A new test for vCJD?February 4, 2011
Over the last few days there have been headlines about the development of a new test for vCJD. I thought I would look beyond the headlines and see exactly what the researchers have done.
Creutzfeldt-Jakob disease (CJD) is a type of prion disease. Prion diseases involve misfolding of a protein in our bodies. This misfolded protein can then cause the misfolding of further proteins of the same type leading to disease. Prion diseases generally result in neurological problems by causing the death of brain and nerve cells. CJD itself causes symptoms including memory loss, personality changes, problems with balance and coordination and eventually the brain damage leads to death.
In the 1990’s in the UK a new variant of it emerged, vCJD, that is linked to the consumption of BSE (bovine spongiform encephalopathy – another prion disease)-infected cattle.
A large number of cases of BSE in cattle have been identified in the UK, and although only a small number of cases of vCJD in people have been identified there is the worry that because there is a long preincubation period in prion diseases, many more people may be infected than have currently shown symptoms.
Because of the risk of secondary infection (that is, an infected person donating blood to an uninfected person and thus infecting that person), there are now strict rules on blood products in the UK to try to minimise this risk: all plasma is imported from the USA (where there have been very few cases of BSE) and the UK imports whole blood from low-risk countries for the transfusion of young children.
Ideally there would be a test that could be used on donated blood from people in the UK to screen out infected blood, and this is what the researchers from UCL have started to develop.
What they did:
On the right is a (very) rough diagram of what the researchers did (you can see why I dropped art as soon as possible at school!). It was already known that prions can bind to metal surfaces and so the researchers created a ‘capture matrix’ consisting of stainless steel particles (grey circles). They basically mashed up some brain tissue from either normal or vCJD infected brain and added this to blood and then added the mixture to the capture matrix, with the hypothesis that the prions in the blood mixture would stick to the matrix. After this step they isolated the capture matrix using a magnet and got rid of the remaining blood mixture.
To find out if the prions were there or not they added an antibody (the upside-down Y shape) to the matrix. This (primary) antibody binds to the prions on the matrix and sticks. They then washed away any unbound antibody. Then they added a second antibody that would bind to the first antibody. This secondary antibody was conjugated to an enzyme called horseradish peroxidise (HRP). Again they washed away any unbound antibody. Finally they added a substrate that when acted on by the HRP makes light (the yellow lines – I mean it about my lack of drawing ability!).
What they found:
Using the brain-spiked blood mixtures the researchers found they could distinguish the vCJD samples from the normal brain-spiked samples (because they released more light)– a very good sign!
Because it is not known yet what level of prions make an infective dose and using brain-spiked blood could possibly be creating artificially high levels of them, they then tested the blood of some vCJD sufferers. Alongside this blood they also ran blood from patients of other neurological diseases that present similarly to vCJD and some normal samples. They ran the test twice and said that anything that came up as positive twice was a positive result. None of the negative samples came up positive on both assays (although a small number came up as positive on one or the other) and so they got no false positives. They did get some false negatives: 6 out of 21 vCJD samples came back as not positive overall – 3 because they only came up positive on one test and 3 because they didn’t come up at all.
What they concluded:
The researchers concluded that the capture matrix they used was capturing the prions from the blood sample, bringing them together. Because the prions are now concentrated in a smaller area it is much easier to detect them and so samples can be identified as positive with far lower prion levels than any previous tests have allowed, making the test much more sensitive.
The researchers point out that this test will be difficult to validate as vCJD is so rare that there are few positive samples to test. Any test like this also needs to be tested on lots of negative samples, to check that it doesn’t produce a lot of false positives and this is also difficult because much of the population in the UK has been exposed to BSE and so is possibly carrying the prion without yet displaying any symptoms of vCJD , meaning we cannot yet know if a blood sample from the UK is truly negative. Large numbers of blood samples from countries with very low vCJD risk (because they had few or no BSE cases) will need to be screened to get around this problem.
Although this is obviously a preliminary result it is very exciting because it hints at the possibility of a blood test for vCJD that would not only allow the UK to use UK-donated blood for more patients but could potentially be used to test people for the disease in the future (and would be a much less invasive technique than the current test which is a brain biopsy).
Edgeworth JA et al. (2011.) Detection of prion infection in variant Creutzfeldt-Jakob disease: a blood-based assay. The Lancet. DOI:10.1016/S0140-6736(10)62308-2
Hilton DA. (2006.) Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease. Journal of Pathology 208, 134-141. DOI: 10.1002/path.1880
Collinge J et al. (1996.) Molecular analysis of prion strain variation and the aetiology of ‘new variant’ CJD. Nature. 383, 685-690