US 11,698,342 B2
Method and system for analysing fluorospot assays
Christian Smedman, Stockholm (SE); Joakim Jalden, Stollentuna (SE); Daniel Pelikan, Vange (SE); Pol Del Aguila Pla, Solna (SE); and Klas Magnusson, Bandhagen (SE)
Assigned to MABTECH AB, Nacka Strand (SE)
Appl. No. 16/627,029
Filed by MABTECH PRODUCTION AB, Nacka Strad (SE)
PCT Filed Jun. 27, 2018, PCT No. PCT/SE2018/050691
§ 371(c)(1), (2) Date Dec. 27, 2019,
PCT Pub. No. WO2019/004913, PCT Pub. Date Jan. 3, 2019.
Claims priority of application No. 1730177-1 (SE), filed on Jun. 29, 2017.
Prior Publication US 2020/0116636 A1, Apr. 16, 2020
Int. Cl. G01N 21/64 (2006.01)
CPC G01N 21/6428 (2013.01) [G01N 2021/6439 (2013.01); G01N 2021/6471 (2013.01)] 13 Claims
OG exemplary drawing
 
1. A method for analysing fluorospot assays, the method comprising:
(i) illuminating a well of an assay plate having a plurality of wells, with a plurality of excitation lights, wherein at a given time, the well of the assay plate is illuminated by only one excitation light;
(ii) capturing at least one image of the well, in raw image format, for each of the plurality of excitation lights;
(iii) generating a model of analyte release distribution in the well for each of the plurality of excitation lights, wherein the generation of the model of analyte release distribution in the well for a given excitation light comprises:
(a) deconvolving the captured at least one image of the well for the given excitation light to estimate a pre-diffusion analyte distribution; and
(b) detecting at least one potential analyte release site based on local maxima in the pre-diffusion analyte distribution; and
(iv) for the plurality of excitation lights employed to illuminate the well of the assay plate, clustering a plurality of co-positioned fluorospots as a multiple secretion fluorospot, wherein the clustering is performed for all generated models of analyte release distribution, and wherein the clustering determines at least one multiple secretion fluorospot, wherein the method includes:
generating the model of analyte release distribution in the well for each of the plurality of excitation lights comprises optimizing the pre-diffusion analyte distribution based on the detected at least one potential release site; and
modifying the model of analyte release distribution in the well to analyse at least one fluorospot therein, wherein optimizing the pre-diffusion analyte distribution, based on the detected at least one potential analyte release site, is implemented iteratively at least once, and comprises employing at least one of: alternating direction methods, multiplicative update rules, forward-backward proximal gradient algorithms.