G immunoglobulins biotechnologically fused to the peroxidase enzyme

Abstract

The present invention describes immunoglobulins biotechnologically fused with the peroxidase enzyme for use in immunoenzymatic assays.

Description

[0001] "G immunoglobulins biotechnologically fused to the peroxidase enzyme"

[0002] DESCRIPTION

[0003] Peroxidase enzymes , and horseradish peroxidase (HRP ) in particular, are widely used as markers since they can produce , when incubated with an appropriate substrate , a colored, fluorometric or luminescent derivative of the conj ugated molecule , allowing the detection and quanti fication thereof .

[0004] These features make this enzyme a diagnostic tool with a very wide application domain, from the agri- food industry to medical diagnostics .

[0005] More in particular, peroxidase is an enzyme containing a heme group, which uses hydrogen peroxide to oxidi ze a wide variety of organic and inorganic compounds .

[0006] Peroxidases are widely distributed in a large variety of organisms , from microorganisms to mammals to plants , but that isolated from horseradish (HRP ) is the most commonly used peroxidase as a biochemical reagent .

[0007] Horseradish (Armoracia rusti cana ) is a rustic perennial plant grown in temperate regions of the world primarily for the culinary value of its roots .

[0008] This plant contains a series of distinct peroxidase isoenzymes , of which isoenzyme C (HRP C ) is the most abundant .

[0009] HRP C is a 44173 . 9 Dalton glycoprotein with 6 lysine residues which can be exploited to chemically bind the enzyme to a molecule of interest , such as an antibody . The enzyme produces a colored, fluorometric, or luminescent derivative when incubated with an appropriate substrate, allowing the detection and quantification of the conjugated molecule.

[0010] Commercially available HRP is purified from horseradish roots and is commonly used in techniques such as ELISA and immunohistochemistry by virtue of the monomeric nature thereof and the ease with which it produces, by means of the activity thereof in the presence of appropriate substrates, colored products.

[0011] The primary or secondary antibodies bound to HRP which are commercially available are obtained by chemical conjugation.

[0012] However, this approach has several disadvantages, namely: a. the need to extract and purify peroxidase C as a functional enzyme, with resulting costs and procedural difficulties (e.g., presence of impurities, co-extraction of similar enzymes etc.) b. variability in the composition of the conjugation product (variable stoichiometric ratios of enzyme to antibody molecules) , resulting in variability between production batches and thus difficult standardization of the derived industrial products (diagnostic kits) , c. risk of affecting the biochemical activity of the peroxidase enzyme and / or the chemically conjugated antibody, as a consequence of the chemical bonding reaction of the two proteins.

[0013] All of these factors influence the immunoenzymatic tests in which such conjugates are employed.

[0014] With the genetic fusion, the expression of antibodies biotechnologically conjugated to the enzyme and recombinantly produced, these problems can be completely overcome. Some attempts to express fusion proteins in E. coli have been made , but the fusion product was found to be insoluble .

[0015] Genetically fused recombinant HRP antibodies , such as the Fab fragment of an antibody against atrazine (Koliasnikov et al 2011 ) , were produced in yeast cells ( Pi chia pastori s) to solve the solubility problem .

[0016] In this case the recombinant antibody is produced and secreted, but it is hyper-glycosylated by the yeast cells , and this modi fication af fects the peroxidase activity, limiting the enzymatic action to only one type of substrate .

[0017] Other recombinant antibody formats , always fused to HRP, have been expressed in mammalian cells , for example an antibody against the IBV-N protein of an avian virus expressed in HEK293T cells by trans fection (Ku et al . 2022 ) ; however, the sensitivity of the developed assay was not very high .

[0018] Other similar works have been published, however all provided the expression of recombinant single-chain antibody formats ( scFv, minibody, nanobody) or Fab, which have the limit of losing avidity and sensitivity in the recognition of the antigen of interest .

[0019] For example , the patent application US 2020 / 339963 discloses an scFv construct fused with a peroxidase enzyme or the fusion thereof with the constant portion of a human type 1 light or heavy chain (minibody) ; in the described fusion between scFv and HRP, the stoichiometric ratio of antibody to enzyme is 1 : 1 .

[0020] The publication by Joosten V et al ( Journal of Biotechnology, vol . 12 , no . 4 , pages 347-359 ) discloses the lama variable domain (VHH, also known as nanobody) fused with a peroxidase (which is not horseradish peroxidase) , with a stoichiometric antibody : enzyme ratio of 1:1; the production is obtained by expression in the fungus Aspergillus .

[0021] The publication by Koliasnikov 0. V. et al (Acta Naturae, vol. no.3, pages 85-92) describes the fusion of peroxidase with the recombinant Fab fragment, obtained by expression in the yeast Pichia pastoris, in a stoichiometric antibody : enzyme ratio of 1:1.

[0022] The publication of Gu Kui et al (International Journal of Molecular Sciences, vol.23, no. 14, pages 7589) and the publication by Sheng Yamin et al. (Journal of Nanobiotechnology, vol. 17, no. 1) describe the fusion of a camelid VHH (nanobody) variable domain with horseradish peroxidase in an antibody: enzyme ratio of 1:1, obtained by expression in mammalian cells; the product is then described for application in an ELISA assay.

[0023] Summary of the invention

[0024] The inventors of the present patent application have surprisingly developed a complete IgG antibody (bivalent, comprising the two heavy chains associated with the two light chains) to which two active peroxidase enzyme molecules are fused, one for each heavy chain. The product obtained has surprisingly shown to maintain the affinity properties and catalytic properties unchanged and, at the same time, to be extremely stable.

[0025] Object of the invention

[0026] In a first object, the present invention describes a G-type immunoglobulin fused to the peroxidase enzyme.

[0027] In a second object, the present invention describes a method for preparing a G-type immunoglobulin fused to the peroxidase enzyme. In a third obj ect an immunoenzymatic method is described, comprising the use of the immunoglobulin fused to the peroxidase enzyme according to the present invention .

[0028] Brief description of the drawings

[0029] Figure 1 shows a diagrammatic depiction of the gene constructs of the recombinant heavy chain (HC) fused to peroxidase and that of the relative light chain ( LC ) .

[0030] Figure 2 shows the results of Western bl ot analysis of IgGl-HRP expression in N. benthamiana plants by agroinfiltration .

[0031] Figure 3 shows the results of puri fication by G-protein af finity of IgGl-HRP from N. benthamiana plants .

[0032] Figure 4 shows the results of the comparative functional ELISA assay between mAb and mAb-HRP .

[0033] Detailed description of the invention

[0034] In a first obj ect , the present invention describes a G-type immunoglobulin fused to a peroxidase enzyme .

[0035] For the purposes of the present invention, said G-type immunoglobulin is complete (HC + LC ) .

[0036] More in particular, said complete G-type immunoglobulin is not represented by a fragment selected from the group comprising : scFv, minibody, VHH (nanobody) , Fab .

[0037] More preferably, said IgG can be an IgGl .

[0038] For the purposes of the present invention, said IgG is from mice .

[0039] For the purposes of the present invention, the peroxidase enzyme is preferably represented by horseradish peroxidase ( Armoracia rusti cana ) . According to a preferred aspect, said enzyme is the horseradish peroxidase enzyme C.

[0040] According to a preferred aspect of the invention, such an enzyme is characterized by the amino acid sequence corresponding to SEQ. ID no . 2.

[0041] The horseradish (Armoracia rusticana) peroxidase enzyme C is encoded by the nucleotide sequence corresponding to SEQ. ID no. 1.

[0042] Since each type-G immunoglobulin heavy chain can bind one enzyme, this can comprise two peroxidase enzyme polypeptides.

[0043] In fact, according to an aspect of the present invention, the stoichiometric ratio of immunoglobulin to peroxidase can be 1:2.

[0044] In particular, said G-type immunoglobulin is fused to the peroxidase enzyme by a linker.

[0045] For the purposes of the present invention, the linker is selected from the group comprising:

[0046] - a peptide bond,

[0047] - PGPEF (SEQ. ID no. 15) ,

[0048] - (GGGGS)3(SEQ. ID no. 16)

[0049] - (EAAK)2-5° (SEQ. ID no. 17) .

[0050] In a second object, the present invention describes a method for preparing a G-type immunoglobulin fused to the peroxidase enzyme.

[0051] In particular, the method of the present invention for preparing a G-type immunoglobulin fused to the peroxidase enzyme comprises the steps of:

[0052] 1) preparing a first vector comprising the coding sequence for the heavy chain (HC) of an IgG, the sequence coding a peptide linker (L) and the sequence coding the peroxidase enzyme; 2) preparing a second vector comprising the coding sequence for the light chain (LC) of an IgG;

[0053] 3) transforming Agrobacterium tumefaciens with said first vector;

[0054] 4) transforming Agrobacterium tumefaciens with said second vector;

[0055] 5) transforming a host plant cell by means of the first transformation agent and by means of the second transformation agent;

[0056] 6) expressing said fused immunoglobulin to the peroxidase enzyme in said host plant cell.

[0057] For the purposes of the present invention, said IgG can be an IgGl .

[0058] For the purposes of the present invention, said IgG can be murine .

[0059] In an aspect of the present invention, the peroxidase enzyme is preferably represented by horseradish peroxidase (Armoracia rusticana) .

[0060] According to a preferred aspect, said enzyme is the horseradish peroxidase enzyme C.

[0061] According to an aspect of the present invention, in step 1) said first vector is prepared by the sequence corresponding to SEQ. ID no. 5.

[0062] The amino acid sequence of IgGlmouse-HRP is characterized by the amino acid sequence corresponding to SEQ. ID. no. 6:

[0063] According to a preferred aspect of the invention, the abovedescribed vector can comprise a suitable expression promoter.

[0064] Both vectors of step 1) and 2) are objects of the present invention .

[0065] For the purposes of the present invention, the transformation of steps 3) and 4) is carried out by electroporation.

[0066] Agrobacterium tumefaciens cells transformed with the first and second vectors, respectively, are further objects of the present invention .

[0067] For the purposes of the present invention, in step 5) a plant cell of Nicotiana sp. and, preferably, of Nicotiana benthamianaris transformed.

[0068] A plant cell transformed as described above is a still further object of the present invention.

[0069] According to an alternative aspect of the present invention, in step 2) the second vector comprises a coding sequence for the light chain (LG) of an IgG, the sequence coding a peptide linker (L) and the sequence coding the peroxidase enzyme.

[0070] For the purposes of the present invention, the peroxidase enzyme is preferably represented by horseradish peroxidase (Armoracia rusticana) .

[0071] According to a preferred aspect, said enzyme is the horseradish peroxidase enzyme C. Thereby, the expressed G-type immunoglobulin comprises four peroxidase enzymes , two of which are linked to heavy chains and two to light chains .

[0072] According to an aspect of the present invention, the stoichiometric ratio of immunoglobulin to peroxidase can be 1 : 2 .

[0073] According to an aspect of the present invention, the stoichiometric ratio of immunoglobulin to peroxidase can be 1 : 4 .

[0074] A vector thus prepared, the trans formation agent comprising it and the plant cell trans formed in accordance with said alternative aspect are further obj ects of the present invention .

[0075] In a third obj ect an immunoenzymatic method is described, comprising the use of the immunoglobulin fused to the peroxidase enzyme according to the present invention .

[0076] In particular, such an immunoenzymatic method is a method which finds application in one or more of the medical , veterinary or agricultural fields .

[0077] Experimental part

[0078] To obtain the fusion protein, a synthetic gene was constructed by cloning the sequence coding horseradish peroxidase C ( from horseradish cell cDNA) and fusing it to the sequence coding for the heavy chain of a mouse IgGl immunoglobulin by ampli fication reactions with appropriate oligonucleotides and subsequent DNA restriction and ligation reactions . The nucleotide sequence encoding the recombinant gamma chain was then inserted into a vector suitable for expression in plant cells under the control of the promoter CaMV35S . The vector obtained and that containing the sequence coding the light chain of the original murine antibody, schemati zed in Figure 1 , were inserted by electroporation into cells of Agrobacteri um tumefaci ens .

[0079] IgG-HRP expression was obtained by agroinfiltration of Ni cotiana benthamiana plants using the agrobacteria trans formed with the constructs described above . With the same procedure , 2 di f ferent recombinant antibodies were expressed to veri fy the validity and applicability of the methodology .

[0080] The expression of the recombinant IgG-HRP heavy chain was veri fied by Western blot analysis under reducing conditions of the extracts of the agroinfiltrated N. benthamiana leaves ( Figure 2 ) using a secondary mouse anti- IgG antibody ( Figure 2A) and a horseradish antiperoxidase antibody ( Figure 2B ) for the detection thereof .

[0081] Extracts of plants infiltrated with untrans formed agrobacteria (WT ) were also analyzed as a control .

[0082] For both recombinant antibodies , a molecular weight band higher than 50 kDa of the control unmodi fied heavy chain is visible , and corresponding to the weight of about 86 kDa expected for the IgGl heavy chain fused to the HRP . In the case of detection with the anti- HRP antibody, several additional bands are visible which are probably attributable to the presence of other plant peroxidases , however, the band of the correct molecular weight ( 86 kDa ) is present only in the extracts of the plants infiltrated with the agrobacteria containing the constructs for the expression of IgG-HRP with respect to the related controls .

[0083] The peroxidase functionality was veri fied by preliminary immunoenzymatic assays . The extracts of the total soluble proteins of the agroinfiltrated leaves were tested both by putting them directly in a chromogenic substrate and by ELISA assay on the immobili zed antigen . Both recombinant antibodies were puri fied from the leaves of plants agroinfiltrated by protein G chromatography, analyzed by SDS-PAGE under reducing conditions ( 10% Gel ) and characteri zed . The analysis of the puri fied antibodies ( Figure 3 ) confirms the presence of the band of approximately 86 kDa corresponding to the heavy chain fused to the HRP in addition to that of 25 kDa corresponding to the light chain . In addition to the marker (M) , bovine serum albumin, the band of which is about 60 kDa, was also in the gel .

[0084] Consistent additional bands are not visible , as well as apparent antibody degradation phenomena which can sometimes occur in the plant production of classic immunoglobulins .

[0085] The antibodies were quanti fied by DAS ELISA using one anti-mouse antibody for IgG-HRP capture and a second anti-mouse conj ugated to alkaline phosphatase for colorimetric detection in a di f ferent substrate not usable by HRP . To demonstrate the ef fectiveness of our IgG-HRP, a comparative ELISA assay was carried out . The unmodi fied antibody, IgGl , and the IgGl -HRP fusion antibody were incubated on the immobili zed antigen (AFB1-BSA) , while a second incubation with an anti-mouse HRP was necessary for the detection of the first ( 2 di f ferent secondary ones were used) , the chromogenic substrate was used directly for the detection of the second .

[0086] The result of the assay is shown in figure 4 , and it can be noted that the use of IgG-HRP has a double advantage : the time for carrying out the assay is halved and at the same time a greater analytical sensitivity is obtained . In fact , the same absorbance value is achieved with a lower amount of antibody and by eliminating a biochemical reagent from the system (cost reduction) . Both of these elements are crucial in the development of an immunodiagnostic assay because they allow the implementation of more performing assays in analytical terms by linking the sensitivity of the system to the specificity of the primary antibody, which is that responsible for the recognition of the antigen of interest, and allow reducing the procedural steps of the operator, increasing the assay robustness, minimizing errors and diagnostic times.

[0087] Construction of the HCIgGl-HRP fusion gene

[0088] To obtain the fusion protein, a gene construct was created by cloning the sequence coding horseradish peroxidase C (Armoracia rusticana) and fusing it to the sequence coding the heavy chain of a mouse immunoglobulin G (IgGl) . Initially the peroxidase C (POXCI) coding sequence was amplified by RT-PCR: starting from cDNA, obtained from total RNA extracted from horseradish leaves using oligo dT and Superscript III reverse transcriptase (Invitrogen, Thermofisher) , a PCR was carried out using the oligonucleotides HPlfor and HPlrev (Table 1) , which appear at the beginning and end of the gene, respectively. The amplified sequence was cloned in the vector pGEM4Z. In parallel, the sequence coding a portion of the constant region (CH) of the heavy chain of a mouse IgGl, the nucleotide sequence of which corresponds to SEQ. ID no. 3 and the amino acid sequence thereof corresponds to SEQ. ID no. 4, was amplified by PCR, using the oligonucleotides CH2for and CH3Lrev (Table 1) , and cloned into the vector pBluescriptSK using the restriction sites EcoRV and EcoRI . The sequence coding the mature protein POXCI was then amplified by PCR, using the oligonucleotides HP2for and HP2rev (Table 1) , digested with the restriction enzymes EcoRI and Sad, and fused to the sequence coding the portion of HCIgGl, contained in the vector pBS digested with the same enzymes. The nucleotide sequence encoding the obtained recombinant gamma chain was then digested with restriction enzymes Xma I and Sac I and inserted into a vector suitable for expression in plant cells, containing the missing initial portion of the heavy chain, digested with the same restriction enzymes. The vector (pBI-HCIgGl) obtained is that containing the sequence encoding the light chain of the original murine antibody (pBI- LCIgGl) , the nucleotide sequence of which, relative to the constant portion (CL) , corresponds to SEQ. ID no. 7 and the amino acid sequence of which corresponds to SEQ. ID no. 8, are schematized in Figure 1; both genes are placed under the control of the promoter CaMV35S and the terminator Nos for plant expression, and of a signal peptide for cell sorting, the nucleotide sequence of which corresponds to SEQ. ID no. 18 and the amino acid sequence of which corresponds to SEQ. ID no. 19. All the DNA amplifications for cloning were carried out using Pfx AccuPrime DNA polymerase (Invitrogen, Thermofisher) following the manufacturer's instructions. The same cloning strategy was used for the fusion of both heavy chains of the two monoclonal antibodies used (Abl and Ab2) .

[0089] Table 1 Expression of IgGl-HRP in Nicotiana benthamiana plants

[0090] The transient expression of the two recombinant antibodies fused to peroxidase (Abl and Ab2) was carried out by infiltration of N. benthamiana plants with Agrobacterium tumefaciens. The agrobacteria (strain LBA 4404) transformed, by electroporation, with pBI-HCIgGl and pBI-LCIgGl of each antibody were grown separately O / N at 28°C, centrifuged and suspended in 10 mM MES, pH 5.5 (each) . To obtain complete IgGs, suitable volumes of agrobacterial suspension were mixed together to have a final concentration of each of 0.6 O.D.

[0091] (600 nm) (ratio 1:1) . Six-week-old N. benthamiana plants (at the 6- 7 leaf stage) , grown at 24°C with a 16-hour light and 8-hour dark cycle, were infiltrated with this suspension using a syringe, for a preliminary assessment of antibody expression (Western blot analysis) , or a vacuum chamber, for antibody purification. In the latter case, the plants were infiltrated by completely immersing each plant in the solution containing the agrobacterium mixture inside a vacuum chamber. The vacuum was applied and then quickly released. The infiltrated leaves were harvested 6 days after infiltration and immediately processed, or frozen in liquid N2 and stored at -80°C before use.

[0092] Western blot analysis of infiltrated plant extracts

[0093] To verify the correct expression of the IgGl-HRP fusion proteins (Abl and Ab2) , leaf disks (approximately 0.5 cm in diameter) taken from agroinfiltrated leaves were homogenized, in a tube using a pestle, in PBS (2:1 v / w) and then centrifuged at 13000 xg for 15 minutes at 4°C to extract the total soluble proteins (TSP) . Five micrograms of TSP were resolved by 10% SDS-PAGE and transferred to PVDF using a semi-dry transfer system (Hoefer) . To detect the heavy chain of the antibodies, an IgG gamma HRP anti-mouse antibody was used (KPL, Cat. 474-1802) diluted 1:10000 (Figure 2A) or an anti- HRP produced in goat (Rockland) diluted 1:10000 and then an antigoat HRP secondary antibody (AbClonal) diluted 1:20000 (Figure 2B) . The incubations were carried out for one hour at RT in PBS containing 2% (w / v) skim milk. After washes in PBS + 0.05% (v / v) Tween, the membranes were developed using a chemiluminescent peroxidase substrate (Millipore, Merck) . Extracts of plants infiltrated with unprocessed agrobacteria (WT) were also analyzed as a negative control .

[0094] Purification of IgGl-HRP

[0095] 20 g of agroinfiltrated leaves of N. benthamiana were pulverized in liquid nitrogen with pestle and mortar and the powder obtained was resuspended and homogenized in 2 volumes of phosphate buffer (PBS) at pH 7.2. The homogenate was pre-filtered using a commercial filtering material of synthetic origin (Miracloth, Millipore, Merck, Darmstadt, Germany) and centrifuged for 20 minutes at 40,000 xg. The supernatant was recovered and passed over a 0.45 pm filter and loaded onto a gel filtration column packed with 1 ml of protein G-conjugated Sepharose™ 4 FF resin (Cytiva, Merck, Darmstadt, Germany) , following the manufacturer's instructions. After loading the column with the supernatant, it was washed with 10 volumes of phosphate buffer and the recombinant antibody was eluted with 100 mM glycine buffer at pH 2.7. The eluted sample fractions were immediately buffered at pH 7.2 using 1 M Tris-HCl pH 9.0 to minimize antibody degradation and / or aggregation. Fractions containing purified recombinant antibody were collected and dialyzed into phosphate buffer by filtration using

[0096] Amicon Ultra-15, 50 kDa MWCO filters (Millipore, Merck, Darmstadt, Germany) . The samples obtained from the purification procedure were then analyzed by acrylamide gel electrophoresis (SDS-PAGE, Figure 3) .

[0097] Functional indirect ELISA and antibody quantification DAS-ELISA

[0098] 100 ng per well of BSA-conj ugated antigen (BSA-AFM1 or BSA-AFB1) was immobilized on the O / N solid phase at 4°C in phosphate buffer (PBS) at pH 7.2. The wells were then washed with 4 x 300 pl of washing buffer (PBS + 0.05% (v / v) Tween 20, pH 7.2) and blocked for 2 hours at 37°C with 1% BSA in PBS (w / v) at pH 7.2. The recombinant monoclonal antibody produced in the plant was used as a reference for the quantification of the corresponding antibody fused to peroxidase. After 4 washes with PBS + Tween 20, 100 pl per well in PBS (pH 7.2) of 1:1 serial dilutions of mAbs (from 80 to 5 ng / well) were incubated for 30 minutes at room temperature, to construct a calibration line from which to calculate the amount of antibody fused to the peroxidase analyzed in parallel. After washing, the wells were incubated with 100 pl per well of alkaline phosphatase-conj ugated anti-mouse antibody diluted 1:5000 in PBS + 0.2% (w / v) BSA (A3562 - Merck, Darmstadt, Germany) for 1 hour at 37 °C. After further washing, 100 pl per well of p-Nitrophenyl phosphate disodium salt (pNPP) was used as a chromogenic substrate for the alkaline phosphatase. The absorbance was measured at 405 nm with a microplate reader (F50, TECAN, Mannedorf, Switzerland) .

[0099] The DAS-ELISA included immobilizing a goat anti-mouse lambda light chain antibody (A90-121A, Bethyl, FORTIS Life Sciences) on the solid phase of the assay. For each well, 100 pl of a 1:500 O / N dilution was incubated at 4°C in phosphate buffer (PBS) at pH 7.2. The blocking and washing procedures were the same as described for the previous test. The same amounts of reference mAb and peroxidase- fused antibody as described above were incubated for 1 hour at 37 °C. The remainder of the assay was conducted under the same conditions previously described for the indirect ELISA assay.

[0100] Comparative ELISA assay between Ab-HRP and Ab + anti -mouse

[0101] 65 ng / well of antigen (AFB1-BSA) in 100 pl PBS pH 7.2 were immobilized on the solid phase of the O / N microplate at 4°C. The wells were washed with 4 x 300 pl of washing buffer (PBS with 0.05% (v / v) Tween 20, pH 7.2) and blocked for 2 hours at 37°C with 1% BSA in PBS (w / v) at pH 7.2. The wells were again washed and then incubated with 100 pl of 1:1 serial dilutions of mAb (from 40 to 0.625 ng / well) and mAb-HRP (from 5 to 0.078 ng / well) in PBS (pH 7.2) for 30 minutes at room temperature (RT) . After a further washing step of the wells, those containing the mAb-HRP were directly developed for 15 minutes with 100 pl per well of 3, 3 ', 5, 5 ' -tetramethylbenzidine (TMB) and the colorimetric reaction was blocked with 50 pl of 3 M HC1 per well before reading at 450 nm on microplate reader (F50, TECAN, Mannedorf, Switzerland) . The wells containing unconjugated mAb were incubated with 100 pl of dilutions of two different secondary antibodies. Specifically, an anti-mouse IgG (H+L) (ABclonal, Cat. A5003) diluted 1:5000 in PBS + 0.2% (w / v) BSA or an anti-mouse IgG gamma HRP (KPL, Cat. 474-1802) diluted 1:2000 in PBS + 0.2% (w / v) BSA were used. Both incubations were carried out for 45 minutes at 37°C. After washing the wells, the development procedures with TMB and acquisition of absorbance values were the same as those described for the mAb biotechnologically conj ugated to the peroxidase .

[0102] From the above description the advantages of fered by the present invention are immediately clear .

[0103] The product obtained has high ef ficiency, excellent performance and considerable stability .

[0104] In fact , the technology used allows obtaining a product which maintains an unchanged ability to recogni ze the antigen and the avidity of the original antibody; at the same time , by virtue of the fact that the expression occurs in plant cells , the peroxidase assumes the conformation and glycosylation of the original enzyme , fully maintaining the functionality thereof .

[0105] These features result in a wide versatility of the described methodology, as it is potentially trans ferable on any antibody already in use in existing diagnostic systems , allowing the methodology to be implemented in existing diagnostic systems , in a short time ( a few months ) and at low costs .

Claims

CLAIMS1 . A complete G-type immunoglobulin (HC + LC ) biotechnologically fused to a horseradish peroxidase enzyme .2 . A G-type immunoglobulin biotechnologically fused to the peroxidase enzyme according to the preceding claim, wherein said G- type immunoglobulin comprises heavy chains and light chains , wherein each of said heavy chains is linked by a peptide linker to a polypeptide represented by the horseradish peroxidase enzyme .

3. A G-type immunoglobulin biotechnologically fused to the peroxidase enzyme according to preceding claim 1 or 2 , wherein said G immunoglobulin comprises heavy chains and light chains , wherein each of said light chains is linked by a peptide linker to a polypeptide represented by the horseradish peroxidase enzyme .4 . A G-type immunoglobulin biotechnologically fused to the horseradish peroxidase enzyme comprising a stoichiometric immunoglobulin : enzyme ratio of 1 : 4 or 1 : 2 .5 . A G-type immunoglobulin biotechnologically fused to the peroxidase enzyme according to the preceding claim, wherein said peroxidase enzyme is a horseradish peroxidase enzyme C .

6. A G-type immunoglobulin biotechnologically fused to the peroxidase enzyme according to any one of the preceding claims 1 to5 , wherein said G-type immunoglobulin is an IgGl .7 . A G-type immunoglobulin biotechnologically fused to the peroxidase enzyme according to any one of the preceding claims 1 to6 , wherein said G-type immunoglobulin is murine .

8. A method for preparing the G-type immunoglobulin biotechnologically fused to a peroxidase enzyme, comprising the steps of:1) preparing a first vector comprising the coding sequence for a heavy chain (HC) of an IgG, the sequence coding a peptide linker (L) and the sequence coding the peroxidase enzyme;2) preparing a second vector comprising the coding sequence for a light chain (LG) of an IgG;3) preparing a first transformation agent, transforming Agrobacterium tumefaciens with said first vector by electroporation;4) preparing a second transformation agent, transforming Agrobacterium tumefaciens with said second vector by electroporation;5) transforming a host plant cell of Nicotiana benthamiana by means of said first transformation agent and by means of said second transformation agent;6) expressing a complete G-type immunoglobulin biotechnologically fused to a peroxidase enzyme in said host plant cell .

9. A method according to the preceding claim, wherein in step 2) said second vector comprises the coding sequence for a light chain (LG) of an IgG, the sequence coding a peptide linker (L) and the sequence coding the peroxidase enzyme.

10. A method according to claim 8 or 9, wherein said peroxidase enzyme is a horseradish peroxidase enzyme.

11. A method according to any one of claims 8 to 10, wherein said peroxidase enzyme is a horseradish peroxidase enzyme C.

12. A method according to any one of claims 8 to 11, wherein said G-type immunoglobulin is an IgGl.

13. A method according to any one of claims 8 to 12, wherein said IgG is murine.

14. A G-type immunoglobulin biotechnologically fused to a peroxidase enzyme obtainable by the method according to any one of claims 8 to 13.

15. A vector comprising a nucleotide sequence encoding a heavy chain of IgG biotechnologically fused to a peroxidase enzyme.

16. A vector comprising a nucleotide sequence encoding a light chain of IgG biotechnologically fused to a peroxidase enzyme.

17. A vector according to claim 15 or 16, wherein said peroxidase enzyme is a horseradish peroxidase enzyme.

18. A vector according to the preceding claim, wherein said enzyme is a horseradish peroxidase enzyme C.

19. A vector according to any one of claims 15 to 18, wherein said IgG is an IgGl.

20. A vector according to any one of claims 15 to 19, wherein said IgG is murine.

21. A transformation agent represented by Agrobacterium tumefaciens transformed with the vector according to any one of claims 15 or 17 to 20.

22. A transformation agent comprising the vector according to any one of claims 16 to 20.

23. A host cell transformed by means of the transformation agent of claim 21 and by means of a transformation agent comprising a nucleotide sequence encoding a light chain of IgG.24 . A host cell trans formed by means of the trans formation agent of claim 21 and by means of the trans formation agent of claim 22 .25 . A direct immunoenzymatic method comprising the use of the G-type immunoglobulin biotechnologically fused to a peroxidase enzyme according to any one of claims 1 to 7 or obtained according to the method of any one of claims 8 to 13 .

26. A G-type immunoglobulin biotechnologically fused to a peroxidase enzyme .

Images