WO2022015131A1 - Vnar domain sequences of potamotrygon schroederi and compositions for recognising human glycated haemoglobin - Google Patents

Vnar domain sequences of potamotrygon schroederi and compositions for recognising human glycated haemoglobin Download PDF

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WO2022015131A1
WO2022015131A1 PCT/MX2020/000015 MX2020000015W WO2022015131A1 WO 2022015131 A1 WO2022015131 A1 WO 2022015131A1 MX 2020000015 W MX2020000015 W MX 2020000015W WO 2022015131 A1 WO2022015131 A1 WO 2022015131A1
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vnar
seq
glycated hemoglobin
amino acid
protein
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PCT/MX2020/000015
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Spanish (es)
French (fr)
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Tanya Amanda Camacho Villegas
Nayeli Areli PÉREZ PADILLA
Elia REZA ESCOBAR
Mario Alberto FLORES VALDEZ
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Centro De Investigación Y Asistencia En Tecnología Y Diseño Del Estado De Jalisco A.C.
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Publication of WO2022015131A1 publication Critical patent/WO2022015131A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood

Definitions

  • the present invention is related to the technical field of Biotechnology and Biomedicine, since it provides a set of DNA sequences and their translation to proteins of vNAR domains isolated from ( Potamotrygon schroederi), these sequences specifically recognize biomarkers associated with metabolic diseases such as diabetes, in the particular case of human glycated hemoglobin for the monitoring and control of the disease in diagnosed and undiagnosed patients.
  • diabetes is one of the leading causes of death and it is estimated that 1 in 10 people has diabetes, and only 1 in 4 receives adequate control and monitoring of the disease.
  • Complications associated with diabetes and its concomitant diseases generate high health costs, ranging between 673,000 - 1197,000 million dollars (2015), and are projected to increase 1.2 times by 2040.
  • the prevalence of diabetes has been increasing and projects the same trend in the coming years.
  • Diabetes is a global health problem, so the monitoring and control of the disease have become a priority.
  • the metabolic lack of control and the consequences of diabetes complications are aggravated when detection, control and follow-up are not carried out in a timely and efficient manner. Therefore, patient surveillance prevents complications, decreases deaths in productive ages and lowers the costs of care in the health sector, as well as the hospitalization rate.
  • Currently, diabetes monitoring by detecting glycated hemoglobin is widely used. with which an approximation of blood glucose levels of about 3 months can be obtained, in addition, sequences or fragments known as variable domains with antigen recognition capacity (vNAR) have been described.
  • vNAR variable domains with antigen recognition capacity
  • vNAR sequences are obtained from shark (WO2011056056 A2) but they have the disadvantage that they could not recognize human glycated hemoglobin biomarkers, since they are selected against soluble proteins of another nature.
  • Ray vNARs can be obtained from freshwater organisms, reducing the cost associated with the capture or maintenance of sharks. It should be considered that there are biotechnology-based companies that develop research, development and innovation basing their technology on shark vNAR sequences, which generates competition between them. In the case of freshwater rays, there is no such precedent, therefore, there are wide possibilities for research, development, innovation and technology transfer.
  • the present invention aims to counteract the aforementioned disadvantages, by providing a set of DNA sequences and their translation into proteins of vNAR domains isolated from the sweet ray Potamotrygon schroederi, these sequences specifically recognize the human glycated hemoglobin biomarker and its variants, mainly glucose in the amino terminal lysine or valine of glycated hemoglobin and can be used for the monitoring and control of metabolic diseases.
  • Figure 2 is a polyacrylamide SDS-PAGE photograph of the extraction and purification of a vNAR R007 protein.
  • Figure 2 is a polyacrylamide SDS-PAGE photograph of the extraction and purification of a vNAR R016 protein.
  • Figure 3 is a photograph of a transfer membrane showing the detection of the recombinant proteins vNAR R007 and R016, by Western Blot.
  • Figure 4 is a graphic comparison of the human glycated hemoglobin recognition ELISA test and its variants for each vNAR protein (*P ⁇ 0.05, ***P ⁇ 0.001).
  • Figure 5 is a graphical comparison of the ELISA test for recognition of glycated hemoglobin in whole blood and serum samples, by vNAR proteins ( ⁇ *P ⁇ 0.001).
  • Figure 6 is a graph illustrating the detection limit determination of glycated hemoglobin by ELISA protein vNAR R016 of insoluble and soluble origin (**P ⁇ 0.01, ***P ⁇ 0.001).
  • Figure 7 is a graph of an ELISA test for selective recognition of glycated hemoglobin in a sample of non-glycated hemoglobin mixed with glycated hemoglobin (***P ⁇ 0.001).
  • Figure 8 is a graph of an ELISA test for recognition of glycated hemoglobin in whole blood using vNAR protein conjugated (AuNP's-R016) and unconjugated (R016) to gold nanoparticles.
  • Figure 9 A) is a graph showing the percentage curves of glycated hemoglobin and Mixture 1, 2 of vNAR R016 and Mixture 3 of AuNPs-R016,
  • B) is a graph of recognition stability evaluation of AuNPs R016 by glycated hemoglobin, at different times.
  • Figure 10 A) is a photograph of the macroscopic reaction of the AuNP's-R007 mixture with Used blood.
  • B) is a graph illustrating the RGB color profile of AuNP's-R007 mixtures with Used blood.
  • the present invention has as its first object an isolated and purified vNAR protein, which comprises: an amino acid sequence of the first complementarity determining region (CDR1) that is selected from the following group of amino acid sequences: DTSHILSGTK (SEQ. ID. NO 1), DTSCGLYSTS (SEQ. ID. NO. 5), or substantially similar variants thereof; and a third complementary determining region (CDR3) amino acid sequence selected from the following group of amino acid sequences: QTIGRRQTLHTGIGAMWDSTSD (SEQ. ID. NO. 2). QAGGRLCVGGGNY (SEQ. ID NO.6), or substantially similar variants thereof.
  • CDR1 first complementarity determining region
  • DTSHILSGTK SEQ. ID. NO 1
  • DTSCGLYSTS SEQ. ID. NO. 5
  • CDR3 third complementary determining region
  • QAGGRLCVGGGNY SEQ. ID NO.6
  • An embodiment of the vNAR protein of the present invention is when the CDR1 is the amino acid sequence SEQ. ID NO. one; and CDR3 is the amino acid sequence SEQ. ID NO. two.
  • Another embodiment of the protein according to the present invention is when the CDR1 is the amino acid sequence SEQ. ID NO. 5; and CDR3 is the amino acid sequence SEQ. ID NO. 6.
  • One more embodiment of the protein according to the present invention is when it comprises one of the following amino acid sequences: SEQ. ID No. 4, SEQ. ID NO. 8, or substantially similar variants thereof.
  • the vNAR protein according to this invention is of Potamotrygon schroederi origin, and it was found to have the ability to recognize the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin.
  • a second object of the present invention is an AON sequence, which comprises a nucleotide sequence that encodes the vNAR protein described above. In a preferred embodiment of the present invention, it is when the DNA sequence comprises one of the following nucleotide sequences: SEQ. ID NO. 3, or SEQ. ID NO. 7.
  • a third object of the Invention in question is that it also refers to a recombinant DNA sequence, which includes: the AON sequences according to the present invention.
  • the fourth object of said invention is a composition that recognizes the target or biomarker of the beta subunit of human glycated hemoglobin, where said composition comprises: at least one vNAR protein, as proposed by the present invention.
  • One embodiment of said composition is when it also comprises: at least one substance that increases its efficiency of use in the recognition of the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin.
  • a substance that increases the efficiency of use is selected from the following group: nanoparticles, chromophores, proteins and/or a combination between them.
  • the nanoparticles can be gold, latex, and/or a combination of them, to name a few examples.
  • a fifth object of the present invention is an ex vivo method for the recognition of the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin, said method comprises the following steps: provide a blood sample from a patient suspected of diabetes ; contacting the blood sample with the composition that recognizes the human glycated hemoglobin beta subunit target or biomarker, in accordance with the present invention; Y detecting the presence of glycated hemoglobin, wherein the vNAR protein of said composition binds to the beta subunit of human glycated hemoglobin; where said detection is by means of the following immunoassays: ELISA, DotBlot, Western blot, agglutination, immunoturbidimetry, immunofluorescence, lateral flow strips (LFA), point-of-care tests (POC), among others.
  • a sixth object of the present invention is the use of the vNAR protein, and the composition, as described in the present invention, for the recognition of the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin.
  • Example 1 A protocol was carried out in order to obtain a vNAR variable domain with recognition capacity for glycated hemoglobin in a whole blood sample, said protocol was carried out as follows.
  • RNA extraction and obtaining vNAR fragments 1.
  • tRNA total RNA
  • Reverse transcription reactions were performed with tRNA and Oligo(dT)18.
  • Fragment amplification was performed by polymerase chain reaction (POR) using specific oligonucleotides that include restriction sites (W02011056058A2). The Fragments were analyzed by 1.5% agarose gel.
  • the pComb3x phagemid was used and the amplification protocol was carried out in accordance with what was written in Barbas et al., 2001. 2. Selection and characterization of vNAR.
  • Phage display rounds of selection.
  • vNAR coding fragments were amplified by PCR with specific oligonucleotides including restriction sites for Ncol and Xhol. Plasmid pET-28a+ and vNAR fragments were digested, ligation was performed with T4 ligase in a 1:3 ratio (insert vector). They were transformed into E. coli strain TG1 cells and BL21(DE) cells. Subsequently, ligation was verified by colony PCR, using specific oligonucleotides for the T7 sequence of plasmid pET-28a+. Plasmid was obtained and after sequencing, an in silico analysis was performed with the CLC viewer 8.0 program. Induction was performed with 1 mM IPTG when the DOeoo was 0.8 and incubated at 37 ° C for 5 300 rpm. 2.2.2. Expression, purification and detection of recombinant vNAR proteins.
  • each vNAR protein was transferred to a nitrocellulose membrane. After blocking, it was incubated with anti-HIs-HRP (1:5,000) at 4 oC for 16 h. After three washes, the development solution was added containing 2.7 mg of HRP substrate diluted in 1 mL of cold methanol, 4.5 mL of 1X PBS and 15 ⁇ L of hydrogen peroxide. Developing solution was added and incubated for 10 min.
  • vNAR by recognition analysis of commercial glycated hemoglobin and in whole blood by ELISA. 500 ng of commercial HbA1c, HbAGIy, HbAlab, HbAO (Exocefl) were placed. BSA3% was used as a negative control. All plate incubations were performed for 1 h at 37 °C. After blocking 500 ng of vNAR R007 and R016 were added. After washing, 50 ⁇ L of anti-Hb (1:5,000) in 1% BSA-1x PBS were added. All washes were repeated 3 times and incubations were 1 h at 37 °C.
  • the plate was revealed with 50 ⁇ L of TMB Ultra, incubated at 37 *C for 15 min and analyzed in the microplate reader for absorbance at 652 nm.
  • the samples were used by freezing at -20 °C according to the protocol described by SeMn et al., 2005 and Stoviter, 1962, and a 1:5,000 dilution with water was used.
  • the assay was performed under the same conditions described above. The percentage of glycated hemoglobin contained in the samples was 9.6 and 5.2%.
  • vNAR R016 To analyze the selectivity for vNAR R016, mixtures of commercial HbAGIy and HbAO were used. A total of 500 ng of the hemoglobin mixture was used, 375, 250 and 125 ng of the hemoglobins were mixed in the ratio of HbAGIy/HbAO, remaining 3:1, 1:1 and 1:3. As a negative control, 3% BSA-1x PBS was used. After blocking, 500 ng of vNAR R016 were added. After washing, 50 ⁇ L of anti-Hb-HRP (1:5000) were added. Washing and development were repeated as stated above.
  • the nanoparticles were generated with chloroauric acid (HAuCM, SIGMA cat. 254169) at 0.1 M and sodium cirate at 0.5 M with boiling water under constant stirring and based on what is described by Panlkar ef a/., 2019; Sitó et al , 2012. Once the AuNP's were stabilized, they were prepared for conjugation by diluting to 50% with MiliQ water and adding NaOH to a final concentration of 0.4 mM for a final pH of 6.1.
  • the vNAR R007 protein was previously dialyzed with conjugation buffer (25 mM NaH 2 PO 4 , 75 mM NaCl, pH 7) and the vNAR R015 protein was dialyzed with HEPES buffer. (15 mM HEPES, 10 mM NaCl, 1% glycerol, pH 9).
  • the aggregation of the AuNP's was calculated with the ratio of the absorbances obtained at 690/560 nm, where the optimal result is close to zero. Subsequently, for R007, 50 ⁇ L of vNAR R007 (2 ng/pL) were added and for R016, 20 ⁇ L of vNAR R016 (0.45 ⁇ g/pL) were added to 1 mL of AuNP's at OD520nm of 1 (pH 6.1), incubated for 5 minutes To verify the conjugation, an absorbance spectrum from 450 to 700 nm was performed in Nanodrop 2000 and compared with unconjugated AuNP's.
  • vNAR R016 conjugated and not conjugated to AuNP's blood lysed with glycated hemoglobin of 9.6, 7.5 and 4.9% diluted 1:1000 in water was placed. BSA was used as negative recognition control, per well and in triplicate. After blocking, 50 ⁇ L of unconjugated R016 vNAR (10 ng/pL) and conjugated AuNP's-R016 diluted 1:13 in HEPES buffer were added. After washing, 50 ⁇ L of antl-His-HRP (1:5000) was added. Washing and development were repeated as stated above.
  • vNAR R016 whole blood sample (containing 9.6, 7.0 and 5.2% glycated hemoglobin) was diluted vNAR R016 was used in: Mix 1) vNAR R016 0.7 mg/mL with anti-His (0.001 mg/mL) and anti -mouse (0.001 mg/mL), Mix 2) the vNAR R0160.14 mg/mL with anti-His (0.001 mg/mL) and anti-mouse (0.001 mg/mL) or, Mix 3) the AuNP's - vNAR R016 without adding any antibody. The results were analyzed at 660 nm. III. AuNP's-vNAR R007 recognition analysis by color change.
  • RNA was obtained at a concentration of 537.8 ng/pL. with a 260/280 ratio of 2.02.
  • the library was generated and 3.26 x 10* were obtained on phage display. 7.32 x 10* and 5 x 10* colony forming units (CFU/mL) for rounds 1, 2 and 3, respectively. 36 colonies were chosen at random and analyzed by colony PCR to corroborate the presence of vNAR fragments, 24 positive colonies were obtained.
  • the plasmid was extracted and the sequence obtained.
  • Clones R007 and R016 have a percentage of Identity of 85 and 68%, respectively, with the vNARs reported from different elasmobranchs (for example, the NCBI accession number sequences of AAX10140.1 and AAT02204.1, respectively). Isolated vNARs have not been reported with anteriority.
  • vNARs are classified into 4 types (Zieionka et al., 2015), therefore, R007 corresponds to a type IV vNAR and R016 is type II.
  • Figure 1B first wash (L1, 20 mM imidazole), second wash (L2, 50 mM imidazole), elution 1 (E1, 150 mM imidazole), elution 2 (E2, 200 mM imidazole), elution 3 ( E3, 250 mM Imidazole), elution 4 (E4, 300 mM imidazole), and elution 5 (E5, 500 mM imidazole).
  • the purified protein is indicated in the table.
  • Molecular Weight Marker (MPM) Biorad).
  • B vNAR R016 was denaturingly extracted with guanidine and renatured on a urea gradient column in the presence of ⁇ -mercaptoethanoi.
  • SA sonicated extract with buffer A
  • SB1 first sonicated extract with buffer B
  • Second sonicated extract with buffer B SB2
  • denatured extract ED
  • non-retained denatured extract NR
  • wash LU, 6 M urea
  • renaturing buffer 5 - 0 M urea R5 - R0
  • Figure 2B Wash I_ 50 mM imidazole
  • Elution 1 E1, 150 mM imidazole
  • Elution 2 E2, 200 mM imidazole
  • Elution 3 E3, 250 mM imidazole
  • Elution 4 E4, 300 mM imidazole
  • elution 5 E5, 500 mM imidazole
  • the purified protein is enclosed in the box.
  • Molecular weight marker MPM
  • the approximate weight is 14.2 kDa for R007 and 13.2 kDa for R016.
  • Figure 3 the analysis of the vNARs by Western blot is shown. Lane 1 corresponds to vNAR R007, lane 2 is the molecular weight marker and lane 3 to vNAR R016. The arrows indicate the band corresponding to each protein.
  • Figure 5 shows that there is a significant difference between the recognition of blood and serum samples, including blood samples with 9.2 and 5.2% glycated hemoglobin (***P ⁇ 0.001) for both vNARs, demonstrating that donuts R007 (Figure 5A) and R016 ( Figure 5B) have specific recognition capacity for glycated hemoglobin in a whole blood sample.
  • the detection limit of glycated hemoglobin was determined using the extracted vNAR R016 in soluble and insoluble form.
  • Figure 6 shows a better recognition of glycated hemoglobin by the vNAR obtained by insoluble with respect to soluble conditions, having a detection limit
  • the R016 vNAR obtained in soluble form has a lower detection limit of 500 ng of vNAR for the recognition of glycated hemoglobin in ELISA. 5. Selectivity analysis of vNAR R016 to glycated hemoglobin.
  • Figure 7 shows that vNAR R016 has specific recognition of glycated hemoglobin in a mixture of glycated and non-glycated hemoglobin, discriminating the presence of non-glycated hemoglobin (HhAO). This makes the use of vNAR feasible in complex samples and with different proportions of glycated hemoglobin.
  • vNAR R016 Agglutination and absorbance recognition analysis of vNAR R016.
  • the SPINREACT® cano control Kit was used and mixtures 1, 2 and 3 were used to evaluate the use of vNAR R016.
  • Figure 9A shows that vNAR R016 behaves similarly to the kit control even when the various Mixes 1, 2 and 3. Showing that the R016 vNAR either conjugated to AuNP's (Mix 3) or unconjugated (Mix 1 and 2) has specificity and selectivity for glycated hemoglobin when a qualitative technique is used.
  • vNAR R016 is capable of differentiating between percentages of glycated hemoglobin.
  • the CV of the control with respect to time was 74.77%, showing that the reaction is affected with time.
  • This same analysis was performed using the AuNP's-R016 conjugate where the coefficient of variation was 4.81%. Additionally, the determination coefficient R 2 for the AuNP * s-R016 conjugate is 0.8516 at 5 min, 0.9271 and 0.8315 at 10 and 15 min, respectively.
  • the use of AuNP's-R016 is projected as a strategy that can accurately and stably determine the percentage of glycated hemoglobin in qualitative methods up to 15 min of reaction.
  • Table 1 shows the percentages of hemoglobin obtained in a clinical laboratory against those obtained with the SPINREACT kit and with the AuNP s-R016, obtaining greater accuracy with the AuNP's-R016 conjugate with (if average error of 6% compared to the 15.7% with the commercial kit.
  • Figure 10A shows the macroscopic reaction of the AuNP's-R007 mixture with blood used from a non-diabetic, diabetic and diabetic patient. hyperglycemic, with 4.9, 7.0 and 9.6% glycated hemoglobin, respectively. At first glance it is possible to see a difference in color and saturation between the samples due to the agglomeration of gold nanoparticles associated with the increase in the percentage of glycated hemoglobin.
  • Figure 10B shows the graph of the RGB color profile for the AuNP's-RG07 mixtures with blood used with 4.9, 7.0 and 9.6% of glycated hemoglobin, evaluating each color separately.
  • the vNAR domains obtained by the present invention function as detection molecules for glycated hemoglobin and its variants with application in the control or monitoring of metabolic diseases in humans, for example, diabetes and its concomitant diseases (retinopathy, nephropathy), cardiovascular diseases, obesity among other.
  • metabolic diseases for example, diabetes and its concomitant diseases (retinopathy, nephropathy), cardiovascular diseases, obesity among other.
  • the amino acid sequence of clone RQ07 is as follows: TSDYDGAGTVLTVN: where the amino acids in bold correspond to the
  • CDR1 the underlined amino acids correspond to CDR3
  • the double underlined amino acids are the canonical dsteines.
  • This protein sequence is SEQ. ID NO. 4
  • the amino acid sequence of CDR1 corresponds to SEQ. ID NO. 1
  • the amino acid sequence of CDR3 is SEQ. ID NO.
  • the nucleotide sequence encoding the R007 protein (SEQ. ID. NO. 4) is:
  • the amino acid sequence of clone R016 is as follows: ATRVDQTPREATKQPGETLTINfiVLRDTSCGLYSTSWFVQRPGRSAWDRLSI GGRYAESVNKPAKSFSLRISNLIAEDSATYFCKAQAGGRLCVGGGNYYGGAG TVLTVN; where the amino acids in bold correspond to CDR1, the underlined amino acids correspond to CDR3, and the double underlined amino acids are the canonical dsteines.
  • This protein sequence is SEQ. ID NO. 8
  • the amino acid sequence of CDR1 corresponds to SEQ. ID NO. 5
  • the amino acid sequence of CDR3 is SEQ. ID NO. 6.
  • the nucleotide sequence encoding the R016 protein (SEQ. ID. NO. 8) is: CCCTGCGGATCAGCAATCTAATTGCTGAAGACTCAGCCACGTACTTTTGCA AAGCACAAGCGGGAGGGCGACTATGTGTGGGGGGGTAACTACTACGG TGGAGCTGGCACCGTGCTGACTGTGAAC, and corresponds to SEQ. ID NO.
  • sequences described above have the ability to recognize the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin. Therefore, the amino acid sequences encoded by the AON R007 and AON R016 sequences together and separately can be used as a complement or substitute for the monodonal antibodies on which the commercial tests for the detection and quantification of glycated hemoglobin are based. developing new screening tests. Likewise, it can be used in new technologies alone or in combination, for example, tests at the point of care (POC). Therefore, various types of products and/or compositions can be formulated.
  • POC point of care
  • vNARs can be conjugated to various nanoparticles (gold, latex, among others), to chromophores or other proteins to increase their efficiency of use in the application for the control and monitoring of metabolic diseases. They can also be produced in the form of monies, trimers or tetramers to increase the avidity towards their antigen. So far, only mammalian-derived IgG-type monodonal antibodies have been developed for this specific application. No variable vNAR fragments of any type or species have been found for the detection of hemoglobin and its glycated variants. LITERATURE CITED
  • HbA1c haemoglobin A1c

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Abstract

Disclosed is an isolated vNAR protein comprising: a CDR1 comprising an amino acid sequence selected from the following group: SEQ. ID. NO. 1, SEQ. ID. NO. 5 or variants thereof substantially similar to same; and a CDR3 comprising an amino acid sequence selected from the following group: SEQ. ID. NO. 2, SEQ. ID. NO. 6 or variants thereof substantially similar to same. Also disclosed is a composition that recognises a target or biomarker of the beta subunit of human glycated haemoglobin, which comprises: at least one vNAR protein according to the invention. The invention further relates to an ex-vivo method for recognising the target or biomarker of the beta subunit of human glycated haemoglobin, which comprises: providing a blood sample of a suspected diabetes patient; bringing the blood sample into contact with the composition of the invention; and detecting the presence of glycated haemoglobin.

Description

SECUENCIAS DE DOMINIOS vNAR DE Potamotrygon schroederi Y COMPOSICIONES PARA EL RECONOCIMIENTO DE HEMOGLOBINA Potamotrygon schroederi vNAR DOMAIN SEQUENCES AND COMPOSITIONS FOR HEMOGLOBIN RECOGNITION
GLICADA HUMANA HUMAN GLYCAD
CAMPO TÉCNICO DE LA INVENCIÓN TECHNICAL FIELD OF THE INVENTION
La presente invención se relaciona con el campo técnico de la Biotecnología y Biomedicina, ya que proporciona un conjunto de secuencias de ADN y su traducción a proteínas de dominios vNAR aisladas de ( Potamotrygon schroederi), estas secuencias reconocen específicamente biomarcadores asociados a enfermedades metabólicas como la diabetes, en el caso particular de la hemoglobina glicada humana para el monitoreo y control de la enfermedad en pacientes diagnosticados y no diagnosticados. The present invention is related to the technical field of Biotechnology and Biomedicine, since it provides a set of DNA sequences and their translation to proteins of vNAR domains isolated from ( Potamotrygon schroederi), these sequences specifically recognize biomarkers associated with metabolic diseases such as diabetes, in the particular case of human glycated hemoglobin for the monitoring and control of the disease in diagnosed and undiagnosed patients.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
Actualmente, la diabetes es una de las primeras causas de muerte y se contempla que 1 de cada 10 personas tiene diabetes, además que sólo 1 de cada 4 recibe control y monitoreo adecuado de la enfermedad. Las complicaciones asociadas a diabetes y sus enfermedades concomitantes, generan gastos sanitarios altos, oscilando entre 673,000 - 1197,000 millones de dólares (2015), y se proyecta que aumente 1.2 veces para el 2040. La prevalencia de diabetes ha ido en incremento y se proyecta la misma tendencia ene los próximos años. Currently, diabetes is one of the leading causes of death and it is estimated that 1 in 10 people has diabetes, and only 1 in 4 receives adequate control and monitoring of the disease. Complications associated with diabetes and its concomitant diseases generate high health costs, ranging between 673,000 - 1197,000 million dollars (2015), and are projected to increase 1.2 times by 2040. The prevalence of diabetes has been increasing and projects the same trend in the coming years.
La diabetes es un problema de salud a nivel mundial, por lo que el monitoreo y control de la enfermedad se han vuelto prioritarios. El descontrol metabólico y las consecuencias por complicaciones de la diabetes se agravan cuando la detección, control y seguimiento no se realizan de forma oportuna y eficiente. Por lo tanto, la vigilancia de los pacientes evita complicaciones, la disminución de muertes en edades productivas y disminuye tos costos de atención del sector salud, así como la tasa de hospitalización. En la actualidad, es ampliamente utilizado el monitoreo de diabetes mediante la detección de hemoglobina glicada, con la cual puede obtenerse un aproximado de los niveles glucómicos sanguíneos de alrededor de 3 meses, además, se han descrito secuencias o fragmentos conocidos como dominios variables con capacidad de reconocimiento a un antfgeno (vNAR). Diabetes is a global health problem, so the monitoring and control of the disease have become a priority. The metabolic lack of control and the consequences of diabetes complications are aggravated when detection, control and follow-up are not carried out in a timely and efficient manner. Therefore, patient surveillance prevents complications, decreases deaths in productive ages and lowers the costs of care in the health sector, as well as the hospitalization rate. Currently, diabetes monitoring by detecting glycated hemoglobin is widely used. with which an approximation of blood glucose levels of about 3 months can be obtained, in addition, sequences or fragments known as variable domains with antigen recognition capacity (vNAR) have been described.
Actualmente, las secuencias de vNAR conocidas son obtenidas de tiburón (WO2011056056 A2) pero tienen la desventaja de que no podrían reconocer biomarcadores de hemoglobina glicada de humano, puesto que están seleccionadas contra proteínas solubles de otra índole. Los vNAR de raya pueden obtenerse de organismos de agua dulce disminuyendo el costo asociado a la captura o mantenimiento de tiburones. Se debe considerar que existen empresas de base biotecnológica que desarrollan Investigación, desarrollo e innovación basando su tecnología en secuencias vNAR de tiburón, lo cual genera competencia entre ellas. En el caso de las rayas de agua dulce, no existe tal antecedente, por lo tanto, existen amplias posibilidades de investigación, desarrollo, innovación y transferencia tecnológica. Currently, the known vNAR sequences are obtained from shark (WO2011056056 A2) but they have the disadvantage that they could not recognize human glycated hemoglobin biomarkers, since they are selected against soluble proteins of another nature. Ray vNARs can be obtained from freshwater organisms, reducing the cost associated with the capture or maintenance of sharks. It should be considered that there are biotechnology-based companies that develop research, development and innovation basing their technology on shark vNAR sequences, which generates competition between them. In the case of freshwater rays, there is no such precedent, therefore, there are wide possibilities for research, development, innovation and technology transfer.
Los fragmentos de anticuerpos reportados en los documentos de patentes EP3133085A1, CN106255704A, WO2019148089A1, WO2018141964A1 y US20100092470A1 están dirigidos principalmente a terapia de cáncer y no se ha reportado ningún anticuerpo de características semejantes a los vNAR con capacidad de reconocimiento a hemoglobina glicada humana y sus variantes. The antibody fragments reported in patent documents EP3133085A1, CN106255704A, WO2019148089A1, WO2018141964A1 and US20100092470A1 are mainly aimed at cancer therapy and no antibodies with characteristics similar to vNARs with the ability to recognize human glycated hemoglobin and its variants have been reported. .
La presente invención pretende contrarrestar las desventajas antes mencionadas, mediante la aportación de un conjunto de secuencias de ADN y su traducción a proteínas de dominios vNAR aislados de raya dulce Potamotrygon schroederi, estas secuencias reconocen específicamente el biomarcador de hemoglobina glicada humana y sus variantes, principalmente glucosa en la lisina o valina del amino terminal de la hemoglobina glicada y se puede emplear para el monitoreo y control de enfermedades metabólicas. The present invention aims to counteract the aforementioned disadvantages, by providing a set of DNA sequences and their translation into proteins of vNAR domains isolated from the sweet ray Potamotrygon schroederi, these sequences specifically recognize the human glycated hemoglobin biomarker and its variants, mainly glucose in the amino terminal lysine or valine of glycated hemoglobin and can be used for the monitoring and control of metabolic diseases.
Los detalles característicos de la presente invención se muestran claramente en la siguiente descripción detallada de algunas de sus realizaciones preferentes, respaldada con los ejemplos y figuras que se acompañan de manara ilustrativa, mas no limitativa, en donde: The characteristic details of the present invention are clearly shown in the following detailed description of some of its preferred embodiments, supported with the examples and figures that are accompanied by an illustrative, but not limiting, manner, where:
Las figural es una fotografía de los geies de poliacrilamida SDS-PAGE de la extracción y purificación de una proteína vNAR R007 La figura 2 es una fotografía de los goles de po8acrilamkla SDS-PAGE de la extracción y purificación de una proteína vNAR R016. Figure 2 is a polyacrylamide SDS-PAGE photograph of the extraction and purification of a vNAR R007 protein. Figure 2 is a polyacrylamide SDS-PAGE photograph of the extraction and purification of a vNAR R016 protein.
La figura 3 es una fotografía de una membrana de transferencia donde se muestra la detección de las proteínas recombinantes vNAR R007 y R016, por Western Blot. Figure 3 is a photograph of a transfer membrane showing the detection of the recombinant proteins vNAR R007 and R016, by Western Blot.
La figura 4 es un comparativo gráfico de la prueba de ELISA de reconocimiento a hemoglobina glicada humana y sus variantes porcada proteína vNAR (*P < 0.05, ***P < 0.001). Figure 4 is a graphic comparison of the human glycated hemoglobin recognition ELISA test and its variants for each vNAR protein (*P < 0.05, ***P < 0.001).
La figura 5 es un comparativo gráfico de la prueba de ELISA de reconocimiento a hemoglobina glicada en muestras de sangre total y suero, por las proteínas vNAR (~*P < 0.001). Figure 5 is a graphical comparison of the ELISA test for recognition of glycated hemoglobin in whole blood and serum samples, by vNAR proteins (~*P < 0.001).
La figura 6 es una gráfica donde se ilustra la determinación de límite de detección de hemoglobina glicada por ELISA proteína vNAR R016 de origen insoluble y soluble (**P < 0.01, ***P < 0.001). La figura 7 es un gráfico de una prueba de ELISA de reconocimiento selectivo a hemoglobina glicada en una muestra de hemoglobina sin gllcar mezclada con hemoglobina glcada (***P < 0.001). Figure 6 is a graph illustrating the detection limit determination of glycated hemoglobin by ELISA protein vNAR R016 of insoluble and soluble origin (**P < 0.01, ***P < 0.001). Figure 7 is a graph of an ELISA test for selective recognition of glycated hemoglobin in a sample of non-glycated hemoglobin mixed with glycated hemoglobin (***P < 0.001).
La figura 8 es una gráfica de una prueba de ELISA de reconocimiento a hemoglobina glicada en sangre total usando la proteína vNAR conjugada (AuNP's-R016) y sin conjugar (R016) a nanopartículas de oro. Figure 8 is a graph of an ELISA test for recognition of glycated hemoglobin in whole blood using vNAR protein conjugated (AuNP's-R016) and unconjugated (R016) to gold nanoparticles.
La figura 9 A) es un gráfico donde se observan las curvas de porcentaje de hemoglobina glicada y las Mezcla 1, 2 de vNAR R016 y Mezcla 3 de AuNPs- R016, B) es un gráfico de evaluación de estabilidad de reconocimiento de AuNPs· R016 por hemoglobina glicada, en diferentes tiempos. La figura 10 A) es una fotografía de la reacción macroscópica de la mezcla AuNP's-R007 con sangre Usada. B) es un gráfico que lustra el Perfil de color RGB de las mezclas de AuNP's-R007 con sangre Usada. Figure 9 A) is a graph showing the percentage curves of glycated hemoglobin and Mixture 1, 2 of vNAR R016 and Mixture 3 of AuNPs-R016, B) is a graph of recognition stability evaluation of AuNPs R016 by glycated hemoglobin, at different times. Figure 10 A) is a photograph of the macroscopic reaction of the AuNP's-R007 mixture with Used blood. B) is a graph illustrating the RGB color profile of AuNP's-R007 mixtures with Used blood.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
La presente invención tiene como primer objeto una proteína vNAR aislada y purificada, la cual comprende: una secuencia de aminoácidos de la primera región determinante de complementariedad (CDR1) que es seleccionada del siguiente grupo de secuencias de aminoácidos: DTSHILSGTK (SEQ. ID. NO. 1 ), DTSCGLYSTS (SEQ. ID. NO. 5), o sus variantes sustancialmente similares a ellas; y una secuencia de aminoácidos de la tercera región determinante de compiementariedad (CDR3) seleccionada del siguiente grupo de secuencias de aminoácidos: QTIGRRQTLHTGIGAMWDSTSD (SEQ. ID. NO. 2). QAGGRLCVGGGNY (SEQ. ID NO.6), o sus variantes sustancialmente similares a ellas. The present invention has as its first object an isolated and purified vNAR protein, which comprises: an amino acid sequence of the first complementarity determining region (CDR1) that is selected from the following group of amino acid sequences: DTSHILSGTK (SEQ. ID. NO 1), DTSCGLYSTS (SEQ. ID. NO. 5), or substantially similar variants thereof; and a third complementary determining region (CDR3) amino acid sequence selected from the following group of amino acid sequences: QTIGRRQTLHTGIGAMWDSTSD (SEQ. ID. NO. 2). QAGGRLCVGGGNY (SEQ. ID NO.6), or substantially similar variants thereof.
Una modalidad de la proteína vNAR de la presente invención es cuando, la CDR1 es la secuencia de aminoácidos SEQ. ID. NO. 1; y la CDR3 es la secuencia de aminoácidos SEQ. ID. NO. 2. An embodiment of the vNAR protein of the present invention is when the CDR1 is the amino acid sequence SEQ. ID NO. one; and CDR3 is the amino acid sequence SEQ. ID NO. two.
Otra modalidad de la proteína según la presente Invención es cuando la CDR1 es la secuencia de aminoácidos SEQ. ID. NO. 5; y la CDR3 es la secuencia de aminoácidos SEQ. ID. NO. 6. Another embodiment of the protein according to the present invention is when the CDR1 is the amino acid sequence SEQ. ID NO. 5; and CDR3 is the amino acid sequence SEQ. ID NO. 6.
Una modalidad más de la proteína de acuerdo con la presente Invención es cuando comprende, una de las siguientes secuencias de aminoácidos: SEQ. ID. No. 4, SEQ. ID. NO. 8, o sus variantes sustancialmente similares a ellas. La proteína vNAR de acuerdo con esta Invención, es de origen Potamotrygon schroederi, y se encontró que tiene la capacidad de reconocimiento del blanco o biomarcador que corresponde a la subunidad beta de la hemoglobina glicada de humano. Un segundo objeto de la presente Invención es una secuencia de AON, la cual comprende una secuencia de nudeótídos que codifica a la proteína vNAR antes descrita. En una realización preferente de la presente invención, es cuando la secuencia de ADN, comprende una de las siguientes secuencias de nucteótidos: SEQ. ID NO. 3, o SEQ. ID. NO. 7. One more embodiment of the protein according to the present invention is when it comprises one of the following amino acid sequences: SEQ. ID No. 4, SEQ. ID NO. 8, or substantially similar variants thereof. The vNAR protein according to this invention is of Potamotrygon schroederi origin, and it was found to have the ability to recognize the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin. A second object of the present invention is an AON sequence, which comprises a nucleotide sequence that encodes the vNAR protein described above. In a preferred embodiment of the present invention, it is when the DNA sequence comprises one of the following nucleotide sequences: SEQ. ID NO. 3, or SEQ. ID NO. 7.
Un tercer objeto de la Invención en cuestión, es que también se refiere a una secuencia de ADN recombinante, la cual comprende: las secuencias de AON de conformidad con la presente invención. A third object of the Invention in question is that it also refers to a recombinant DNA sequence, which includes: the AON sequences according to the present invention.
Dicha invención tiene como cuarto objeto, una composición que reconoce el blanco o blomarcador de la subunidad beta de la hemoglobina glicada humana, donde dicha composición comprende: al menos, una proteína vNAR, tal y como lo propone la presente Invención. Una realización de dicha composición es cuando además comprende: al menos, una sustancia que aumente su eficiencia de uso en el reconocimiento del blanco o blomarcador que corresponde a la subunidad beta de la hemoglobina glicada de humano. Tal sustancia que aumenta la eficiencia de uso es seleccionada del siguiente grupo: nanopartículas, cromóforos, proteínas y/o una combinación entre ellas. Las nanopartículas pueden ser oro, látex, y/o su combinación entre ellos, por citar algunos ejemplos. Como quinto objeto de la presente invención es un método ex vivo para el reconocimiento del blanco o blomarcador que corresponde a la subunidad beta de la hemoglobina glicada de humano, dicho método comprende las siguientes etapas: proveer una muestra de sangre de un páctente sospechoso de diabetes; contactar la muestra de sangre con la composición que reconoce el blanco o blomarcador de la subunidad beta de la hemoglobina glicada humana, de conformidad con la presente invención; y detectar la presencia de hemoglobina glicada, en donde la proteína vNAR de dicha composición, se une a la subunidad beta de la hemoglobina glicada humana; donde dicha detección es mediante los siguientes inmunoensayos: ELISA, DotBlot, Western blot, aglutinación, inmunoturbidimetría, inmunofluorescencia, tiras de flujo lateral (LFA), pruebas de punto de atención (POC), entre otros. The fourth object of said invention is a composition that recognizes the target or biomarker of the beta subunit of human glycated hemoglobin, where said composition comprises: at least one vNAR protein, as proposed by the present invention. One embodiment of said composition is when it also comprises: at least one substance that increases its efficiency of use in the recognition of the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin. Such a substance that increases the efficiency of use is selected from the following group: nanoparticles, chromophores, proteins and/or a combination between them. The nanoparticles can be gold, latex, and/or a combination of them, to name a few examples. As a fifth object of the present invention is an ex vivo method for the recognition of the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin, said method comprises the following steps: provide a blood sample from a patient suspected of diabetes ; contacting the blood sample with the composition that recognizes the human glycated hemoglobin beta subunit target or biomarker, in accordance with the present invention; Y detecting the presence of glycated hemoglobin, wherein the vNAR protein of said composition binds to the beta subunit of human glycated hemoglobin; where said detection is by means of the following immunoassays: ELISA, DotBlot, Western blot, agglutination, immunoturbidimetry, immunofluorescence, lateral flow strips (LFA), point-of-care tests (POC), among others.
Un sexto objeto de la presente invención es el uso de la proteína vNAR, y la composición, tal y como se describe en la presente invención, para el reconocimiento del blanco o biomarcador que corresponde a la subunidad beta de la hemoglobina glicada de humano. A sixth object of the present invention is the use of the vNAR protein, and the composition, as described in the present invention, for the recognition of the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin.
EJEMPLOS EXAMPLES
Los siguientes ejemplos, Ilustran algunas de las realizaciones y obtenciones de la presente invención, los cuales deben ser considerados meramente ilustrativos y no limitativos a los alcances de la presente invención. The following examples illustrate some of the embodiments and obtainments of the present invention, which should be considered merely illustrative and not limiting to the scope of the present invention.
Eiemplo 1. Se llevé acabo un protocolo con la finalidad da obtener un dominio variable vNAR con capacidad de reconocimiento a la hemoglobina glicada en una muestra de sangre total, dicho protocolo se llevó acabo da la manera siguiente. Example 1. A protocol was carried out in order to obtain a vNAR variable domain with recognition capacity for glycated hemoglobin in a whole blood sample, said protocol was carried out as follows.
1. Generación de una biblioteca génlca no inmune, a partir del bazo de una raya de agua dulce de la especie Potamotrygon schroederi 1. Generation of a non-immune gene library from the spleen of a freshwater stingray of the species Potamotrygon schroederi
1.1. Extracción de ARN y obtención de fragmentos vNAR. 1.1. RNA extraction and obtaining vNAR fragments.
Se pesó el bazo de una raya de agua dulce de la especie Potamotrygon schroederi no inmunizado, se realizó ia extracción de ARNt (ARNtotal) empleando reactivos comerciales. Las reacciones de retrotranscrlpdón se realizaron con el ARNt y Oligo(dT)18. La amplificación de fragmentos se realizó por reacción en cadena de la polimerasa (POR) empleando los oligonucleótidos específicos que Incluyen sitios de restricción (W02011056058A2). Los fragmentos se analizaron por gel de agarosa al 1.5%. Se empleó el fagómido pComb3x y se realizó el protocolo para amplificación confórme a lo escrito en Barbas et al., 2001. 2. Selección y caracterización de vNAR. The spleen of a non-immunized freshwater stingray of the species Potamotrygon schroederi was weighed, and tRNA (total RNA) was extracted using commercial reagents. Reverse transcription reactions were performed with tRNA and Oligo(dT)18. Fragment amplification was performed by polymerase chain reaction (POR) using specific oligonucleotides that include restriction sites (W02011056058A2). The Fragments were analyzed by 1.5% agarose gel. The pComb3x phagemid was used and the amplification protocol was carried out in accordance with what was written in Barbas et al., 2001. 2. Selection and characterization of vNAR.
2.1. Despliegue en fagos: rondas de selección. 2.1. Phage display: rounds of selection.
Se inmovilizaron 500 pg de hemoglobina glicada comercial en placa de ELISA toda la noche a 4 °C. Este procedimiento se realizó en cada ronda, confórme a lo descrito en Barbas ef al., 2001. Para la búsqueda de clonas con fragmento vNAR se partió de las placas obtenidas, se seleccionaron colonias y se amplificaron los fragmentos por PCR empleando los oligonudeótidos específicos Ompseq y Gback (Barbas et al., 2001). Se extrajo plásmklo usando un kit comercial. Se realizó un análisis /n SÍ V/CO de las secuencias obtenidas, se seleccionaron las secuencias en correcto marco de lectura. También, se realizó un alineamiento de las clonas seleccionadas con las secuencias reportadas en el National Center for Btotechrtdogy Information (NCBI). 500 pg of commercial glycated hemoglobin was immobilized in an ELISA plate overnight at 4 °C. This procedure was performed in each round, as described in Barbas et al., 2001. To search for clones with vNAR fragments, the plates obtained were used as starting points, colonies were selected and the fragments were amplified by PCR using the specific oligonucleotides Ompseq and Gback (Barbas et al., 2001). Plasma was extracted using a commercial kit. An /n YES V/CO analysis of the obtained sequences was performed, the sequences in the correct reading frame were selected. Also, an alignment of the selected clones with the sequences reported in the National Center for Biology Information (NCBI) was performed.
2.2. Expresión recombinante de proteína vNAR contra hemoglobina glicada. 2.2. Recombinant expression of vNAR protein against glycated hemoglobin.
2.2.1. Clonación y transformación de fragmentos vNAR en un vector de expresión. 2.2.1. Cloning and transformation of vNAR fragments into an expression vector.
Los fragmentos codificantes de vNAR se amplificaron por PCR con oligonudeótidos específicos incluyendo con sitios de restricción para Ncol y Xhol. Se digirieron el plásmido pET-28a+ y los fragmentos vNAR, la ligación se realizó con T4 ligase en una proporción 1:3 (vector inserto). Se transformaron en células E. coli cepa TG1 y células BL21(DE). Posteriormente, se verificó la ligación por PCR de colonia, empleando oligonudeótidos específicos para la secuencia T7 del plásmido pET-28a+. Se obtuvo plásmido y después de su secuendación se realizó un análisis in silico con ei programa CLC viewer 8.0. La inducción se realizó con IPTG 1mM cuando la DOeoo fue de 0.8 y se incubaron a 37 ºC durante 5 h a 300 rpm. 2.2.2. Expresión, purificación y detección de proteínas recombinantes vNAR.The vNAR coding fragments were amplified by PCR with specific oligonucleotides including restriction sites for Ncol and Xhol. Plasmid pET-28a+ and vNAR fragments were digested, ligation was performed with T4 ligase in a 1:3 ratio (insert vector). They were transformed into E. coli strain TG1 cells and BL21(DE) cells. Subsequently, ligation was verified by colony PCR, using specific oligonucleotides for the T7 sequence of plasmid pET-28a+. Plasmid was obtained and after sequencing, an in silico analysis was performed with the CLC viewer 8.0 program. Induction was performed with 1 mM IPTG when the DOeoo was 0.8 and incubated at 37 ° C for 5 300 rpm. 2.2.2. Expression, purification and detection of recombinant vNAR proteins.
Se evaluaron tres métodos para la extracción de proteínas recombinantes a partir de cuerpos de Inclusión, para cada proteína, en seguida se describen los métodos seleccionados para cada vNAR (Pérez y Camacho, 2019; y Camacho y Pérez, 2019). El pelet bacteriano de 100 mL de cultivo se resuspendió en 4 mL para vNAR R007 o en 5 mL para vNAR R016 del amortiguador de sonicado, el sobrenadante corresponde a proteína soluble. Para la extracción desnaturalizante del vNAR R007 se empleó el método de Maggi & Scottf, 2017. Para vNAR R016 se empleó el replegado en columna del manual GE Healthcare, 1999. Three methods for the extraction of recombinant proteins from inclusion bodies were evaluated for each protein; the methods selected for each vNAR are described below (Pérez and Camacho, 2019; and Camacho and Pérez, 2019). The bacterial pellet of 100 mL of culture was resuspended in 4 mL for vNAR R007 or in 5 mL for vNAR R016 of the sonicated buffer, the supernatant corresponding to soluble protein. For the denaturing extraction of vNAR R007, the method of Maggi & Scottf, 2017 was used. For vNAR R016, the column folding of the GE Healthcare manual, 1999, was used.
La purificación se realizó por gradiente de imidazol. La resina se lavó con 10 volúmenes de columna de amortiguador de lavado (50 mM NaHaPOK, 300 mM NaCI, 20 mM de imidazol pH 8), seguido de un lavado con 10 volúmenes de columna del amortiguador anterior, pero conteniendo 50 mM de Imidazol. Se eluyó por gradiente de Imidazol, adicionado con 150, 200, 250 y 300 mM de imidazol respectivamente al amortiguador Inicial. Posteriormente, cada fracción fue analizada por SDS-PAGE, tomando 10 μL de cada fracción. Las eluciones de mayor pureza se mezclaron y se dializaron en una membrana con poro de 3.5 kDa contra amortiguador de diálisis (50 mM NaH2PO4, 300 mM NaCI, 10 mM de Imidazol pH 8), se realizaron varios recambios. Se filtró (0.22 pm) y se cuantiflcó por kit comercial según las indicaciones del proveedor. Purification was performed by imidazole gradient. The resin was washed with 10 column volumes of wash buffer (50 mM NaHaPOK, 300 mM NaCl, 20 mM imidazole pH 8), followed by a wash with 10 column volumes of the above buffer, but containing 50 mM Imidazole. It was eluted by Imidazole gradient, added with 150, 200, 250 and 300 mM of imidazole respectively to the Initial buffer. Subsequently, each fraction was analyzed by SDS-PAGE, taking 10 μL of each fraction. The highest purity elutions were pooled and dialyzed on a 3.5 kDa pore membrane against dialysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM Imidazole pH 8), several exchanges were performed. It was filtered (0.22 pm) and quantified using a commercial kit according to the supplier's instructions.
Por electrotransferencia semiseca se transfirieron 5 pg de cada proteína vNAR a una membrana de nitrocelulosa. Después del bloqueo se Incubó con anti-HIs- HRP (1:5,000) a 4 ºC durante 16 h, después de tres lavados se agregó la solución de revelado conteniendo 2.7 mg de sustrato HRP diluido en 1 mL de metanol frío, 4.5 mL de PBS 1X y 15 μL de peróxido de hidrógeno. Se agregó la solución de revelado y se Incubó durante 10 mln. By semi-dry electroblotting, 5 pg of each vNAR protein was transferred to a nitrocellulose membrane. After blocking, it was incubated with anti-HIs-HRP (1:5,000) at 4 ºC for 16 h. After three washes, the development solution was added containing 2.7 mg of HRP substrate diluted in 1 mL of cold methanol, 4.5 mL of 1X PBS and 15 µL of hydrogen peroxide. Developing solution was added and incubated for 10 min.
2.3. Selección de vNAR por analisis de reconocimiento a hemoglobina glicada comercial y en sangre total por ELISA. Se colocaron 500 ng de HbA1c, HbAGIy, HbAlab, HbAO (Exocefl) comerciales. Se empleó BSA3% como control negativo. Todas las incubaciones de la placa se realizaron por 1h a 37 °C. Después del bloqueo se agregaron 500 ng de vNAR R007 y R016. Después de los lavados se agregaron 50 μL de anti-Hb (1:5,000) en BSA 1%-PBS 1x. Todose los lavados se repitieron 3 veces y las Incubaciones fueron de 1 h a 37 °C. La placa se reveló con 50 μL de TMB Ultra, se incubó a 37 *C por 15 min y se analizó en el lector de microptacas para absorbencia a 652 nm. Para evaluar el reconocimiento a sangre total, las muestres fueron Usadas por congelación a -20 °C según el protocolo descrito por SeMn et al., 2005 y Stoviter, 1962, y se empleó a una dilución 1 :5,000 con agua. El ensayo se realizó bajo las mismas condiciones anteriormente descritas. El porcentaje de hemoglobina glicada que contenían las muestras fueron de 9.6 y 5.2%. 23. Selection of vNAR by recognition analysis of commercial glycated hemoglobin and in whole blood by ELISA. 500 ng of commercial HbA1c, HbAGIy, HbAlab, HbAO (Exocefl) were placed. BSA3% was used as a negative control. All plate incubations were performed for 1 h at 37 °C. After blocking 500 ng of vNAR R007 and R016 were added. After washing, 50 μL of anti-Hb (1:5,000) in 1% BSA-1x PBS were added. All washes were repeated 3 times and incubations were 1 h at 37 °C. The plate was revealed with 50 μL of TMB Ultra, incubated at 37 *C for 15 min and analyzed in the microplate reader for absorbance at 652 nm. To evaluate the recognition of whole blood, the samples were used by freezing at -20 °C according to the protocol described by SeMn et al., 2005 and Stoviter, 1962, and a 1:5,000 dilution with water was used. The assay was performed under the same conditions described above. The percentage of glycated hemoglobin contained in the samples was 9.6 and 5.2%.
2.4. Determinación de límite de detección de hemoglobina glicada por el vNAR R016. 2.4. Determination of detection limit of glycated hemoglobin by vNAR R016.
Se analizó con una ELISA con 50 μL de hemoglobina glicada (10 pg/mL), después del bloqueo se agregaron 1000, 500, 250, 50 y 0 ng del vNAR R016 diluido en 50 μL, extraído de manera soluble e insoluble. Se lavaron los pozos y se agregaron 50 μL de antí-Hb-HRP (1:5000) Se repitió el lavado y el revelado como se declaró anteriormente. It was analyzed with an ELISA with 50 μL of glycated hemoglobin (10 pg/mL), after blocking 1000, 500, 250, 50 and 0 ng of vNAR R016 diluted in 50 μL, extracted in soluble and insoluble form, were added. Wells were washed and 50 µL of anti-Hb-HRP (1:5000) was added. Washing and development were repeated as stated above.
2.5. Anállsto de selectividad a hemoglobina glicada por el vNAR R016. 2.5. Selectivity analysis for glycated hemoglobin by vNAR R016.
Para analizar la selectividad por el vNAR R016, se utilizaron mezclas de HbAGIy y HbAO comerciales. Se utilizaron 500 ng totales de la mezcla de hemoglobinas, se mezclaron 375, 250 y 125 ng de las hemoglobinas en relación de HbAGIy/HbAO quedando de 3:1 , 1:1 y 1:3. Como control negativo se empleó BSA 3%- PBS 1x. Después del bloqueó se agregaron 500 ng del vNAR R016. Después dee los lavados, se agregaron 50 μL de antí-Hb-HRP (1 :5000). Se repitió el lavado y el revelado como se declaró anteriormente. To analyze the selectivity for vNAR R016, mixtures of commercial HbAGIy and HbAO were used. A total of 500 ng of the hemoglobin mixture was used, 375, 250 and 125 ng of the hemoglobins were mixed in the ratio of HbAGIy/HbAO, remaining 3:1, 1:1 and 1:3. As a negative control, 3% BSA-1x PBS was used. After blocking, 500 ng of vNAR R016 were added. After washing, 50 μL of anti-Hb-HRP (1:5000) were added. Washing and development were repeated as stated above.
3. Evaluación de reconocimiento a hemoglobina gibada por vNAR R016 conjugado con nanoparticulas de oro. 3.1. Síntesis y conjugación de proteína vNAR R016 con nanoparticulas de oro.3. Evaluation of recognition of gibbet hemoglobin by vNAR R016 conjugated with gold nanoparticles. 3.1. Synthesis and conjugation of vNAR R016 protein with gold nanoparticles.
Las nanoparticulas se generaron con ácido cloroáurico (HAuCM, SIGMA cat. 254169) a 0.1 M y cirato de sodio a 0.5 M con agua hirviendo en agitación constante y con base en lo descrito por Panlkar ef a/., 2019; Sitó et al , 2012. Una vez estabilizadas las AuNP's, se prepararon para conjugar diluyendo al 50% con agua MiliQ y agregando NaOH a una concentración final de 0.4 mM para un pH final de 6.1. The nanoparticles were generated with chloroauric acid (HAuCM, SIGMA cat. 254169) at 0.1 M and sodium cirate at 0.5 M with boiling water under constant stirring and based on what is described by Panlkar ef a/., 2019; Sitó et al , 2012. Once the AuNP's were stabilized, they were prepared for conjugation by diluting to 50% with MiliQ water and adding NaOH to a final concentration of 0.4 mM for a final pH of 6.1.
Para realizar la conjugación del vNAR con nanoparticulas de oro (AuNP's), la proteína vNAR R007 previamente fue dializada con amortiguador de conjugación (25 mM NaH2PO4, 75 mM NaCI, pH 7) y la protelna vNAR R015 fue dlallzada con amortiguador HEPES (15 mM HEPES, 10 mM NaCI, 1% glicerol, pH 9). To conjugate vNAR with gold nanoparticles (AuNP's), the vNAR R007 protein was previously dialyzed with conjugation buffer (25 mM NaH 2 PO 4 , 75 mM NaCl, pH 7) and the vNAR R015 protein was dialyzed with HEPES buffer. (15 mM HEPES, 10 mM NaCl, 1% glycerol, pH 9).
3.2. Optimización de conjugación de vNAR R016 con nanoparticulas de oro. Se optimizó la conjugación de las AuNP's y el vNAR R016, adaptan*) el protocolo descrito en el kit de Gold Nanopartide Conjugation Optimizatlon Kit (Cytodiagnostics) usando las AuNP's previamente sintetizadas (DO520nm de 1). Se prepararon diluciones de la proteína vNAR R016 correspondientes a 0, 0.15, 0.3, 0.45, 0.6 y 0.7 pg/pL como concentración final en pozo en HEPES (15 mM HEPES, 10 mM NaCI, 1% glicerol) con diferentes pH de 7 hasta 11. Se calculó la agregación de las AuNP's con la relación de las absorbencias obtenidas a 690/560 nm, donde el resultado óptimo es cercano a cero. Posteriormente, para R007 se agregaron 50 μL de vNAR R007 (2 ng/pL) y para R016 se agregaron 20 μL de vNAR R016 (0.45 μg/pL) a 1 mL de AuNP's a DO520nm de 1 (pH 6.1), se incubaron por 5 min. Para verificar la conjugación se realizó un espectro de absorbencia de 450 a 700 nm en Nanodrop 2000 y se comparó con AuNP's sin conjugar. 3.2. Conjugation optimization of vNAR R016 with gold nanoparticles. The conjugation of the AuNP's and the R016 vNAR was optimized, adapt*) the protocol described in the Gold Nanopartide Conjugation Optimization Kit (Cytodiagnostics) using the previously synthesized AuNP's (OD520nm of 1). Dilutions of the vNAR R016 protein corresponding to 0, 0.15, 0.3, 0.45, 0.6 and 0.7 pg/pL were prepared as final concentration in well in HEPES (15 mM HEPES, 10 mM NaCl, 1% glycerol) with different pH from 7 to 11. The aggregation of the AuNP's was calculated with the ratio of the absorbances obtained at 690/560 nm, where the optimal result is close to zero. Subsequently, for R007, 50 μL of vNAR R007 (2 ng/pL) were added and for R016, 20 μL of vNAR R016 (0.45 μg/pL) were added to 1 mL of AuNP's at OD520nm of 1 (pH 6.1), incubated for 5 minutes To verify the conjugation, an absorbance spectrum from 450 to 700 nm was performed in Nanodrop 2000 and compared with unconjugated AuNP's.
3.3 Comparación de reconocimiento a hemoglobina glicada por vNAR R016 conjugado a nanoparticulas de oro y sin conjugar. 3.3 Comparison of recognition of glycated hemoglobin by vNAR R016 conjugated to gold nanoparticles and unconjugated.
I. Análisis de reconocimiento par ELISA Para el vNAR R016 conjugado y no conjugado a AuNP’s se colocó sangre lisada con hemoglobina glicada de 9.6, 7.5 y 4.9% diluida 1:1000 en agua. Se empleó BSA como control negativo de reconocimiento, por pozo y por triplicado. Después del bloqueo se agregó 50 μL del vNAR R016 sin conjugar (10 ng/pL) y del conjugado AuNP‘s-R016 diluido 1 :13 en amortiguador HEPES. Después del lavado, se agregó 50 μL de antl-His-HRP (1:5000). Se repitió el lavado y el revelado como se declaró anteriormente. I. ELISA Recognition Assay For the vNAR R016 conjugated and not conjugated to AuNP's, blood lysed with glycated hemoglobin of 9.6, 7.5 and 4.9% diluted 1:1000 in water was placed. BSA was used as negative recognition control, per well and in triplicate. After blocking, 50 μL of unconjugated R016 vNAR (10 ng/pL) and conjugated AuNP's-R016 diluted 1:13 in HEPES buffer were added. After washing, 50 μL of antl-His-HRP (1:5000) was added. Washing and development were repeated as stated above.
II. Análisis de reconocimiento por aglutinación y absorbencia. Para los ensayos se empleó el kit comercial de "Determinación cuantitativa de la hemoglobina glicosilada” (SPINREACT®) el resultado se midió por absorbencia y se comparó con una curva de calibración realzada en cada ensayo para valdar los resultados siguiendo sus instrucciones de uso. También se diluyó la muestra de sangre total (que contenían 9.6, 7.0 y 5.2% de hemoglobina glicada). Se empleó el vNAR R016 en: Mezcla 1) el vNAR R016 0.7 mg/mL con anti-His (0.001 mg/mL) y anti-ratón (0.001 mg/mL), Mezcla 2) el vNAR R0160.14 mg/mL con anti-His (0.001 mg/mL) y anti-ratón (0.001 mg/mL) o, Mezcla 3) las AuNP's - vNAR R016 sin adicionar ningún anticuerpo. Los resultados se analizaron a 660 nm. iii. Análisis de reconocimiento de la AuNP's- vNAR R007 por cambio de color Se emplearon 40 μL de AuNP’s-R007 y 4 μL de sangre Usada diluida 1:100, con porcentaje de hemoglobina glcada de 4.9, 7.0 y 9.6 %. Se incubó durante 20 minutos y se colocaron 20 μL en discos de fibra de vidrio de 6 mm de diámetro. Inmediatamente se adquirió una fotografía, se analizó el perfil de color RGB y la saturación de las muestras empleando la aplicación Color Grab versión 3.6.1. II. Recognition analysis by agglutination and absorbency. For the tests, the commercial kit for "Quantitative determination of glycosylated hemoglobin" (SPINREACT®) was used. The result was measured by absorbance and compared with a calibration curve carried out in each test to validate the results following its instructions for use. Also whole blood sample (containing 9.6, 7.0 and 5.2% glycated hemoglobin) was diluted vNAR R016 was used in: Mix 1) vNAR R016 0.7 mg/mL with anti-His (0.001 mg/mL) and anti -mouse (0.001 mg/mL), Mix 2) the vNAR R0160.14 mg/mL with anti-His (0.001 mg/mL) and anti-mouse (0.001 mg/mL) or, Mix 3) the AuNP's - vNAR R016 without adding any antibody. The results were analyzed at 660 nm. III. AuNP's-vNAR R007 recognition analysis by color change. 40 μL of AuNP's-R007 and 4 μL of used blood diluted 1:100 were used, with a percentage of glycated hemoglobin of 4.9, 7.0 and 9.6% It was incubated for 20 minutes and 20 μL were placed in glass fiber discs of 6 mm diameter. or. A photograph was immediately acquired, the RGB color profile and saturation of the samples were analyzed using the Color Grab version 3.6.1 application.
4. Análisis estadístico. Los resultados fueron analizados con el programa GradPhad Prism 5 con la prueba post-ANOVA de comparación múltiple Bonfenroni. Ejemplo 2. Con al método antas descrito, aa obtuyieroq los gigujfflteS resultados: 4. Statistical analysis. The results were analyzed with the GradPhad Prism 5 program with the Bonfenroni multiple comparison post-ANOVA test. Example 2. With the previously described method, we obtained the gigujfflteS results:
A. Construcción de la biblioteca de fragmentos vNAR. A. Construction of the vNAR fragment library.
1. Biblioteca de fragmentos vNAR y despliegue en fagos A partir del bazo de una raya de agua dulce de la especie Potamotrygon schroederi, se obtuvo ARN a una concentración de 537.8 ng/pL. con una relación 260/280 de 2.02. Se generó la biblloteca y en el despliegue en fagos se obtuvieron 3.26 x 10*. 7.32 x 10* y 5 x 10* unidades formadoras de colonia (UFC/mL) para las rondas 1, 2 y 3, respectivamente. Se eligieron 36 colonias al azar y se analzaron por PCR de colonia para corroborar la presencia de los fragmentos vNAR, se obtuvieron 24 colonias positivas. Se extrajo el plásmido y se obtuvo la secuencia. 1. Library of vNAR fragments and phage display From the spleen of a freshwater stingray of the species Potamotrygon schroederi, RNA was obtained at a concentration of 537.8 ng/pL. with a 260/280 ratio of 2.02. The library was generated and 3.26 x 10* were obtained on phage display. 7.32 x 10* and 5 x 10* colony forming units (CFU/mL) for rounds 1, 2 and 3, respectively. 36 colonies were chosen at random and analyzed by colony PCR to corroborate the presence of vNAR fragments, 24 positive colonies were obtained. The plasmid was extracted and the sequence obtained.
B. Caracterización de vNAR B. Characterization of vNAR
1. Análisis de secuencias in silico. 1. In silico sequence analysis.
De las donas enviadas a secuenciar, se descartaron las secuencias que tenían poca variabilidad, quedando dos secuencias como candidatos potenciales para su expresión. Estas clonas fueron denominadas como R007 y R016, sus secuencias corresponden a las siguientes secuencias de amlnoáddos: AWVDQTPRTATRETGESLSINfiGLTDTSHILSGTKWFWNNPGSTDWESITIG GRYVESVNNQAKSFSLQIKDLTVEDSGTYYCKAQTIGRRQTLHTGIGAMWDS TSDYDGAGTVLTVN (SEQ. ID. NO. 4) yOf the donuts sent for sequencing, the sequences that had little variability were discarded, leaving two sequences as potential candidates for their expression. These clones were named R007 and R016, their sequences correspond to the following amlnoad sequences: AWVDQTPRTATRETGESLSINfiGLTDTSHILSGTKWFWNNPGSTDWESITIG GRYVESVNNQAKSFSLQIKDLTVEDSGTYYCKAQTIGRRQTLHTGIGAMWDS TSDYDGAGTVLTVN (SEQ. ID. NO. 4) and
ATRVDQTPREATKQPGETLTINfiVLRDTSCGLYSTSWFVQRPGRSAWDRLSI GGRYAESVNKPAKSFSLRISNLIAEDSATYFCKAQAGGRLCVGGGNYYGGAG TVLTVN (SEQ. ID. NO. 8), respectivamente. Las clonas R007 y R016 tienen un porcentaje de Identidad del 85 y 68 %, respectivamente con los vNAR reportados de diferentes elasmobranqulos (por ejemplo, las secuencias con número de acceso NCBI de AAX10140.1 y AAT02204.1, respectivamente). Los vNAR aislados no han sido reportados con anterioridad. Además, sólo tienen coincidencias con los dominios vNAR (Grifflths ef al., 2013) y no con fragmentos variables de otras hmunoglobulinas (por ejemplo, camélidos o humanos). Basado en las dstefnas canónicas y no canónicas los vNAR son clasificados en 4 tipos (Zieionka ef al., 2015), por lo tanto, R007 corresponde a un vNAR tipo IV y R016 es del tipo II. ATRVDQTPREATKQPGETLTINfiVLRDTSCGLYSTSWFVQRPGRSAWDRLSI GGRYAESVNKPAKSFSLRISNLIAEDSATYFCKAQAGGRLCVGGGNYYGGAG TVLTVN (SEQ. ID. NO. 8), respectively. Clones R007 and R016 have a percentage of Identity of 85 and 68%, respectively, with the vNARs reported from different elasmobranchs (for example, the NCBI accession number sequences of AAX10140.1 and AAT02204.1, respectively). Isolated vNARs have not been reported with anteriority. In addition, they only have matches with the vNAR domains (Grifflths et al., 2013) and not with variable fragments of other immunoglobulins (for example, camelids or humans). Based on canonical and non-canonical patterns, vNARs are classified into 4 types (Zieionka et al., 2015), therefore, R007 corresponds to a type IV vNAR and R016 is type II.
2. Expresión de proteínas vNAR. 2. Expression of vNAR proteins.
2.1. Extracción y purificación de proteína recombinante vNAR. Posterior a la transformación de las construcciones pET28a-vNAR en la cepa £ coi/ BL21(DE3) se realizó la inducción, extracción y purificación de vNAR. Para el vNAR R007, se procedió con la desnaturalización con urea y la renaturalización con glutatión oxidado y reducido. En la Figura 1A, primer extracto sonicado (S1 ), segundo extracto sonicado (S2), tercer extracto sonicado (S3), extracto desnaturalizado (ED), extracto renaturalizado (ER), extracto renaturalizado no retenido (NR) y el debris celular de extracto renaturalizado (D). Figura 1B) primer lavado (L1, 20 mM de imidazol), segundo lavado (L2, 50 mM de imidazol), elución 1 (E1, 150 mM de imidazol), elución 2 (E2, 200 mM de Imidazol), elución 3 (E3, 250 mM de Imidazol), elución 4 (E4, 300 mM de imidazol) y elución 5 (E5, 500 mM de imidazol). En el cuadro se indica la proteica purificada. Marcador de peso molecular (MPM) (Biorad). 2.1. Extraction and purification of recombinant vNAR protein. After the transformation of the pET28a-vNAR constructs in the £coi/BL21(DE3) strain, the induction, extraction and purification of vNAR was carried out. For vNAR R007, denaturation with urea and renaturation with oxidized and reduced glutathione were performed. In Figure 1A, first sonicated extract (S1), second sonicated extract (S2), third sonicated extract (S3), denatured extract (DE), renatured extract (ER), renatured non-retained extract (NR) and the cellular debris of renatured extract (D). Figure 1B) first wash (L1, 20 mM imidazole), second wash (L2, 50 mM imidazole), elution 1 (E1, 150 mM imidazole), elution 2 (E2, 200 mM imidazole), elution 3 ( E3, 250 mM Imidazole), elution 4 (E4, 300 mM imidazole), and elution 5 (E5, 500 mM imidazole). The purified protein is indicated in the table. Molecular Weight Marker (MPM) (Biorad).
B vNAR R016 fue extraído de manera desnaturalizante con guanldlna y se renaturalizó en columna por gradiente de urea en presencia de β- mercaptoetanoi. En la Figura 2A, extracto de sonicado con amortiguador A (SA), extracto de primer sonicado con amortiguador B (SB1). extracto de segundo sonicado con amortiguador B (SB2), extracto desnaturalizado (ED), extracto desnaturalizado no retenido (NR), lavado (LU, 6 M de urea), amortiguador renaturalizante 5 - 0 M de urea (R5 - R0), en la Figura 2B lavado (l_ 50 mM de imidazol), elución 1 (E1. 150 mM de imidazol), elución 2 (E2, 200 mM de imidazol), elución 3 (E3, 250 mM de imidazol), elución 4 (E4, 300 mM de imidazol) y elución 5 (E5, 500 mM de imidazol). En el cuadro se encierra la proteína purificada. Marcador de peso molecular (MPM). Después de la cuantificación se obtuvo el rendimiento total para el vNAR R007 (85.75 mg/L) es casi 20 veces mayor respecto al rendimiento de R016 (1.45 mg/L), ski embargo, ambas proteínas son capaces de reconocer la hemoglobina glicada humana. 2.2. Detección de proteína recombinante vNAR por Western blot. B vNAR R016 was denaturingly extracted with guanidine and renatured on a urea gradient column in the presence of β-mercaptoethanoi. In Figure 2A, sonicated extract with buffer A (SA), first sonicated extract with buffer B (SB1). second sonicated extract with buffer B (SB2), denatured extract (ED), non-retained denatured extract (NR), wash (LU, 6 M urea), renaturing buffer 5 - 0 M urea (R5 - R0), in Figure 2B Wash (I_ 50 mM imidazole), Elution 1 (E1, 150 mM imidazole), Elution 2 (E2, 200 mM imidazole), Elution 3 (E3, 250 mM imidazole), Elution 4 (E4, 300 mM imidazole) and elution 5 (E5, 500 mM imidazole). The purified protein is enclosed in the box. Molecular weight marker (MPM). After the quantification, the total yield for vNAR R007 (85.75 mg/L) was almost 20 times higher than the yield of R016 (1.45 mg/L), however, both proteins are capable of recognizing human glycated hemoglobin. 2.2. Detection of recombinant vNAR protein by Western blot.
El peso aproximado es de 14.2 kDa para R007 y de 13.2 kDa para R016. En la Figura 3, se muestra el análisis de los vNAR por Western blot. El carril 1 corresponde al vNAR R007, el carril 2 es el marcador de peso molecular y el carril 3 al vNAR R016. Las flechas indican la banda correspondiente a cada proteína. The approximate weight is 14.2 kDa for R007 and 13.2 kDa for R016. In Figure 3, the analysis of the vNARs by Western blot is shown. Lane 1 corresponds to vNAR R007, lane 2 is the molecular weight marker and lane 3 to vNAR R016. The arrows indicate the band corresponding to each protein.
3. Selección de vNAR por análisis de reconocimiento a hemoglobina glicada. 3. Selection of vNAR by glycated hemoglobin recognition analysis.
Se evaluó por ELISA el reconocimiento a la hemoglobina glicada y sus variantes (HbA1c, HbAGIy. HhAlab. HhAO y BSA) por el vNAR R007 (Figura 4A) y R016 (Figura 4B). Ambas clonas tienen reconocimiento por las hemoglobinas glicadas, siendo la hemoglobina glicada en el N-termlnal (HbA1c) y en las Usinas (HbAGIy) la de mayor reconocimiento (***P < 0.001). The recognition of glycated hemoglobin and its variants (HbA1c, HbAGIy. HhAlab. HhAO and BSA) by vNAR R007 (Figure 4A) and R016 (Figure 4B) was evaluated by ELISA. Both clones are recognized by glycated hemoglobins, being the glycated hemoglobin in the N-terminus (HbA1c) and in the Lysins (HbAGIy) the one with the highest recognition (***P < 0.001).
En la evaluación del reconocimiento con muestras de pedentes no diabético y diabético hlperglucémico (5.2 y 9.6 %, respectivamente) en la Figura 5 se demuestra que hay diferencia significativa entre el reconodmiento de las muestras de sangre y suero, Incluyendo las muestras de sangre con 9.2 y 5.2 % de hemoglobina glicada (***P < 0.001) para ambos vNAR, demostrando que las donas R007 (Figura 5A) y R016 (Figura 5B) tienen capacidad de reconodmiento específico a la hemoglobina glicada en una muestra de sangre completa. In the evaluation of recognition with non-diabetic and hyperglycemic diabetic samples (5.2 and 9.6%, respectively), Figure 5 shows that there is a significant difference between the recognition of blood and serum samples, including blood samples with 9.2 and 5.2% glycated hemoglobin (***P < 0.001) for both vNARs, demonstrating that donuts R007 (Figure 5A) and R016 (Figure 5B) have specific recognition capacity for glycated hemoglobin in a whole blood sample.
4. Determinación de límite de detección de hemoglobina glicada por vNAR R016. 4. Determination of detection limit of glycated hemoglobin by vNAR R016.
Se determinó el límite de detección de hemoglobina glicada empleando el vNAR R016 extraído de forma soluble e insoluble. En la Figura 6 se observa mejor reconocimiento a la hemoglobina glicada por el vNAR obtenido de manera insoluble respecto a las condiciones solubles, teniendo un límite de detecciónThe detection limit of glycated hemoglobin was determined using the extracted vNAR R016 in soluble and insoluble form. Figure 6 shows a better recognition of glycated hemoglobin by the vNAR obtained by insoluble with respect to soluble conditions, having a detection limit
Inferior entre 50 y 250 ng para el vNAR R016 de origen insoluble. Por otra parte, el vNAR R016 obtenido de forma soluble tiene un límite de detección inferior de 500 ng de vNAR para ei reconocimiento de hemoglobina glicada en ELISA. 5. Análsis de selectividad del vNAR R016 a hemoglobina glicada. Lower between 50 and 250 ng for the R016 vNAR of insoluble origin. On the other hand, the R016 vNAR obtained in soluble form has a lower detection limit of 500 ng of vNAR for the recognition of glycated hemoglobin in ELISA. 5. Selectivity analysis of vNAR R016 to glycated hemoglobin.
En la Figura 7, se demuestra que el vNAR R016 tiene reconocimiento específico a la hemoglobina glicada en una mezcla de hemoglobina glicada y sin giicar, discriminando la presencia de la hemoglobina sin giicar (HhAO). Lo anterior hace factible ei empleó del vNAR en muestras complejas y con diversas proporciones de hemoglobina glicada. Figure 7 shows that vNAR R016 has specific recognition of glycated hemoglobin in a mixture of glycated and non-glycated hemoglobin, discriminating the presence of non-glycated hemoglobin (HhAO). This makes the use of vNAR feasible in complex samples and with different proportions of glycated hemoglobin.
C. Reconocimiento a hemoglobina glicada por vNAR R016 y R007, conjugadas con nanoparticulas de oro. C. Recognition of glycated hemoglobin by vNAR R016 and R007, conjugated with gold nanoparticles.
1. Análisis de reconocimiento por ELISA, de vNAR R016 conjugado a nanoparticulas de oro. 1. ELISA recognition analysis of vNAR R016 conjugated to gold nanoparticles.
Una vez sintetizadas las nanoparticulas de oro siguiendo el método de Panikar et al., 2019, se optimizó la conjugación con el vNAR-AuNP's. Se seleccionaron 0.9 pg de vNAR a pH de 8. Se comparó con AuNP's sin conjugar. En la Figura 8, se demuestra que las AuNP*s-R016 tienen reconocimiento a hemoglobina glicada en sangre que contiene diferente porcentaje de hemoglobina glicada (**P < 0.01, **P < 0.001). Lo anterior demuestra que el nanoconjugado se realizó en la orientación adecuada, dejando expuesto el sitio de unión a antígeno del vNAR (CDR1 y CDR3). Once the gold nanoparticles were synthesized following the method of Panikar et al., 2019, the conjugation with the vNAR-AuNP's was optimized. 0.9 pg of vNAR was selected at pH 8. It was compared with unconjugated AuNP's. In Figure 8, it is shown that AuNPs * s-R016 have recognition of glycated hemoglobin in blood containing different percentage of glycated hemoglobin (**P < 0.01, **P < 0.001). This demonstrates that the nanoconjugate was made in the proper orientation, exposing the antigen binding site of the vNAR (CDR1 and CDR3).
1.1. Análisis de reconocimiento por aglutinación y absorbencia del vNAR R016. Se empleó el Kit SPINREACT® cano control y para evaluar el uso del vNAR R016 se emplearon las mezclas 1, 2 y 3. En la Figura 9A se demuestra que el vNAR R016 tiene un comportamiento semejante al control del kit aun cuando se usen las diversas Mezclas 1, 2 y 3. Mostrando que el vNAR R016 ya sea conjugado a AuNP's (Mezcla 3) o sin conjugar (Mezcla 1 y 2) tiene especificidad y selectividad por la hemoglobina glicada cuando se emplea una técnica cualitativa. Además, el vNAR R016 es capaz de diferenciar entre porcentajes de hemoglobina glicada. Con base en estos resultados se empleó la Mazda 3 de AuNP*s-R016 debido a que permite evitar el uso de otros anticuerpos, con lo que se esperarla reducir costos en una aplicación futura. En la Figura 9A) Mezcla 1 y Mezcla 2C de vNAR (0.7 y 0.14 pg, respectivamente) con anticuerpos monodonales anti-HIs y antkatón y Mezcla 3 (AuNP's-R016) sin anticuerpos añadidos. Se realizó el seguimiento de la absorbencia después de 15 min de incubación con la Mezcla 3 (Figura 9B) y diferentes porcentajes de hemoglobina glicada, con la finaüdad de determinar si las lecturas eran estables. El coeficiente de variación (CV) representa la dispersión de los datos para una muestra como se define en Ochoa Azze, 2012. El CV del control respecto al tiempo fue de 74.77%, mostrando que la reacción se ve afectada con el tiempo. Se realizó este mismo análisis empleando el conjugado AuNP’s-R016 donde el coeficiente de variación es fue 4.81%. Adicionalmente, el coeficiente de determinación R2 para el conjugado AuNP*s-R016 es de 0.8516 a los 5 min, de 0.9271 y 0.8315 a los 10 y 15min, respectivamente. El empleo del AuNP's-R016 se proyecta como estrategia que, puede determinar con exactitud y estabilidad el porcentaje de hemoglobina glicada en métodos cualitativos hasta después de 15 min de reacción. 1.1. Agglutination and absorbance recognition analysis of vNAR R016. The SPINREACT® cano control Kit was used and mixtures 1, 2 and 3 were used to evaluate the use of vNAR R016. Figure 9A shows that vNAR R016 behaves similarly to the kit control even when the various Mixes 1, 2 and 3. Showing that the R016 vNAR either conjugated to AuNP's (Mix 3) or unconjugated (Mix 1 and 2) has specificity and selectivity for glycated hemoglobin when a qualitative technique is used. In addition, vNAR R016 is capable of differentiating between percentages of glycated hemoglobin. Based on these results, Mazda 3 of AuNP * s-R016 was used because it avoids the use of other antibodies, which is expected to reduce costs in a future application. In Figure 9A) Mix 1 and Mix 2C of vNAR (0.7 and 0.14 pg, respectively) with monodonal anti-HIs and antkaton antibodies and Mix 3 (AuNP's-R016) without added antibodies. Absorbance was monitored after 15 min of incubation with Mix 3 (Figure 9B) and different percentages of glycated hemoglobin, in order to determine if the readings were stable. The coefficient of variation (CV) represents the dispersion of the data for a sample as defined in Ochoa Azze, 2012. The CV of the control with respect to time was 74.77%, showing that the reaction is affected with time. This same analysis was performed using the AuNP's-R016 conjugate where the coefficient of variation was 4.81%. Additionally, the determination coefficient R 2 for the AuNP * s-R016 conjugate is 0.8516 at 5 min, 0.9271 and 0.8315 at 10 and 15 min, respectively. The use of AuNP's-R016 is projected as a strategy that can accurately and stably determine the percentage of glycated hemoglobin in qualitative methods up to 15 min of reaction.
Para comparar la exactitud de AuNP's-R016 respecto al kit SPINREACT se evaluaron muestras de sangre de pacientes con diferente porcentaje de hemoglobina glicada. En la Tabla 1 , se muestran los porcentajes de hemoglobina obtenidos en un laboratorio clínico contra los obtenidos con el kit SPINREACT y con las AuNP s-R016, obteniendo mayor exactitud con el conjugado AuNP’s- R016 con (si error promedio del 6% respecto al 15.7% con el kit comercial. To compare the accuracy of AuNP's-R016 with respect to the SPINREACT kit, blood samples from patients with different percentages of glycated hemoglobin were evaluated. Table 1 shows the percentages of hemoglobin obtained in a clinical laboratory against those obtained with the SPINREACT kit and with the AuNP s-R016, obtaining greater accuracy with the AuNP's-R016 conjugate with (if average error of 6% compared to the 15.7% with the commercial kit.
1.2. Cambio de color y saturación macroscópica en papel por vNAR R007.1.2. Color shift and macroscopic saturation on paper by vNAR R007.
En la Figura 10A) se demuestra la reacción macroscópica de la mezcla AuNP's- R007 con sangre Usada de un paciente no diabético, diabético y diabético hiperglucémico, con 4.9, 7.0 y 9.6% de hemoglobina glicada, respectivamente, A simple vista es posible ver una diferencia de color y saturación entre las muestras debido a la aglomeración de las nanopartículas de oro asociado al aumento del porcentaje de hemoglobina glicada. En la Figura 10B, se observa la gráfica del perfil de color RGB para las mezclas AuNP's-RG07 con sangre Usada con 4.9, 7.0 y 9.6 % de hemoglobina glicada, evaluando cada color por separado. Con diferencia estadística (*P < 0.05s **P < 0.01 , ***P < 0.001 ) entre las muestras de pacientes no diabético, diabético y diabético hiperglucémico en los tres colores (RGB, rojo, verde y azul). Figure 10A) shows the macroscopic reaction of the AuNP's-R007 mixture with blood used from a non-diabetic, diabetic and diabetic patient. hyperglycemic, with 4.9, 7.0 and 9.6% glycated hemoglobin, respectively. At first glance it is possible to see a difference in color and saturation between the samples due to the agglomeration of gold nanoparticles associated with the increase in the percentage of glycated hemoglobin. Figure 10B shows the graph of the RGB color profile for the AuNP's-RG07 mixtures with blood used with 4.9, 7.0 and 9.6% of glycated hemoglobin, evaluating each color separately. With statistical difference (*P < 0.05 s **P < 0.01 , * **P < 0.001 ) between the samples of non-diabetic, diabetic and hyperglycemic diabetic patients in the three colors (RGB, red, green and blue).
Tabla 1. Comparación del porcentaje de hemoglobina glicada de muestras de sangre con SPINREACT®, el conjugado AuNP’s~R016 y laboratorio clínico.
Figure imgf000019_0002
Ejemplo 3. Composiciones farmacéuticas que contienen a las proteínas vNAR obtenidas Table 1. Comparison of the percentage of glycated hemoglobin in blood samples with SPINREACT®, the AuNP's~R016 conjugate and clinical laboratory.
Figure imgf000019_0002
Example 3. Pharmaceutical compositions containing the vNAR proteins obtained
Los dominios vNAR obtenidos la presente invención, funcionan como moléculas de detección a hemoglobina glicada y sus variantes con aplicación en el control o monitoreo de enfermedades metabólicas en humanos, por ejemplo, diabetes y sus enfermedades concomitantes (retinopatía, nefropatía), enfermedades cardiovasculares, obesidad entre otras. The vNAR domains obtained by the present invention function as detection molecules for glycated hemoglobin and its variants with application in the control or monitoring of metabolic diseases in humans, for example, diabetes and its concomitant diseases (retinopathy, nephropathy), cardiovascular diseases, obesity among other.
La secuencia de aminoácidos de la clona RQ07 es la siguiente:
Figure imgf000019_0001
TSDYDGAGTVLTVN: donde los aminoácidos en negritas corresponden alThe amino acid sequence of clone RQ07 is as follows:
Figure imgf000019_0001
TSDYDGAGTVLTVN: where the amino acids in bold correspond to the
CDR1, los aminoácidos subrayados corresponden al CDR3, y los aminoácidos con doble subrayado son las dsteínas canónicas. Esta secuenda de proteína es la SEQ. ID. NO. 4, la secuencia de aminoácidos de la CDR1 corresponde a la SEQ. ID. NO. 1 , y la secuencia de aminoácidos de la CDR3 es la SEQ. ID. NO. CDR1, the underlined amino acids correspond to CDR3, and the double underlined amino acids are the canonical dsteines. This protein sequence is SEQ. ID NO. 4, the amino acid sequence of CDR1 corresponds to SEQ. ID NO. 1, and the amino acid sequence of CDR3 is SEQ. ID NO.
2. two.
La secuencia de nucteótidos que codifica a la proteína R007 (SEQ. ID. NO. 4) es:
Figure imgf000020_0002
The nucleotide sequence encoding the R007 protein (SEQ. ID. NO. 4) is:
Figure imgf000020_0002
La secuencia de aminoácidos de la clona R016 es la siguiente: ATRVDQTPREATKQPGETLTINfiVLRDTSCGLYSTSWFVQRPGRSAWDRLSI GGRYAESVNKPAKSFSLRISNLIAEDSATYFCKAQAGGRLCVGGGNYYGGAG TVLTVN; donde los aminoácidos en negritas corresponden al CDR1, los aminoácidos subrayados corresponden ai CDR3, y los aminoácidos con doble subrayado son las dsteínas canónicas. Esta secuencia de protefna es la SEQ. ID. NO. 8, la secuenda de aminoácidos de la CDR1 corresponde a la SEQ. ID. NO. 5, y la secuencia de aminoácidos de la CDR3 es la SEQ. ID. NO. 6. The amino acid sequence of clone R016 is as follows: ATRVDQTPREATKQPGETLTINfiVLRDTSCGLYSTSWFVQRPGRSAWDRLSI GGRYAESVNKPAKSFSLRISNLIAEDSATYFCKAQAGGRLCVGGGNYYGGAG TVLTVN; where the amino acids in bold correspond to CDR1, the underlined amino acids correspond to CDR3, and the double underlined amino acids are the canonical dsteines. This protein sequence is SEQ. ID NO. 8, the amino acid sequence of CDR1 corresponds to SEQ. ID NO. 5, and the amino acid sequence of CDR3 is SEQ. ID NO. 6.
La secuenda de nucteótidos que codifica a la proteína R016 (SEQ. ID. NO. 8) es:
Figure imgf000020_0001
CCCTGCGGATCAGCAATCTAATTGCTGAAGACTCAGCCACGTACTTTTGCA AAGCACAAGCGGGAGGGCGACTATGTGTGGGGGGGGGTAACTACTACGG TGGAGCTGGCACCGTGCTGACTGTGAAC, y corresponde a la SEQ. ID. NO. The nucleotide sequence encoding the R016 protein (SEQ. ID. NO. 8) is:
Figure imgf000020_0001
CCCTGCGGATCAGCAATCTAATTGCTGAAGACTCAGCCACGTACTTTTGCA AAGCACAAGCGGGAGGGCGACTATGTGTGGGGGGGGGTAACTACTACGG TGGAGCTGGCACCGTGCTGACTGTGAAC, and corresponds to SEQ. ID NO.
7. 7.
Las secuencias descritas anteriormente tienen la capacidad de reconocimiento del blanco o biomarcador que corresponde a la subunidad beta de la hemoglobina glicada de humano. Por lo tanto, las secuencias de aminoácidos codificadas por las secuencias de AON R007 y AON R016 conjuntamente y por separado, pueden ser utilizadas como complemento o sustituto de los anticuerpos monodonales en los cuales se basan las pruebas comerciales para la detección y cuantificadón de hemoglobina glicada desarrollando nuevas pruebas de detección. Asi mismo, puede ser empleado en nuevas tecnologías solas o en combinación, por ejemplo, pruebas en el punto de atención (POC). Por lo que se pueden formular varios tipos de productos y/o composiciones. The sequences described above have the ability to recognize the target or biomarker that corresponds to the beta subunit of human glycated hemoglobin. Therefore, the amino acid sequences encoded by the AON R007 and AON R016 sequences together and separately can be used as a complement or substitute for the monodonal antibodies on which the commercial tests for the detection and quantification of glycated hemoglobin are based. developing new screening tests. Likewise, it can be used in new technologies alone or in combination, for example, tests at the point of care (POC). Therefore, various types of products and/or compositions can be formulated.
Adicionalmente, estos vNAR pueden ser conjugados a diversas nanoparticulas (oro, látex, entro otros), a cromóforos u otras proteínas para aumentar su eficiencia de uso en la aplicación para el control y monitoreo de enfermedades metabólicas. También pueden produdrse en forma de dineros, trímeros o tetrámeros para aumentar la avidez hacía su antígeno. Hasta el momento sólo se han desarrollado anticuerpos monodonales de tipo IgG derivados de mamíferos para esta aplicación específica. No se ha encontrado fragmentos variables vNAR de ningún tipo o especie para la detección de hemoglobina y sus variantes gl ¡cadas. LITERATURA CITADA Additionally, these vNARs can be conjugated to various nanoparticles (gold, latex, among others), to chromophores or other proteins to increase their efficiency of use in the application for the control and monitoring of metabolic diseases. They can also be produced in the form of monies, trimers or tetramers to increase the avidity towards their antigen. So far, only mammalian-derived IgG-type monodonal antibodies have been developed for this specific application. No variable vNAR fragments of any type or species have been found for the detection of hemoglobin and its glycated variants. LITERATURE CITED
Anniebell, S., & Gopinath, S. C. B. (2018). Polymer Conjugated Gold Nanopartides in Biomedical AppHcations. Current Medicinal Chemistry , 25(12), 1433-1445. https://doi.org/10.2174/09298e7324666170116123e33Anniebell, S., & Gopinath, S.C.B. (2018). Polymer Conjugated Gold Nanoparticles in Biomedical Applications. Current Medicinal Chemistry, 25(12), 1433-1445. https://doi.org/10.2174/09298e7324666170116123e33
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Panlkar, S. S., Ramfrez-Garcfa, G., SWhlk, S., Lopez-Luke, T., Rodríguez- González, C., Ciapara, i. H., De La Rosa, E. (2019). Ultrasensitive SERS Substrato for Labei-Free Therapeutic-Drug Monitoring of Paditaxei and CydophosphamWe in Blood Serum. Analytícal Chemistry , 91(3), 2100- 2111. https://doi.oig/10.1021/acs.arektfiem.8b04523 Pórez-Padllla, N. A., y Camacho-Vllegas, T. A. (2019). Póster Comparison of two methods for obtained variable domains vNAR for indusion bodies. 8th Symposium of the Mexkan Proteomlcs Sodety; 3rd PanAMerican-Human Proteome Organization (Pan-HUPO) Meeting; 2nd Ibero-American Symposium on Mass Spectrometry. October 20th to 23rd, 2019. HeW in Acapulco, Guerrero, México. Panlkar, SS, Ramfrez-Garcfa, G., SWhlk, S., Lopez-Luke, T., Rodríguez-González, C., Ciapara, i. H., De La Rosa, E. (2019). Ultrasensitive SERS Substrate for Labei-Free Therapeutic-Drug Monitoring of Paditaxei and CydophosphamWe in Blood Serum. Analytical Chemistry, 91(3), 2100-2111. https://doi.oig/10.1021/acs.arektfiem.8b04523 Pórez-Padllla, NA, and Camacho-Vllegas, TA (2019). Comparison of two methods for obtained variable domains vNAR for induction bodies. 8th Symposium of the Mexkan Proteomlcs Sodety; 3rd Pan-American-Human Proteome Organization (Pan-HUPO) Meeting; 2nd Ibero-American Symposium on Mass Spectrometry. October 20 th to 23 rd , 2019. HeW in Acapulco, Guerrero, Mexico.
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Claims

REIVINDICACIONES
1. Una proteína vNAR aislada, caracterizada porque comprende: i) una secuencia de aminoácidos de la primera región determinante de complementariedad (CDR1) que es seleccionada del siguiente grupo de secuencias de aminoácidos: SEQ. ID. NO. 1, SEQ. ID. NO. 5, o sus variantes sustancialmente similares a ellas; y ii) una secuencia de aminoácidos de la tercera región determinante de complementariedad (CDR3) seleccionada del siguiente grupo de secuencias de aminoácidos: SEQ. ID. NO. 2, SEQ. ID NO. 6, o sus variantes sustancialmente similares a ellas. 1. An isolated vNAR protein, characterized in that it comprises: i) an amino acid sequence of the first complementarity determining region (CDR1) that is selected from the following group of amino acid sequences: SEQ. ID NO. 1, SEQ. ID NO. 5, or substantially similar variants thereof; and ii) an amino acid sequence of the third complementarity determining region (CDR3) selected from the following group of amino acid sequences: SEQ. ID NO. 2, SEQ. ID NO. 6, or substantially similar variants thereof.
2. La proteína de la reivindicación anterior, donde la CDR1 es la secuencia de aminoácidos SEQ. ID. NO. 1; y la CDR3 es la secuencia de aminoácidos SEQ. ID. NO. 2. 2. The protein of the preceding claim, wherein CDR1 is the amino acid sequence SEQ. ID NO. one; and CDR3 is the amino acid sequence SEQ. ID NO. two.
3. La proteína de la reivindicación 1, donde la CDR1 es la secuencia de aminoácidos SEQ. ID. NO. 5; y la CDR3 es la secuencia de aminoácidos SEQ. ID. NO. 6. 3. The protein of claim 1, wherein CDR1 is the amino acid sequence SEQ. ID NO. 5; and CDR3 is the amino acid sequence SEQ. ID NO. 6.
4. La proteína según las reivindicaciones anteriores, caracterizada porque comprende, una de las siguientes secuencias de aminoácidos: SEQ. ID. No.4, SEQ. ID. NO. 8, o sus variantes sustancialmente similares a ellas. 4. The protein according to the preceding claims, characterized in that it comprises one of the following amino acid sequences: SEQ. ID No.4, SEQ. ID NO. 8, or substantially similar variants thereof.
5. La proteína de acuerdo con las reivindicaciones anteriores, donde dicha proteína vNAR es de origen Potamotrygon schroederi. 5. The protein according to the preceding claims, wherein said vNAR protein is of Potamotrygon schroederi origin.
6. La protefna de conformidad con las reivindicaciones anteriores, caracterizada porque tiene la capacidad de reconocimiento del blanco o biomarcador que corresponde a la subunkted beta de la hemoglobina glicada de humano. 6. The protein according to the preceding claims, characterized in that it has the ability to recognize the target or biomarker that corresponds to the subunkted beta of human glycated hemoglobin.
7. Una secuencia de ADN, caracterizada porque comprende, una secuencia de nucleótidos que codifica a la proteina vNAR de las reivindicaciones anteriores. 7. A DNA sequence, characterized in that it comprises a nucleotide sequence that encodes the vNAR protein of the preceding claims.
8. La secuencia de ADN, de 1a reivindicación anterior, donde te secuencia de nucleótidos es te: SEQ. ID NO. 3, o SEQ. ID. NO. 7. 8. The DNA sequence of the preceding claim, where the nucleotide sequence is the: SEQ. ID NO. 3, or SEQ. ID NO. 7.
9. Una secuencia de ADN recombinante, caracterizada porque comprende: la secuencia de ADN de conformidad con cualquiera de las reivindicaciones9. A recombinant DNA sequence, characterized in that it comprises: the DNA sequence according to any of the claims
7 y 8. 7 and 8.
10. Una composición que reconoce el blanco o biomarcador de la subunidad beta de la hemoglobina glicada de humano, caracterizada porque comprende: al menos, una proteina vNAR de conformidad con cualquiera de las reivindicaciones 1 a la e. 10. A composition that recognizes the target or biomarker of the beta subunit of human glycated hemoglobin, characterized in that it comprises: at least one vNAR protein according to any of claims 1 to e.
11. La composición de la reivindicación anterior, caracterizada porque además comprende: al menos, una sustancia que aumente su eficiencia de uso en el reconocimiento del blanco o biomarcador de la subunidad beta de la hemoglobina glícada de humano. 11. The composition of the preceding claim, characterized in that it further comprises: at least one substance that increases its efficiency of use in the recognition of the target or biomarker of the beta subunit of human glycated hemoglobin.
12. La composición según te reivindicación anterior, donde te sustancia que aumenta te eficiencia de uso es seleccionada del siguiente grupo: nanoparticulas, cromóforos, proteínas y/o una combinación entre ellas. 12. The composition according to the preceding claim, where the substance that increases the efficiency of use is selected from the following group: nanoparticles, chromophores, proteins and/or a combination between them.
13. La composición de acuerdo con la reivindicación precedente, donde las nanoparticulas son de: oro, látex, y/o su combinación entre ellos. 13. The composition according to the preceding claim, where the nanoparticles are: gold, latex, and/or their combination between them.
14. Un método ex vivo para reconocer el blanco o biomarcador de la subunidad beta de la hemoglobina glicada humana, caracterizado porque comprende: i) proveer una muestra de sangre de un paciente sospechoso de diabetes; II) contactar la muestra de sangre con la composición que reconoce el blanco o biomarcador de la subunidad beta de la hemoglobina glicada humana, de conformidad con cualquiera de las reivindicaciones 10 a la 13; y detectar la presencia de hemoglobina glicada, en donde la proteína vNAR de la composición, se une a la subunidad beta de la hemoglobina glicada humana. 14. An ex vivo method to recognize the target or biomarker of the beta subunit of human glycated hemoglobin, characterized in that it comprises: i) providing a blood sample from a suspected diabetes patient; II) contacting the blood sample with the composition that recognizes the target or biomarker of the beta subunit of human glycated hemoglobin, in accordance with any of claims 10 to 13; and detecting the presence of glycated hemoglobin, wherein the vNAR protein of the composition binds to the beta subunit of human glycated hemoglobin.
15. El método de la reivindicación anterior, donde la detección es mediante los siguientes inmunoensayos: ELISA, DotBIot, Western blot, aglutinación, inmunoturbidimetría, Inmunofluorescenda, tiras de flujo lateral (LFA), pruebas de punto de atención (POC). 15. The method of the preceding claim, where the detection is by the following immunoassays: ELISA, DotBIot, Western blot, agglutination, immunoturbidimetry, Immunofluorescenda, lateral flow strips (LFA), point of care tests (POC).
16. Un ueo de la protelna vNAR de conformidad con las reivindicaciones 1 a la 6, útil para el reconocimiento del blanco o biomarcador de la subunidad beta de la hemoglobina glicada humana. 16. A use of the vNAR protein according to claims 1 to 6, useful for the recognition of the target or biomarker of the beta subunit of human glycated hemoglobin.
17. Un uso de la composición de conformidad con cualquiera de las reivindicaciones 10 a la 13, útil para el reconocimiento del blanco o biomarcador de la subunidad beta de la hemoglobina glicada humana. 17. A use of the composition according to any of claims 10 to 13, useful for the recognition of the target or biomarker of the beta subunit of human glycated hemoglobin.
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Citations (1)

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EP0201187A1 (en) * 1985-03-29 1986-11-12 Novo Nordisk A/S Determination of glycated (glycosylated) hemoglobin in blood

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EP0201187A1 (en) * 1985-03-29 1986-11-12 Novo Nordisk A/S Determination of glycated (glycosylated) hemoglobin in blood

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HORBER, S. ET AL.: "Harmonization of immunoassays for biomarkers in diabetes mellitus", BIOTECHNOLOGY ADVANCES, vol. 39, no. 107359, February 2019 (2019-02-01), XP086074099, ISSN: 0734-9750, DOI: 10.1016/j.biotechadv. 2019.02.01 5 *
PYADALA, N. : "Development of a colloidal gold-based lateral flow immunoassay for the rapid detection of glycosylated hemoglobin (HbA1c) in whole blood", INTERNATIONAL JOURNAL OF RESEARCH IN PHARMACEUTICAL SCIENCES, J K WELFARE & PHARMASCOPE FOUNDATION, IN, vol. 9, no. 3, 30 November 2017 (2017-11-30), IN , pages 580 - 588, XP009534194, ISSN: 0975-7538, DOI: 10.26452/ijrps.v9i3.1524 *

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