BR102018076342A2 - single chain variable fragment (scfv) of murine antibody in its recombinant form detects dhea hormone in human samples - Google Patents

Abstract

variable single-chain fragment (scfv) of murine antibody in its recombinant form detects DHEA hormone in human samples". hence the interest in developing a molecule capable of detecting the hormone. To this end, the hormone dhea was conjugated with klh and used as an immunogen to obtain hybridomas, of murine origin, anti-dhea. from hybridomas secreting specific immunoglobulin m (igm) for dhea, messenger rnas (mRNA) encoding the light and heavy chain of the igm antibody called 5b7 were obtained. this rnam was used as a template for the synthesis of complementary dna strand (cdna) which was sequenced and cloned into an expression vector of proteins. As a product, a recombinant protein was obtained consisting of the scfv that recognizes the dhea molecule in a very similar way to the parent molecule. proved to be efficient in detecting DHEA in normal adrenal tissue and in cac cells allowing tissue localization of DHEA.

Description

SINGLE-CHAIN VARIABLE FRAGMENT (SCFV) OF MURINE ANTIBODY IN ITS RECOMBINANT FORM DETECTS DHEA HORMONE IN HUMAN SAMPLES FIELD OF THE INVENTION

[001]. The present invention is related to the field of molecular biology applied to medicine and human health. This document highlights the amino acid sequence of the regions of complementarity of the light and heavy chains, in addition to a junctional peptide, of a recombinant murine monoclonal antibody molecule of IgM class capable of detecting the hormone dehydroepiandrostenedione (DHEA) in human samples. The variable single-chain antibody fragment (scFv), the subject of this document, after being expressed in its recombinant form, can be used for the detection of DHEA in cases of premature puberty and adrenal gland tumors in children, as well as in cases of hirsutism in adults.

FUNDAMENTALS OF THE INVENTION AND STATE OF THE TECHNIQUE

[002] . Dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) is a steroid hormone secreted primarily by the adrenal cortex, although the gonads, brain, adipose tissue, and gastrointestinal tract can also produce it. Easily converted to its sulfated metabolite, dehydroepiandosterone sulfate (DHEA-S), DHEA and DHEA-S are the most abundant circulating steroid hormones in the human bloodstream (1).

[003] In the course of human fetal development, the fetal zone represents about 85% of the adrenal cortex and is responsible for the high production of DHEA (2,3). Normally, within six months after giving birth, the concentration of DHEA and DHEA-S tends to decrease dramatically and start to increase again at the age of 6 to 8 years of age when the adrenarche period starts and the reticular layer of the zone of the adrenal cortex becomes the main source of DHEA (4).

[004] . DHEA-S and DHEA are very sensitive and reliable markers of functional pediatric adrenocortical tumors because they progressively increase when the tumor mass is still undetectable by imaging methods (5). Adrenocortical carcinoma (CAC) is an extremely rare malignant neoplasm in children around the world, but it occurs at least 20 times more often in southern Brazil due to the high frequency (0.27%) of a germline mutation (R337H) of the gene TP53 in the population (6,7).

[005] . The emergence of pulmonary, liver or widespread CAC metastases is an ongoing challenge, given the uncertainty or absence of adequate histopathological evaluation, and the limited tools available for diagnosis and treatment.

[006] . Considering the importance of DHEA and DHEA-S as markers for this type of tumor, a murine monoclonal anti-DHEA antibody of the IgM class was developed and characterized as being able to recognize DHEA in cells of the zona reticularis (RL) of supra -renal, one of the organs where the hormone is produced.

[007] . Subsequently, messenger RNA from the corresponding hybridoma was recovered, transformed into cDNA with primers from the hypervariable region of the antibody light (VL) and heavy (VH) chains. These sequences were cloned and sequenced in order to obtain the single chain antibody fragment or scFv (single chain variable fragment) in recombinant form.

[008] . This document, therefore, aims to protect the amino acid sequence of the complementarity regions (CDR) of the light and heavy chains of this anti DHEA scFv, as well as its use as a detecting agent for DHEA in human tissues.

[009] . When it comes to producing monoclonal antibodies that recognize steroid hormones, there are many commercial products available today. Furthermore, there are several studies, such as the ones mentioned below, that describe these molecules, including their use, but which differ, in terms of production, application and target, from the object of this document. Furthermore, in these articles there is no information on the amino acid sequence of the VH and VL domains of antibodies as highlighted in this document.

[010] . Kohen F, Lichter S, Eshhar Z, Lindner HR. Preparation of monoclonal antibodies able to discriminate between testosterone and 5 alpha-dihydrotestosterone. Steroids. 39, p. 453-459,1982.

[011] . Fantl VE, Wang DY. Characterization of monoclonal antibodies raised against testosterone. J Steroid Biochem. 19, p. 16051610, 1983.

[012] . Crichton D, Grattage L, McDonald A, Corrie JE, Steel CM, Hubbard AL, Al-Dujaili EA, Edwards CR. The production and assessment of monoclonal antibodies to cortisol. Steroids. 45, p. 503-517, 1985. doi 10.1016/0039-128X (85)90016-9.

[013] . White A, Gray C, Corrie JE. Monoclonal antibodies to testosterone: the effect of immunogen structure on specificity. J Steroid Biochem. 22 p. 169-175, 1985.

[014] . Gani M, Coley J, Piron J, Humphreys AS, Arevalo J, Wilson IA, Taussig MJ. Monoclonal antibodies against progesterone: effect of steroid-carrier coupling position on antibody specificity. J Steroid Biochem Mol Biol. v. 48(2-3) p. 277-282, 1994 https://doi.org/10.1016/0960-0760 (94)90156-2.

[015] . Pope A, Pritchard K, Williams A, Roberts A, Hackett JR, Mandecki W, Johnson KS. In vitro selection of a high affinity antibody to oestradiol using a phage display human antibody library. Immunotechnology. 2, p. 209-17, 1996.

[016] . Dörsam H, Rohrbach P, Kürschner T, Kipriyanov S, Renner S, Braunagel M, Welschof M, Little M. Antibodies to steroids from a small human naive IgM library. FEBS Lett. 414, p. 7-13, 1997. https://doi.org/10.1016/S0014-5793(97)00966-6

[017] . Tamate K, Charleton M, Gosling JP, Egan D, Ishikawa M, Fottrell PF, Kane MM. Direct colorimetric monoclonal antibody enzyme immunoassay for estradiol-17β in saliva. Clin Chem. v. 43, p. 1159-64, 1997. http://clinchem.aaccjnls.org/content/43/7/1159.

[018] . As for monoclonal antibodies that recognize the hormone DHEA, there are several trademarks of this product that are used for both research and laboratory diagnosis whose email addresses are listed below. In addition, there are published articles relating to the production of these antibodies. However, these studies do not reveal or do not have data on the amino acid sequence of the light chain (VL) and heavy chain (VH) regions of these molecules as the object of this document.

[019] . Email addresses of anti-DHEA monoclonal antibody suppliers:

[020] . https://www.mybiosource.com/prods/Antibody/Monoclonal/Dehydroep iandrosterone/DHEA/datasheet.php?products_id=1494886

[021] https://www.biocompare.com/9776-Antibodies/675868-anti-Dehydroepiandrosterone/?pda=9776 |675868_0_0 ||4|anti%20DHEA

[022] . https://www.biocompare.com/9776-Antibodies/644093-Mouse-AntiDehydroepiandrosterone-DHEA-Antibody-Unconjugated/?pda=9776 | 644093_0_0 ||6|anti%20DHEA

[023] . Articles describing the production of anti-DHEA monoclonal antibodies:

[024] . Parvaz P, Mathian B, Patricot MC, Garcia I, Revol A, Mappus E, Grenot C, Cuilleron CY. Production of monoclonal antibodies to dehydroepiandrosterone-sulphate after immunization of mouse with dehydroepiandrosterone-bovine serum albumin conjugate. J Steroid Biochem. 32, p. 553-558, 1989.

[025] . Lewis JG, Bason LM, Elder PA. Production and characterization of monoclonal antibodies to dehydroepiandrosterone sulfate: application to direct enzyme-linked immunosorbent assays of dehydroepiandrosterone sulfate and androsterone/epiandrosterone sulfates in plasma. Steroids. 61, p. 682-687, 1996.

[026] . Shrivastav TG, Chaube SK, Kariya KP, Kumari P, Singh R, Kumar D. Influence of different homologous and heterologous combinations of antibodies and enzyme conjugates of dehydroepiandrostosterone on the sensitivity and specificity of DHEA ELISA. J Immunoassay Immunochem. 32, p. 114-127, 2011. doi: 10.1080/15321819.2010.543221.

[027] . With regard to the production of scFv or Fab (Fraction antigen binding) antibody fragments that recognize steroid hormones, the articles below are examples of this study, however, the molecules differ in terms of target, mode of production and application regarding the amino acid sequence documented here .

[028] . Arevalo JH, Stura EA, Taussig MJ, Wilson IA. Three-dimensional structure of an anti-steroid Fab' and progesterone-Fab' complex. J Mol Biol. 231, p.103-118, 1993.

[029] . Kobayashi N, Shibahara K, Ikegashira K, Shibusawa K, Goto J. Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody. Affinity, specificity, and idiotype analysis. Steroids. 67, p. 733742, 2002. https://doi.org/10.1016/S0039-128X(02)00022-3.

[030] . Valjakka J, Takkinenz K, Teerinen T, Soderlund H, Rouvinen J. Structural insights into steroid hormone binding: the crystal structure of a recombinant anti-testosterone Fab fragment in free and testosterone-bound forms. J Biol Chem. 277, p. 4183-4190, 2002. doi: 10.1074/jbc.M 105579200.

[031] Kobayashi N, Kato Y, Oyama H, Taga S, Niwa T, Sun P, Ohtoyo M, Goto J. Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display. Steroids. 73, p. 1485-1499, 2008. doi: 10.1016/j.steroids.2008.08.009.

[032] Kobayashi N, Oyama H, Nakano M, Kanda T, Banzono E, Kato Y, Karibe T, Nishio T, Goto J. "Cleavable" hapten-biotin conjugates: preparation and use for the generation of anti-steroid single-domain antibody fragments . Anal Biochem. 387, p. 257-266, 2009. doi: 10.1016/j.ab.2009.01.004.

[033] . Patents listed below report the development of scFv antibody fragments that differ in the amino acid constitution, origin, specificity, reactivity and properties of the scFv described in this document.

[034] . BR 11 2017 018767 1 A2 Dimers of scfv-fc that bind transforming growth factor beta1 with high affinity, avidity and specificity. PI 9507594-1 Monoclonal antibody with humanized scFv, recombinant nucleic acid molecule and recombinant protein. WO2004041863A Single domain antibodies directed against interferon-gamma and uses therefor. US5844093A Anti-EGFR single-chain Fvs and anti-EGFR antibodies. US7598350B2 Human anti-epidermal growth factor receptor antibody. US20040259156A1 Bispecific immunoglobulin-like antigen binding proteins and method of production.

DESCRIPTION OF THE INVENTION

[035] . The invention to be protected by this document is characterized by being constituted by the amino acid sequences that compose the complementarity regions (CDR) of the light and heavy chains of a single chain antibody variable fragment (scFv) resulting from the cloning of the cDNA encoding an antibody murine monoclonal IgM. Its use is related to its ability to detect the hormone dehydroepiandrostenedione (DHEA) in human samples. This scFv can be used to detect DHEA in cases of premature puberty and adrenal gland tumors in children as well as in cases of hirsutism in adults.

DETAILED DESCRIPTION OF THE INVENTION

[036] . Preparation of DHEA Conjugates. DHEA-17-CMO (Steraloids Inc. USA) was coupled to keyhole limpet hemocyanin (KLH) snail hemocyanin based on the previously described protocol (8). Briefly, for 10 mg of DHEA-17-CMO, 400 µl of dioxane and dimethylformamide were added. To this solution, 100 µl of water containing 20 mg of NHS and 40 mg of EDAC was added. The resulting solution was mixed using vortexing and kept overnight at 4°C. The activated steroid was added slowly to the aqueous KLH solution (3.3 mg mL-1) with stirring. The DHEA-17-CMO-KLH conjugate was then dialyzed against 50 mmol L-1 phosphate buffered saline (PBS, pH 7.4) and centrifuged. The protein concentration of the supernatant was determined by the Bradford method. Aliquots were stored at -20°C until further use. DHEA conjugated to bovine serum albumin (DHEA: BSA) was obtained commercially (Steraloids Inc., USA).

[037] . Hybridoma generation. After approval by the animal ethics committee (CEUA-UFPR no. 643-2014). Four BALB/c mice were immunized with DHEA:KLH by subcutaneous injection (15 μg DHEA: KLH / animal) a total of 4 times at intervals three, two and one week respectively. Freund's complete adjuvant was used in the first immunization and incomplete in each subsequent immunization. Three days before cell fusion, DHEA-KLH (5 µg) in PBS was injected intravenously. Three days after the last immunization, splenocytes from the hyperimmunized mouse were fused with murine myeloma cells (P3X63Ag8.653 cell line) at a ratio of 5:1 and centrifuged. The cell pellet was washed twice with RPMI, resuspended and pre-warmed (37°C) 50% polyethylene glycol (PEG) 1500 added dropwise over a period of 1 min. Cells were then centrifuged at 20 °C for 5 min at 500 g. Hypoxanthine-aminopterin-thymidine (HAT) medium was added and cells were cloned by limiting dilutions. Hybridomas were allowed to grow for 15 days prior to screening by ELISA using DHEA: BSA, BSA, KLH and human serum albumin (HSA). Clones positive for DHEA: BSA but not for the other antigens were recloned and kept in culture until stabilization.

[038] . Anti-DHEA antibody isotyping and purification was performed using the hybridoma cell supernatant and IsoStrip® kit (11493027001, Roche Diagnostics). Antibodies from clone 5B7B3C6 were purified by a HiTrap IgM Purification HP column pre-packed with Sepharose High Performance (GE Healthcare Life Sciences).

[039] . Cloning of cDNAs encoding antibody V domains and in silico analysis. Total RNA was isolated from the subcloned hybridoma. Two independent batches were prepared to ensure accuracy. The cDNA encoding the IGHV domain was amplified using universal degenerate primers VHRevU (5'-GAG GTS MAR CTG CAG SAG TCW GG) and VHForU (5'-GAC AGT GGA TAR ACM GAT GG), the last starting for the gamma domain CH1 ( Fields et al., 2013). The primers IGH-Rev (5'-CA GGT CAA GCT GCA GCA GTC AGG) and GH-For (5'-AGA GTG GTG CCT TGG CCC CAG) were also used, which ring at the 5'end of the murine IGHV FR1 and in the 3' of IGHV FR4, respectively (9). In order to amplify the cDNA encoding the mouse IGKV domain, the VkForU primer was used which pairs the constant region of the kappa chain with one of each 5' kappa chain-specific primer (VkRev1 to 9) as recommended (10). PCR products were purified using the PCRapid Invisorb Spin kit (Invitek, Berlin, Germany). Sequencing of PCR products was performed and sequence accuracy was confirmed by amplified cDNA sequencing after cloning in pGEM-T Easy vector (Promega). The sequences were analyzed in silico using the IMGT tools of the Web interface and databases (IMGT / DomainGapAlign) (11).

[040] . Construction, expression and purification of scFv anti DHEA. The synthetic gene encoding the heavy chain variable of monoclonal antibody 5B7B3C6 fused to IGKV through a peptide linker (Gly4Ser) 3, containing a C-terminal His6 tag was designed with optimized codon sequence in Genscript (Piscataway, NJ) for prokaryotic expression and terminal restriction sites (Pst1/XhoI) chosen for cloning into pSW1 expression vector (Ward et al., 1989; Fields et al., 2013). The scFv was expressed in the periplasm of HB2151 bacteria grown in 2XTY medium with ampicillin (0.05 g / L) and induced at A600nm = 0.6-0.8 with IPTG (0.8 mmol.L-1) for 16 hours at 16°C. under agitation (75 rpm). After centrifugation (5000 g, 20 min at 4°C, periplasmic proteins were recovered after mild osmotic shock (TES buffer) and buffer exchange dialysis via dialysis against PBS (pH 7.4) Purification of (His-tagged) scFvs ) 6 was performed using nickel column chromatography The periplasmic extract (20 ml) was incubated with 0.5 ml of Ni2 + -NTA-Agarose (Qiagen, Les Ulis, Fran) for 2 hours After washing with PBS, pH 7.4, containing 20 mmol L-1 imidazole, bound proteins were eluted with 150 mmol L-1 imidazole in 0.5 mL fractions. The protein-containing fractions were pooled and extensively dialyzed against PBS, pH 7.4 The preparations were analyzed by SDS-PAGE on a 12.5% gel under non-reducing conditions.For Western blotting, the monoclonal antibody Tag 6x-His (Thermo Fisher Antibody MA1-21315) was used.This step was followed by incubation with an HRP-conjugated anti-mouse antibody antibody (A4416, Sigma-Aldrich, Saint-Louis, MO, USA). Immune complexes were then detected using chemiluminescence or diaminobenzidine/chloronaphthol substrate (GE Healthcare Life Sciences).

[041] ELISA. A polystyrene plate (Nunc™ MaxiSorp Thermo Scientific) was sensitized at 4 °C overnight with 100 µL of DHEA:BSA (0.312 to 5 µg.mL-1) in 0.1 mol.L-1 carbonate buffer, pH 9.6. Alternatively, BSA, KLH and HSA were used for sensitization to check for non-specific recognition. An amount of 150 μl of blocking solution (PBS / 2% BSA) was added and the microplate was incubated for 1 hour at 37°C. Then, monoclonal antibody 5B7B3C6 at different concentrations (2.5, 5, 10 and 20 μg /mL-1 in PBS / 0.5% BSA) was added to the wells and the microplate was incubated for 1 hour at 37° Ç. Bound antibodies were detected by the addition of anti-mouse antibody conjugated to HRP for 1 hour at 37°C. Finally, 100 μl of chromogenic substrate solution (50 mmol L-1 citrate-phosphate buffer pH 5.0, 0.2 mg mL-1 o-phenylenediamine and 0.02% (v/v) hydrogen peroxide ) have been added. After 20 minutes, the reaction was stopped with 20 ul of 1.0 mol L-1 sulfuric acid and the absorbance was read at 490 nm in an ELISA microplate reader (Bio-Rad 550). At least three washes in PBS/0.05% Tween 20 were performed between each step. The ELISA having scFv as primary antibody was performed under similar conditions of sensitization, blocking and addition of primary antibody (successive dilutions of periplasmic extract). Immunocomplexes were revealed after incubation with monoclonal antibody Tag 6x-His (Thermo Fisher Antibody MA1-21315) for 90 min at 37°C, followed by 1 hour of incubation with HRP-conjugated anti-mouse antibody antibody (A4416, Sigma, Saint-Louis, MO, USA) and colorimetric development using (50 mmol L-1 citrate-phosphate buffer pH 5.0, 0.2 mg mL-1 o-phenylenediamine and 0.02% (v/v) hydrogen peroxide). The reaction was stopped with 20 ul of 1.0 mol L-1 sulfuric acid and the absorbance was read at 490 nm in an ELISA microplate reader (Bio-Rad 550). At least three washing steps in PBS/0.05% Tween 20 were performed between each intermediate step.

[042] Surface plasmon resonance (SPR) analysis. The BIAcore T100 instrument and all reagents were obtained from GE Healthcare Life Sciences, Europe. BSA and DHEA:BSA were covalently linked to a CM5 sensor (approximately 2545 and 1306 RU, respectively). To screen for antibody-producing hybridoma, culture supernatants diluted 1:2 in PBS 150 mM NaCl (pH 7.4) were passed over the immobilized targets at a flow rate of 30 μL min-1 at 25 °C for 1 min. Chip regeneration was performed after injection of 6M guanidine followed by injection of 10 mmol L-1 NaOH (40 μL/min, 30s) between samples. The binding kinetics of monoclonal antibody or scFv to DHEA:BSA were evaluated under similar conditions but using the single cycle kinetics mode. Kinetic constants (kon, koff) were deduced from the analysis of the association and dissociation rates of at least four different antibody concentrations. The dissociation constant KD was calculated from KD = koff / kon. The models were analyzed using BIAevaluation software version 2.0.2.

[043] Table 1 shows the SPR analysis of the interaction of scFv5B7 and DHEA-BSA immobilized on the chip. Data analysis using heterogeneous model.
Table 1.

[044] . Immunohistochemistry. The specificity of monoclonal antibody 5B7B3C6 and scFv was assessed by their staining pattern found in immunohistochemistry using the Spring Bioscience kit. Different tissues embedded in paraffin were used: normal adrenal from a 41-year-old man submitted to adrenalectomy for clear cell cancer, virilizing adrenocortical carcinoma (ACC) and adult liver (negative control for DHEA expression). Tissue samples were obtained and approved by the Ethics Committee of Hospital Pequeno Príncipe (ACC and liver, CAAE: 50622315.0.0000.0097) and by the Ethics Committee of the Health Sciences Center of UFPR (CAAE: 78692617.4.0000.0102). After deparaffinization and rehydration of the sectioned tissues (4 μm thick), the endogenous peroxidase was inactivated with a 5% hydrogen peroxide solution. Antigen retrieval was performed by leaving the samples immersed in Immuno Retriever in the bath at 99 °C for 40 min. After cooling and washing, samples were incubated with 5B7B3C6 hybridoma supernatant or scFv overnight at 4°C in the humid chamber. All slides were washed and incubated with Enzyme Advance HRP Link for 30 min at room temperature, washed and stained with 3,3-diamino-benzidine and hematoxylin. With the exception of assays using scFv, tissue samples were also incubated with anti-histidine antibody (1:800, Thermo Fisher) for 15 min at room temperature. The micrograph images used to quantify anti-DHEA IgM and scFv labeling were obtained with the Axio Scan Z1 (ZEISS ©) with a 40x magnification lens and ZEN Software v 2.3 (ZEISS ©). Anti-DHEA reactivity was quantified in 20 random fields sized 64 χ 64 μm 2 per adrenal area using Image Pro Plus software. The Shapiro-Wilk test was used to verify the normality of the results. The Kruskal-Wallis and Dunn test was used to verify the difference in marking between the adrenal zones. p<0.05 was considered significant. Statistical analysis was performed using GraphPad Prism v 6.05.

DESCRIPTION OF THE FIGURES

[045] . Figure 1 shows the selection of hybridomas that secrete anti-DHEA antibodies. (A) Selection, by ELISA, of hybridoma supernatants using various antigens immobilized on the plate (BSA, DHEA: BSA, KLH, HSA). Bars from left to right: 2H12, 2F5B7, 5B7C2 and 5B7B3 hybridoma. (B) Western blot analysis of hybridoma supernatants after SDS-PAGE of BSA (1) or DHEA:BSA (2). M: Molecular weight marker. SPR analysis of hybridoma supernatants using DHEA: immobilized BSA in which (C) 5B7B3 (blue), 5B7C2 (red), 2F5B7 (green), 2H12 (purple) supernatants (1:2), and purified IgM 5B7B3C6 (40 nM) in green are shown. Antibody samples were injected at t = 0 s and (D) corrected signal expression.

[046] . Figure 2 shows the cloning and characterization of V 5B7 domains. (A) IGHV domain analysis: mRNA amplification using primers VHForU and VHRevU (channel 2) or IGH-Rev and IGH-For (channel 3) after reverse transcription or not (-). (B) IGKV domain analysis: mRNA amplification using VkForU and VkRevl for 9 primers (channels 1 to 9). M: 100 bp, standard base pairs; (C) "Collier de Perle" of the deduced protein sequences and the most similar mouse immunoglobulin identity. CDR H1, H2 and H3 are indicated in red, orange and purple, respectively. CDRs L1, L2 and L3 are indicated in blue, light green and forest green, respectively. (D) CDR identity and related canonical structure. Residues, numbered according to "Collier de Perle", which differ between the two domains of the IGHV are indicated.

[047] Figure 3 shows the functional characterization of the 5B7 monoclonal antibody. (A) Western blot analysis. 1: BSA and 2: BSA-DHEA. (B) Direct ELISA performed with immobilized BSA-DHEA (0.25 μg) and different concentrations of IgM (20-0.16 μg mL-1). (C) Analysis of the interaction of 5B7 injected at 5, 10, 20, 40 nmol L-1 with immobilized BSA-DHEA (red) and BSA (blue).

[048] . Figure 4 shows the expression and evaluation of recombinant scFv 5B7 by ELISA. (A) Schematic representation of the expression cassette. The open reading frame is under the control of a T7 promoter and contains a pelB signal sequence for periplasmic expression, cDNA encoding VH and VL were fused via a linker sequence (Gly4Ser) and cloned into the pelB sequence. This allows expression of scFv with a C term tag (His). (B) Functional analysis of periplasmic extracts containing scFv 5B7 using increasing concentrations of immobilized DHEA-BSA. (C) Electrophoresis (left) and Western blotting (right) of scFv5B7. M: molecular mass markers; (1) flow fraction (2) periplasmic extract; (3) scFv purified by nickel column.

[049] . Figure 5 shows the SPR analysis of the interaction between DHEA-BSA and purified scFv 5B7. (A) The chip was coated with BSA-DHEA (orange) or BSA (blue). (B) Corrected sign fitted with heterogeneous model. (C) scFv 5B7 was pre-incubated with increasing concentrations of free cortisol and then injected at 150, 300, 600, 1200 and 2400 nmol L-1 in continuous mode.

[050]. Figure 6 shows the Immunohistochemistry using anti-DHEA molecules in a tissue section of the adrenal cortex. Normal adrenal from a 41 year old male (A) tested with anti-DHEA IgM (left) and anti-DHEA scFv (right). The dotted square indicates the magnified area shown in (B) confirming the specificity in detecting DHEA in the reticular zone (ZR) (C) converted to red dots (D and E). The quadrants shown in (D) correspond to areas of average quantification for each adrenal zone (ZG, GF, ZR and M) (E). Tissue samples were counterstained using hematoxylin. ZG: zona glomerulosa; ZF: fasciculated zone; ZR: zona reticularis and M: adrenal medulla. White bar: 200 μm, black bar: 500 μm.

Claims (7)

Single-chain variable (scFv) fragment of an antibody that recognizes the human hormone dehydroepiandrosterone (DHEA), characterized by: i) a variable light chain (VL) ii) a variable heavy chain (VH, variable light chain) heavy chain) iii) a junctional peptide between the variable heavy chain and the light chain. Single-chain variable fragment (scFv) according to claim 1, in which the so-called heavy chain (VH) comprises the complementarity determining regions or CDRs 1, 2 and 3 (CDR1, CDR2 and CDR3), characterized in that they are constituted by the amino acid sequences SEQ ID N° 1, SEQ ID N° 2 and SEQ ID N° 3, respectively, or sequences showing at least 95% identity. Single-chain variable fragment (scFv), according to claims 1 and 2, in which the so-called light chain (VL) comprises the complementarity determining regions or CDRs 1, 2 and 3 ( CDR1, CDR2 and CDR3), characterized in that they are constituted by the amino acid sequences SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively, or sequences showing at least 95% identity. Single-chain variable fragment (scFv) according to claims 1, 2 and 3, in which the so-called junctional peptide is characterized by the amino acid sequence SEQ ID N° 7. Single-chain variable fragment (scFv) according to claims 1, 2, 3 and 4, characterized in that it constitutes recombinant forms of antibody, whether Fab', Fab'2 or VH and VL combinations through recombinant production. Single-chain variable fragment (scFv) according to claims 1, 2, 3 and 4, characterized in that it is conjugated to a colorimetric marker molecule for use in the detection of DHEA in human serum or tissue biopsy. Single-chain variable fragment (scFv) according to claims 1, 2, 3 and 4, characterized in that it is present in a pharmaceutical composition comprising the recombinant protein coupled to a chemotherapeutic or radiotherapeutic agent for use in the recognition of human DHEA and a pharmaceutical carrier acceptable.

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