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: Introduction to the landscapes and soils of the Hamilton Basin and South Waikato: field notes

authors: Lowe, David J.

abstract: These notes are an updated version (revised 25 Jan 2021) of those prepared for a field trip held in conjunction with the World Soils Congress in Brisbane in 2010:
Lowe, D.J. 2010. Introduction to the landscapes and soils of the Hamilton Basin. In: Lowe, D.J., Neall, V.E., Hedley, M., Clothier, B., Mackay, A. 2010. Guidebook for Pre-conference North Island, New Zealand “Volcanoes to Oceans” field tour (27-30 July). 19th World Soils Congress, International Union of Soil Sciences, Brisbane. Soil and Earth Sciences Occasional Publication No. 3, Massey University, Palmerston North, pp. 1.24-1.61.

text: Introduction to the landscapes and soils of the Hamilton Basin and South Waikato: field notes These notes are an updated version (revised 25 Jan 2021) of those prepared for a field trip held in conjunction with the World Soils Congress in Brisbane in 2010:
Lowe, D.J. 2010. Introduction to the landscapes and soils of the Hamilton Basin. In: Lowe, D.J., Neall, V.E., Hedley, M., Clothier, B., Mackay, A. 2010. Guidebook for Pre-conference North Island, New Zealand “Volcanoes to Oceans” field tour (27-30 July). 19th World Soils Congress, International Union of Soil Sciences, Brisbane. Soil and Earth Sciences Occasional Publication No. 3, Massey University, Palmerston North, pp. 1.24-1.61.

year: 2021

title: Māori leaders wellbeing: A self-determination perspective

authors: Roche, Maree A.

subjects: Positive psychology

abstract: This research draws on interviews with 18 Māori leaders from various leadership positions within business, community, political and marae organisations, to garner an understanding of how their leadership roles interact with their own well-being. Analysis of interviews revealed that crosscultural developments in self-determination theory could be gained by incorporating Māori tikanga and values into a model of well-being for Māori leaders. Largely, the principles of tino rangatiratanga (autonomy and self-determination), mana (respect and influence), whānau (extended family), whakapapa (shared history) and whanaungatanga (kin relations, consultation and engagement), were united into a model of leader well-being. This ensured that mātauranga Māori (Māori knowledge) informed our model of Māori leader well-being, while also drawing on the burgeoning Western research in the area of well-being, specifically self-determination theory. Overall, we find that similarities exist with self-determination theory and Māori tikanga and values. However, in contrast to self-determination theory, autonomy and competence are developed within relationships, which means that ‘others’ underpin Māori leaders’ well-being. From this perspective, we present a view of the psychological and well-being resources that Māori leaders draw on to guide them through complex times.

text: Māori leaders wellbeing: A self-determination perspective This research draws on interviews with 18 Māori leaders from various leadership positions within business, community, political and marae organisations, to garner an understanding of how their leadership roles interact with their own well-being. Analysis of interviews revealed that crosscultural developments in self-determination theory could be gained by incorporating Māori tikanga and values into a model of well-being for Māori leaders. Largely, the principles of tino rangatiratanga (autonomy and self-determination), mana (respect and influence), whānau (extended family), whakapapa (shared history) and whanaungatanga (kin relations, consultation and engagement), were united into a model of leader well-being. This ensured that mātauranga Māori (Māori knowledge) informed our model of Māori leader well-being, while also drawing on the burgeoning Western research in the area of well-being, specifically self-determination theory. Overall, we find that similarities exist with self-determination theory and Māori tikanga and values. However, in contrast to self-determination theory, autonomy and competence are developed within relationships, which means that ‘others’ underpin Māori leaders’ well-being. From this perspective, we present a view of the psychological and well-being resources that Māori leaders draw on to guide them through complex times.

year: 2015