Detection of transmissible spongiform encephalopathy (TSE) protein employing a highly sensitive Western blot assay and chemiluminescent detection system
View Patent ↗The invention provides a rapid, sensitive immunoassay capable of detecting and quantitating pathogenic protein to a level of 3 to 5 logs. The preferred immunoassay utilized is a chemiluminescent endpoint for a Western blot immunoassay. The invention has been successfully applied to track the clearance of pathogenic protein during production of proteins derived from plasma. It is particularly applicable and has been confirmed by bioassay to relate TSE infectivity to quantitative results on prion protein.
1. An immunoassay method useful for quantitating transmissible spongiform encephalopathy (TSE) protein in a biological sample, comprising the steps of:
a) preparing a relevant biological sample for a Western blot assay, including preparing buffered dilutions of the sample using a physiologically compatible buffer and treating the buffered dilutions with proteinase-K;
b) concentrating the buffered dilutions to form concentrated proteinase-K samples;
c) preparing a spiked control sample comprising TSE protein for the Western blot assay, using a physiological compatible buffer and treating the spiked control sample with proteinase-K;
d) performing the Western blot assay for TSE protein on the concentrated proteinase-K samples and the control sample; and
e) quantitating the TSE protein results by quantifying the results of the Western blot assay by (i) determining the number of immunoreactive positions that contain a signal attributable to the presence of TSE protein in the relevant biological sample; (ii) determining the number of immunoreactive positions that contain a signal attributable to the presence of TSE protein in the control sample; and (iii) approximating the amount of TSE protein present in the relevant biological sample to the limits of detection by comparison to results obtained for the control sample,
wherein the signal attributable to the presence of TSE protein is visualized using a chemiluminescent technique, and
wherein the method can detect as low as 5 pg of TSE protein in the relevant biological sample.
2. The method of claim 1 wherein the biological sample is chosen from the group consisting of a homogenized tissue, plasma, and a plasma processing fraction sample.
3. The method of claim 1 wherein the biological sample is diluted serially, up to nine logs.
4. The method of claim 1 wherein the physiologically compatible buffer is a buffered saline solution.
5. The method of claim 4 wherein the buffer component of the buffered saline solution is chosen from the group consisting of PBS, 0.1% BSA in PBS, and Tris buffered saline.
6. The method of claim 1 wherein the buffered dilutions are concentrated by centrifugation or filtration.
7. The method of claim 1 wherein the Western blot immunoassay comprises the steps of:
a) separating the proteinase-K treated samples electrophoretically;
b) transferring the separated samples to a membrane;
c) adding a blocking agent to the membrane containing the separated samples;
d) incubating the membranes with a first antibody capable of binding to TSE protein;
e) washing the incubated membrane with a low salt buffer to remove any non-binding antibodies and proteins;
f) incubating the washed membrane with a second antibody capable of recognizing the first antibody, which second antibody contains a reporter group capable of providing a measurable signal; and
g) measuring the signal produced by counting the number of lanes with a detectable signal.
8. The method of claim 7 wherein the Western blot immunoassay utilizes the monoclonal antibody, 3F4, as the first antibody for the identification of the TSE protein on the membrane.
9. The method of claim 8 wherein the results of the Western blot are quantified by:
a) determining the number of immunoreactive lanes on the membrane that contain a signal attributable to the presence of TSE protein; and
b) approximating the amount of TSE protein present to the limits of detection.