Development of Complement assay - Optimization of buffers for a complement ELISA kit and development of a new homogenous complement assay
(2015) KBK820 20151Pure 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.
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/7870232
- author
- Karlsson, Sara LU
- supervisor
- organization
- course
- KBK820 20151
- year
- 2015
- 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}}, 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}}, }