Ultrasensitivity detection system of different nature molecules to early cancer detection

La técnica ELISA (del inglés “enzyme-linked immunosorbent assay”) es el método de rutina más utilizado y fiable para la detección de marcadores proteicos relevantes en la asistencia sanitaria. Aumentar la sensibilidad de este ensayo es crucial para detectar biomoléculas relacionadas con trastornos de salud y facilitar el diagnóstico en las primeras etapas de enfermedades como el cáncer, permitiendo de esta manera una posible disminución de la mortalidad. Así, se han desarrollado varios métodos para mejorar la sensibilidad del ELISA mediante la inmovilización de anticuerpos en las placas de poliestireno. En este trabajo hemos desarrollado dos estrategias de inmovilización de anticuerpos a un sustrato sólido con el fin de aumentar la sensibilidad en el ensayo ELISA. En una de las estrategias hemos involucrado la reacción de alta eficiencia y especificidad entre la tetracina y el trans-cicloocteno con el fin de aumentar la densidad del anticuerpo en la superficie; y en la otra, la afinidad del dominio de unión a quitina por su sustrato como método para lograr la correcta orientación del anticuerpo inmovilizado. Para la primera estrategia se prepararon superficies con tetracina, mientras que el anticuerpo fue modificado con TCO. La preparación de las superficies se llevó a cabo de tres formas diferentes: 1) partiendo de placas aminadas comerciales tratadas con glutaraldehído y lisina para aumentar el número de grupos NH2 en superficie, 2) partiendo de placas aminadas mediante un proceso de química en mojado y 3) recubriendo placas MaxiSorp con la proteína BSA marcada con 20 tetracinas por molécula. Para la evaluación de las superficies se utilizaron tres modelos de ELISA, dos de ellos recubriendo con el anticuerpo de baja afinidad anti c-myc (9E10) marcado con TCO, donde se detecta la proteína c-myc-GST-IL8h o c-myc-IL6h, y como anticuerpo de detección el específico para la citoquina correspondiente. El otro ELISA, para la evaluación de las superficies, se utilizó para detectar la proteína CEA, relacionada con el diagnóstico del cáncer colorrectal. En todos los casos se observó un aumento de la sensibilidad del ELISA desarrollado en las superficies preparadas con tetracina comparado con la superficie estándar. En la detección de c-myc-GST-IL8h se obtuvo un aumento de sensibilidad del ELISA, comparado con las superficies estándar, de 2,6 veces para las superficies preparadas a partir de placas aminadas comerciales, de 11 veces partiendo de placas aminadas mediante química en mojado y de 13,7 veces en placas previamente recubiertas con BSA-tetracina. Del mismo modo, en el ELISA de detección de c-myc IL6h se aumentó la sensibilidad 9 veces en placas con tetracina a partir de placas aminadas mediante química en mojado y, por último, el ELISA de detección de CEA tuvo un aumento de 12 veces en placas previamente recubiertas con BSA-tetracina. En la segunda estrategia fueron preparadas superficies de poliestireno con quitosano acetilado mediante un proceso de acetilación in situ, mientras que, por otro lado, fue preparado el anticuerpo anti cmyc (9E10) unido a ChBD. Esta unión fue realizada de forma química a través de una reacción de tipo química clic y de forma recombinante fusionando ChBD al carboxilo terminal del anticuerpo. Ambos anticuerpos fueron evaluados mediante el ELISA de detección de la proteína c-myc-GST-IL8h, en las placas de quitosano acetilado, observándose un aumento de la sensibilidad de 6 veces (p<0,0001) con respecto al ELISA realizado en la superficie estándar usando el anticuerpo obtenido de forma recombinante. Asimismo, se diseñó un método de detección de dos moléculas de distinta naturaleza en un ensayo tipo multiplex de spot en placas de ELISA. El objetivo fue determinar la concentración de dos moléculas relacionadas con el cáncer colorrectal, el miRNA-29b y la proteína CEA. Para la detección del miRNA mediante un ensayo inmunoenzimático, se desarrolló un método directo de hibridación in situ y otro basado en la técnica PCR-ELISA. Con esta última técnica de detección de ácidos nucleicos se logró la detección del miRNA-29b con un LOD de 1358 moléculas totales simultáneamente con la proteína CEA, con un LOD de 2,54 ng/ml. En este trabajo se demuestra, a través de la reacción tetracina - TCO, que la sensibilidad del ensayo ELISA mejora de forma significativa usando un sistema de inmovilización de anticuerpos que permita que estos queden fijados mediante un enlace covalente a la superficie. Además, la correcta orientación de los anticuerpos en la superficie de un ensayo de ELISA mejora la sensibilidad de la técnica. Se demuestra también la detección simultánea de dos moléculas de distinta naturaleza (un miRNA y una proteína) en un ensayo inmunoenzimático.The enzyme-linked immunosorbent assay (ELISA) is the most widely used and reliable clinical routine method for the detection of essential protein markers in healthcare. Improving ELISAs is crucial to detect biomolecules related to health disorders and facilitating diagnosis at the early disease stages such as cancer, thus allowing the possibility of a decrease in mortality. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. In this work we have developed two strategies of antibodies immobilization on a solid support to improve the ELISA sensitivity. To increase antibody density on the surface, one strategy was related to the highly efficient and specific reaction between tetrazine and trans cyclooctene. The second strategy was based on the affinity between chitin binding domain and its substrate to promote the adequate capture antibody orientation. To develop the first strategy, surfaces with exposed tetrazine groups were prepared, while antibody was modified with TCO. The surfaces with tetrazine were carried out in three different ways: 1) from commercial aminated plates treated with glutaraldehyde and lysine to increase the NH2 groups, 2) from aminated plates after a wet chemical process and 3) from Maxisorp plates coated with BSA modified with 20 tetrazine per molecule. The surfaces were evaluated using three ELISA models, two of them using the low affinity antibody anti c-myc (9E10) modified with TCO as a capture antibody. This procedure was carried out to detect the recombinant proteins c-myc-GST-IL8h and/or c-myc-IL6h, and the detection was performed with the corresponding specific cytokine biotinylated antibody. The third ELISA for the evaluation of surfaces was a CEA detection ELISA, a protein related with colorectal cancer. The sensitivity increased in all the surfaces treated with tetrazine in comparison with the standard unmodified surfaces. Thus, in commercial aminated plates treated with tetrazine, the detection sensitivity of c-myc-GST-human IL8 was 2.6-fold increase, on tetrazine surfaces previously aminated with a wet chemical process 11-fold and 13.7-fold on plates coated with BSA-tetrazine. On the other hand, the sensitivity of c-myc IL6h detection ELISA increased 9-fold in tetrazine treated surfaces using aminated plates after a wet chemical process, while CEA ELISA detection increased 12-fold on surfaces plates previously coated with BSA-tetrazine. For the second strategy, we combined polystyrene surfaces coated with acetylated chitosan prepared by an in-situ acetylation process. In this case, the anti-cmyc antibody (9E10) was linked to ChBD. The anti c-myc-ChBD antibody was obtained using a click chemistry reaction or cloning a recombinant protein with ChBD attached to the end carboxyl of anti c-myc. Both anti cmyc- ChBD antibodies were evaluated on chitosan acetylated surfaces using a sandwich detection ELISA to detect c-myc-GST-IL8h. The ELISA assays developed on chitin surfaces were 6-fold more sensitive (p<0.0001) than those performed on standard surface. Besides, a spot multiplex ELISA was designed to detect two molecules of different nature in the same assay: the CEA protein and the miRNA-29b, both related with colorectal cancer. The immunoenzymatic detection of miRNA was developed through in situ hybridization or by a PCRELISA technique. Using a PCR-ELISA a LOD of 1.358 molecules to miRNA-29b was obtained, while the LOD of CEA detection was 2.54 ng/ml, both molecules were simultaneously detected in the same well. In this work we show that it is possible to improve the ELISA sensitivity using an immobilization system through a tetrazine-TCO reaction, which allow the capture antibodies to bind covalently on to surfaces. Moreover, the adequate capture antibody orientation on surfaces also improves the ELISA technique sensitivity. On the other hand, we also demonstrate, the simultaneous detection of a miRNA and a protein, two different nature molecules, by mean of an immunoenzymatic assay

Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique

This research was funded by Compra Publica Precomercial, Reference 2012/000069, Ministerio de Economia y Competitividad, Espana. ONCOVER project: Volatile compound detection system for early cancer diagnosis.Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.Compra Publica Precomercial, Ministerio de Economia y Competitividad, Espana 2012/00006

Development of a High-Throughput Calcium Mobilization Assay for CCR6 Receptor Coupled to Hydrolase Activity Readout

CCR6 is a chemokine receptor highly implicated in inflammatory diseases and could be a potential therapeutic target; however, no therapeutic agents targeting CCR6 have progressed into clinical evaluation. Development of a high-throughput screening assay for CCR6 should facilitate the identification of novel compounds against CCR6. To develop a cell-based assay, RBL-2H3 cells were transfected with plasmids encoding β-hexosaminidase and CCR6. Intracellular calcium mobilization of transfected cells was measured with a fluorescent substrate using the activity of released hexosaminidase as readout of the assay. This stable, transfected cell showed a specific signal to the background ratio of 19.1 with low variability of the signal along the time. The assay was validated and optimized for high-throughput screening. The cell-based calcium mobilization assay responded to the specific CCR6 ligand, CCL20, in a dose-dependent manner with an EC50 value of 10.72 nM. Furthermore, the assay was deemed robust and reproducible with a Z’ factor of 0.63 and a signal window of 7.75. We have established a cell-based high-throughput calcium mobilization assay for CCR6 receptor. This assay monitors calcium mobilization, due to CCR6h activation by CCL20, using hexosaminidase activity as readout. This assay was proved to be robust, easy to automate and could be used as method for screening of CCR6 modulators

Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces

Amino terminal recognition by a CCR6 chemokine receptor antibody blocks CCL20 signaling and IL-17 expression via β-arrestin.

CCR6 chemokine receptor is an important target in inflammatory diseases. Th17 cells express CCR6 and a number of inflammatory cytokines, including IL-17 and IL-22, which are involved in the propagation of inflammatory immune responses. CCR6 antagonist would be a potential treatment for inflammatory diseases such as psoriasis or rheumatoid arthritis. The aim of this study is to develop an antagonistic monoclonal antibody (mAb) against human CCR6 receptor (hCCR6). We generate monoclonal antibodies against hCCR6 immunizing Balb/c mice with hCCR6 overexpressing cells. The antibodies were tested by flow cytometry for specific binding to hCCR6, cloned by limiting dilution and resulted in the isolation and purification monoclonal antibody 1C6. By ELISA and flow cytometry, was determined that the antibody obtained binds to hCCR6 N-terminal domain. The ability of 1C6 to neutralize hCCR6 signaling was tested and we determined that 1C6 antibody were able to block response in β-arrestin recruitment assay with IC50 10.23 nM, but did not inhibit calcium mobilization. In addition, we found in a chemotaxis assay that 1C6 reduces the migration of hCCR6 cells to their ligand CCL20. Finally, we determined by RT-qPCR that the expression of IL-17A in Th17 cells treated with 1C6 was inhibited. In the present study, we applied whole cell immunization for successfully obtain an antibody that is capable to neutralize hCCR6 signaling and to reduce hCCR6 cells migration and IL-17 expression. These results provide an efficient approach to obtain therapeutic potential antibodies in the treatment of CCR6-mediated inflammatory diseases

A multinational Delphi consensus to end the COVID-19 public health threat

Despite notable scientific and medical advances, broader political, socioeconomic, and behavioural factors continue to undercut the response to the coronavirus disease 2019 (COVID-19) pandemic1,2. This Delphi study convened a diverse, multidisciplinary panel of 386 academic, health, NGO, government and other experts in COVID-19 response from 112 countries and territories to recommend specific actions to end this persistent global public health threat. The panel developed a set of 41 consensus statements and 57 recommendations to governments, health systems, industry, and other key stakeholders across six domains: communication; health systems; vaccination; prevention; treatment and care; and inequities. In the wake of nearly three years of ragmented global and national responses, it is instructive to note that three of the highest-ranked recommendations call for the adoption of whole-of-society and whole-of-government approaches1, while maintaining proven prevention measures using a vaccines-plus approach2 that employs a range of public health and financial support measures to complement vaccination. Other recommendations with at least 99% combined agreement advise governments and other stakeholders to improve communication, rebuild public trust, and engage communities3 in the management of pandemic responses. The findings of the study, which have been further endorsed by organisations globally, include points of unanimous agreement, as well as six recommendations with >5% disagreement, that provide health and social policy actions to address inadequacies in the pandemic response and help bring this public health threat to an end

A multinational Delphi consensus to end the COVID-19 public health threat

Abstract Despite notable scientific and medical advances, broader political, socioeconomic and behavioural factors continue to undercut the response to the COVID-19 pandemic 1,2 . Here we convened, as part of this Delphi study, a diverse, multidisciplinary panel of 386 academic, health, non-governmental organization, government and other experts in COVID-19 response from 112 countries and territories to recommend specific actions to end this persistent global threat to public health. The panel developed a set of 41 consensus statements and 57 recommendations to governments, health systems, industry and other key stakeholders across six domains: communication; health systems; vaccination; prevention; treatment and care; and inequities. In the wake of nearly three years of fragmented global and national responses, it is instructive to note that three of the highest-ranked recommendations call for the adoption of whole-of-society and whole-of-government approaches 1 , while maintaining proven prevention measures using a vaccines-plus approach 2 that employs a range of public health and financial support measures to complement vaccination. Other recommendations with at least 99% combined agreement advise governments and other stakeholders to improve communication, rebuild public trust and engage communities 3 in the management of pandemic responses. The findings of the study, which have been further endorsed by 184 organizations globally, include points of unanimous agreement, as well as six recommendations with >5% disagreement, that provide health and social policy actions to address inadequacies in the pandemic response and help to bring this public health threat to an end

A multinational Delphi consensus to end the COVID-19 public health threat

Abstract Despite notable scientific and medical advances, broader political, socioeconomic and behavioural factors continue to undercut the response to the COVID-19 pandemic . Here we convened, as part of this Delphi study, a diverse, multidisciplinary panel of 386 academic, health, non-governmental organization, government and other experts in COVID-19 response from 112 countries and territories to recommend specific actions to end this persistent global threat to public health. The panel developed a set of 41 consensus statements and 57 recommendations to governments, health systems, industry and other key stakeholders across six domains: communication; health systems; vaccination; prevention; treatment and care; and inequities. In the wake of nearly three years of fragmented global and national responses, it is instructive to note that three of the highest-ranked recommendations call for the adoption of whole-of-society and whole-of-government approaches , while maintaining proven prevention measures using a vaccines-plus approach that employs a range of public health and financial support measures to complement vaccination. Other recommendations with at least 99% combined agreement advise governments and other stakeholders to improve communication, rebuild public trust and engage communities in the management of pandemic responses. The findings of the study, which have been further endorsed by 184 organizations globally, include points of unanimous agreement, as well as six recommendations with >5% disagreement, that provide health and social policy actions to address inadequacies in the pandemic response and help to bring this public health threat to an end

A multinational Delphi consensus to end the COVID-19 public health threat

Abstract Despite notable scientific and medical advances, broader political, socioeconomic and behavioural factors continue to undercut the response to the COVID-19 pandemic 1,2 . Here we convened, as part of this Delphi study, a diverse, multidisciplinary panel of 386 academic, health, non-governmental organization, government and other experts in COVID-19 response from 112 countries and territories to recommend specific actions to end this persistent global threat to public health. The panel developed a set of 41 consensus statements and 57 recommendations to governments, health systems, industry and other key stakeholders across six domains: communication; health systems; vaccination; prevention; treatment and care; and inequities. In the wake of nearly three years of fragmented global and national responses, it is instructive to note that three of the highest-ranked recommendations call for the adoption of whole-of-society and whole-of-government approaches 1 , while maintaining proven prevention measures using a vaccines-plus approach 2 that employs a range of public health and financial support measures to complement vaccination. Other recommendations with at least 99% combined agreement advise governments and other stakeholders to improve communication, rebuild public trust and engage communities 3 in the management of pandemic responses. The findings of the study, which have been further endorsed by 184 organizations globally, include points of unanimous agreement, as well as six recommendations with >5% disagreement, that provide health and social policy actions to address inadequacies in the pandemic response and help to bring this public health threat to an end