Production of soluble recombinant complement receptor (CR1) antigens to detect or inhibit antibodies to Knops (KN) blood group system antigens
The purpose of this study was to produce a reagent to use in investigation of antibodies directed against the Knops blood group system antigens. A novel reagent based on sr-proteins was produced and used in a new test to inhibit these antibodies.
Current investigation of patients with alloantibodies directed against Knops blood group system antigens can be a difficult, time-consuming process and the provision of blood for transfusion of these patients can often be delayed. This is because these antibodies are hard to identify and the most commonly found anti-Knops antibodies react with most reagent or donor cells that they are tested with because the corresponding Knops antigens are found at high frequency in most populations. The presence of Knops related antibodies can mask underlying antibodies that are clinically significant.
The Knops antigens are carried on Complement Receptor 1 (CR1) located on the red blood cell membrane. Two DNA constructs encoding different parts of CR1 termed long homologous repeat (LHR) C and D were used to transfect human embryonic kidney (HEK293) cells. The cells were grown in different culture systems. Cell culture supernatant containing soluble recombinant (sr)-LHRC or sr-LHR-D was harvested and purified by affinity gel chromatography. The production and purification processes were optimised in terms of protein yield and cost.
The resulting purified sr-LHR-C and sr-LHR-D proteins were used to create a novel reagent containing both proteins. This reagent was used in a new inhibition test based on an indirect antiglobulin technique using commercial gel cards. Using the reagent all examples of previously identified Knops antibodies were inhibited. In addition once these antibodies had been inhibited, underlying antibodies were then detected and identified in some samples.
For the first time Knops specific antibodies can be detected and identified using one unique test. Any underlying clinically significant antibodies will be rapidly identified if present due to inhibition of the KN antibodies. Introduction of the inhibition test into nine NHSBT patient testing laboratories will reduce the time taken for investigation of these patients and make provision of blood for patients a safer, faster process.
Etheridge, W. Production of soluble recombinant complement receptor (CR1) antigens to detect or inhibit antibodies to Knops (KN) blood group system antigens. (Thesis). University of the West of England
|Keywords||blood group antigens, Knops blood group system, complement receptor 1, soluble recombinant proteins, haemagglutination inhibition|
Wd2-etheridge Final project report _Redacted.pdf