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Development of Complement assay - Optimization of buffers for a complement ELISA kit and development of a new homogenous complement assay

Karlsson, Sara LU (2015) KBK820 20151
Pure and Applied Biochemistry
Abstract
The complement system can be activated by three different pathways in the body. It is wanted to be able to detect deficiencies of these activation pathways and Bioporto A/S has acquired the commercial rights to a set of ELISA based assays. There is a need in these commercial assays for optimisation of dilution and wash buffers since the idea with the ELISA kit is to sell it as a “ready-to-use kit” and the dilution and wash buffer will be stored in small, concentrated volumes. In the concentrated volumes the reagent Tween-20 was a problem since it precipitated with CaCl2 in the buffers. The Tween-20 needed to be replaced and it was found that emulfogen had the same ability as Tween-20 to prevent unwanted binding in the wells without... (More)
The complement system can be activated by three different pathways in the body. It is wanted to be able to detect deficiencies of these activation pathways and Bioporto A/S has acquired the commercial rights to a set of ELISA based assays. There is a need in these commercial assays for optimisation of dilution and wash buffers since the idea with the ELISA kit is to sell it as a “ready-to-use kit” and the dilution and wash buffer will be stored in small, concentrated volumes. In the concentrated volumes the reagent Tween-20 was a problem since it precipitated with CaCl2 in the buffers. The Tween-20 needed to be replaced and it was found that emulfogen had the same ability as Tween-20 to prevent unwanted binding in the wells without precipitating with CaCl2. Instead of 0,5% Tween-20 in the dilution buffer 0,1% emulfogen could be used and in the wash buffer 0,01% of emulfogen could be used instead of 0,05% Tween-20. Bioporto has also initiated to convert the complement ELISA kit to a homogenous assay, in this project for the lectin pathway. Different methods were tried out but in the end the best way, both experimentally and by cost was to biotinylate mannan and coat it on streptavidin beads. Most time of the project was used to optimise the biotinylation of mannan where the activation of the mannan was a crucial step. When mannan was activated too much it was not able to bind the protein MBL and the complement cannot be activated. (Less)
Popular Abstract
People around the world are suffering of deficiencies in the complement, causing attacks from the immune system on the bodies on cells. In this project two assays have been developed for detection of these deficiencies.
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author
Karlsson, Sara LU
supervisor
organization
course
KBK820 20151
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Complement, Assays, Deficiencies, Immune system, Development, tillämpad biokemi, applied biochemistry
language
English
id
7870232
date added to LUP
2015-09-30 10:20:57
date last changed
2015-09-30 10:20:57
@misc{7870232,
  abstract     = {The complement system can be activated by three different pathways in the body. It is wanted to be able to detect deficiencies of these activation pathways and Bioporto A/S has acquired the commercial rights to a set of ELISA based assays. There is a need in these commercial assays for optimisation of dilution and wash buffers since the idea with the ELISA kit is to sell it as a “ready-to-use kit” and the dilution and wash buffer will be stored in small, concentrated volumes. In the concentrated volumes the reagent Tween-20 was a problem since it precipitated with CaCl2 in the buffers. The Tween-20 needed to be replaced and it was found that emulfogen had the same ability as Tween-20 to prevent unwanted binding in the wells without precipitating with CaCl2. Instead of 0,5% Tween-20 in the dilution buffer 0,1% emulfogen could be used and in the wash buffer 0,01% of emulfogen could be used instead of 0,05% Tween-20. Bioporto has also initiated to convert the complement ELISA kit to a homogenous assay, in this project for the lectin pathway. Different methods were tried out but in the end the best way, both experimentally and by cost was to biotinylate mannan and coat it on streptavidin beads. Most time of the project was used to optimise the biotinylation of mannan where the activation of the mannan was a crucial step. When mannan was activated too much it was not able to bind the protein MBL and the complement cannot be activated.},
  author       = {Karlsson, Sara},
  keyword      = {Complement,Assays,Deficiencies,Immune system,Development,tillämpad biokemi,applied biochemistry},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of Complement assay - Optimization of buffers for a complement ELISA kit and development of a new homogenous complement assay},
  year         = {2015},
}