ability to specifically react with vNARs. The isotype of both clones were determined as IgG1k. Monoclonal antibodies #370 and #533 specifically detect vNARs and shark serum by ELISA and dot blot. Furthermore, it was tested whereas MAbs 370 and 533 were able to detect any molecule present in the serum from horn shark Heterodontus francisci, and it was shown by an immunoprecipitation assay that a iv molecule of ~80kDa was specifically precipitated and detected by Western blot using the hybridoma #370. Two other shark serum´s proteins were precipitated but not detected by western blot (one of 160 kDa and other in between 160 and 260 kDa). Mass spectrometry analysis of all the identified molecules is currently underway, to determine the identity of them and confirm whereas the 80kDa molecule is indeed a IgNAR molecule. In addition, MAbs 370 and 533 were used to determine if during the course of an immunization protocol of one H. francisci shark, there are any changes in the specific reactivity of serum molecules with an specific antigen. The obtained results using MAb 370 to detect the reactivity indicated that there is an specific-antigen response developed by the immunized sharks then after 10 days post immunization and raising the higher point in between days 26 and 40 post immunization, it was also shown that in the absence of antigen, the levels of reactivity decrease, but soon after boost administration, the specific response increase. This represents the first attempts to investigate if there is a specificantigen response during the immunization of horn sharks and determine whereas an affinity maturation process is taking place by means of an antigen-driven process.