title: The Development of ELISA and SPR-Based Immunoassays for the Detection of Heat Shock Proteins.

authors: Gong, Bei

subjects: ELISA

abstract: Heat shock proteins (Hsps) are highly conserved molecules in all eukaryotes and prokaryotes. They are named on the basis of their molecular weight and their synthesis is up-regulated by stress conditions such as inflammation, oxidative stress and exposure to high temperature. They function by assisting the folding of newly synthesized proteins and thus prevent aggregation or damage to other cellular components. Beside the role in intracellular protein folding, heat shock proteins can particularly act as intercellular signals with a wide variety of biological effects, such as to stimulate cells to produce proinflammatory cytokines, like TNFα, and other proteins involved in immunity and inflammation, or they are also thought to be involved in the pathogenesis of a wide range of diseases such as diabetes mellitus and cardiovascular disease. Thus, screening for aberrant expression of this protein could be an easy and useful tool to detect at risk individuals for developing and those more susceptible towards developing these diseases. 

Currently, the Enzyme-Linked ImmunoSorbent Assay (ELISA) is the main immunoassay method used for the measurement of heat shock proteins levels in both clinical and research laboratories. It is relatively more rapid and sensitive than RadioImmunoassay (RIA), and most importantly, it is safer because enzymes are used as labels instead of harmful radioactive substances. However, a newly developed biosensor method, the Surface Plasmon Resonance (SPR), offers a number of advantageous over ELISA. For example, it is much more simple and rapid because a lot of steps can be set up automatically and no labels are required for SPR immunoassays. 

In this study, a comparison of different types of ELISA assays was made. The results showed that the Sandwich format of ELISA is much more sensitive than Indirect ELISA for detection of the concentrations of Heat Shock Protein 70 (Hsp70), and this sensitivity can be further improved by applying an Avidin-Biotin system together with Sandwich ELISA under certain conditions.

To develop the SPR protocol for the detection of heat shock protein concentrations, two Hsp70 binding curves using different assay formats, the Sandwich SPR immunoassay and the Competitive SPR immunoassay, were set up by using CM 5 sensor chip. Another sensor chip, the mixed Self-Assembled Monolayer (mSAM), was also examined using Sandwich SPR format. No subsequent experimental steps were carried on for SPR studies since the regeneration conditions of these tests for both sensor chips need to be well studied. Thus, this study suggests that in order to set up a SPR immunoassay protocol that is a better choice for the detection of heat shock protein levels in complex matrixes than ELISA, experimental conditions, such as the choice of regeneration buffer and the duration of regeneration cycle, need to be well optimized.

text: The Development of ELISA and SPR-Based Immunoassays for the Detection of Heat Shock Proteins. Heat shock proteins (Hsps) are highly conserved molecules in all eukaryotes and prokaryotes. They are named on the basis of their molecular weight and their synthesis is up-regulated by stress conditions such as inflammation, oxidative stress and exposure to high temperature. They function by assisting the folding of newly synthesized proteins and thus prevent aggregation or damage to other cellular components. Beside the role in intracellular protein folding, heat shock proteins can particularly act as intercellular signals with a wide variety of biological effects, such as to stimulate cells to produce proinflammatory cytokines, like TNFα, and other proteins involved in immunity and inflammation, or they are also thought to be involved in the pathogenesis of a wide range of diseases such as diabetes mellitus and cardiovascular disease. Thus, screening for aberrant expression of this protein could be an easy and useful tool to detect at risk individuals for developing and those more susceptible towards developing these diseases. 

Currently, the Enzyme-Linked ImmunoSorbent Assay (ELISA) is the main immunoassay method used for the measurement of heat shock proteins levels in both clinical and research laboratories. It is relatively more rapid and sensitive than RadioImmunoassay (RIA), and most importantly, it is safer because enzymes are used as labels instead of harmful radioactive substances. However, a newly developed biosensor method, the Surface Plasmon Resonance (SPR), offers a number of advantageous over ELISA. For example, it is much more simple and rapid because a lot of steps can be set up automatically and no labels are required for SPR immunoassays. 

In this study, a comparison of different types of ELISA assays was made. The results showed that the Sandwich format of ELISA is much more sensitive than Indirect ELISA for detection of the concentrations of Heat Shock Protein 70 (Hsp70), and this sensitivity can be further improved by applying an Avidin-Biotin system together with Sandwich ELISA under certain conditions.

To develop the SPR protocol for the detection of heat shock protein concentrations, two Hsp70 binding curves using different assay formats, the Sandwich SPR immunoassay and the Competitive SPR immunoassay, were set up by using CM 5 sensor chip. Another sensor chip, the mixed Self-Assembled Monolayer (mSAM), was also examined using Sandwich SPR format. No subsequent experimental steps were carried on for SPR studies since the regeneration conditions of these tests for both sensor chips need to be well studied. Thus, this study suggests that in order to set up a SPR immunoassay protocol that is a better choice for the detection of heat shock protein levels in complex matrixes than ELISA, experimental conditions, such as the choice of regeneration buffer and the duration of regeneration cycle, need to be well optimized.

year: 2009

title: Anticipatory responses to pulsed resources: An introduction

authors: King, Carolyn M.

abstract: Vigorous exchange of ideas is an essential part of the business of science. What makes that interaction so much more interesting than ordinary conversation is that, in science, ideas always have to be based on documented observation of the real world.No one disputes that requirement, yet it does not automatically ensure agreement, even when all parties are observing the same bits of the same world. That is because what we see in front of our eyes is powerfully influenced by what is behind them. Observations are never free of assumptions, which in turn are never independent of previous knowledge and experience.

text: Anticipatory responses to pulsed resources: An introduction Vigorous exchange of ideas is an essential part of the business of science. What makes that interaction so much more interesting than ordinary conversation is that, in science, ideas always have to be based on documented observation of the real world.No one disputes that requirement, yet it does not automatically ensure agreement, even when all parties are observing the same bits of the same world. That is because what we see in front of our eyes is powerfully influenced by what is behind them. Observations are never free of assumptions, which in turn are never independent of previous knowledge and experience.

year: 2013

title: Riariakina ō rongo hirikapo From kōhanga reo to kura

authors: Hohepa, Margie Kahukura

abstract: Ko te kaupapa tonu, ko te whakatupu i te mahara o te hirikapo kia tiu ai ki te muri, arā, kia puta he whakaaro i pokepokengia nō roto tonu i ngā whakahekenga kāwai o ngā tūpuna o te tamaiti, kua maranga te wā e kaumātua haere ana te tamaiti, ngōna kanohi kua huaki, ngōna taringa kua pīkari, ngā whakaaro kua korikori, tōna wairua hīkaka kua ngawhā, ngērā kaupapa katoa o te tamaiti i taua wā o te tupu. Mā tēnei kōrero hoki hei kawe te taonga o te rongo hirikapo, arā, ngā whakaaro, ngā mahara, nāna ko te waha, nā te waha ko te kupu kōrero e makere iho ai i te ārero tarapepe. Heoi anō he tohutohu anō kei roto e kī ana, manaakingia ngā kai o te hirikapo o te tamaiti, he taonga. Ki te kore e tika te manaakingia e ngā mātua, tērā te wā ka ātitirauhea i te puāwaitanga o te rau. Ngērā kaupapa katoa. (Haki Tuaupiki, 2013)
This project grew out of the desire of a kōhanga and a kura—Te Kōhanga Reo o Ngā Kuaka and Tōku Māpihi Maurea Kura Kaupapa Māori respectively—to enhance transitions between their two sites for tamariki and whānau. Although visits to Tōku Māpihi Maurea before beginning at the kura were available for Ngā Kuaka tamariki on acceptance for enrolment, the instigators of this project believed a more formal transition programme would be beneficial to tamariki and their whānau. The kōhanga and kura were interested in tamariki and whānau experiencing a more effective transition across the two environments and the learning and curriculum in those respective sites.
The kōhanga and kura approached researchers at the University of Waikato Te Kura Toi Tangata Faculty of Education with the research question below and together we developed the proposal. From the outset the project was collaborative, cross-sector, and involved researchers from the kōhanga, the kura and the university.

text: Riariakina ō rongo hirikapo From kōhanga reo to kura Ko te kaupapa tonu, ko te whakatupu i te mahara o te hirikapo kia tiu ai ki te muri, arā, kia puta he whakaaro i pokepokengia nō roto tonu i ngā whakahekenga kāwai o ngā tūpuna o te tamaiti, kua maranga te wā e kaumātua haere ana te tamaiti, ngōna kanohi kua huaki, ngōna taringa kua pīkari, ngā whakaaro kua korikori, tōna wairua hīkaka kua ngawhā, ngērā kaupapa katoa o te tamaiti i taua wā o te tupu. Mā tēnei kōrero hoki hei kawe te taonga o te rongo hirikapo, arā, ngā whakaaro, ngā mahara, nāna ko te waha, nā te waha ko te kupu kōrero e makere iho ai i te ārero tarapepe. Heoi anō he tohutohu anō kei roto e kī ana, manaakingia ngā kai o te hirikapo o te tamaiti, he taonga. Ki te kore e tika te manaakingia e ngā mātua, tērā te wā ka ātitirauhea i te puāwaitanga o te rau. Ngērā kaupapa katoa. (Haki Tuaupiki, 2013)
This project grew out of the desire of a kōhanga and a kura—Te Kōhanga Reo o Ngā Kuaka and Tōku Māpihi Maurea Kura Kaupapa Māori respectively—to enhance transitions between their two sites for tamariki and whānau. Although visits to Tōku Māpihi Maurea before beginning at the kura were available for Ngā Kuaka tamariki on acceptance for enrolment, the instigators of this project believed a more formal transition programme would be beneficial to tamariki and their whānau. The kōhanga and kura were interested in tamariki and whānau experiencing a more effective transition across the two environments and the learning and curriculum in those respective sites.
The kōhanga and kura approached researchers at the University of Waikato Te Kura Toi Tangata Faculty of Education with the research question below and together we developed the proposal. From the outset the project was collaborative, cross-sector, and involved researchers from the kōhanga, the kura and the university.

year: 2017