US 11,714,082 B2
Method for determination of members of the S100 family of calcium binding proteins by immunoturbidimetry
Franz-Paul Armbruster, Bobenheim-Roxheim (DE); Matthias Grimmler, Elz (DE); Pia Schu, Schmelz (DE); Tobias Becker, Elz (DE); and Felix Walzer, Seeheim (DE)
Assigned to Immundiagnostik AG, Bensheim (DE); and DiaSys Diagnostic Systems GmbH, Holzheim (DE)
Appl. No. 16/611,942
Filed by Immundiagnostik AG, Bensheim (DE); and Diasys Diagnostic Systems GmbH, Holzheim (DE)
PCT Filed May 9, 2018, PCT No. PCT/EP2018/062159
§ 371(c)(1), (2) Date Nov. 8, 2019,
PCT Pub. No. WO2018/206737, PCT Pub. Date Nov. 15, 2018.
Claims priority of application No. 17170291 (EP), filed on May 9, 2017.
Prior Publication US 2021/0382044 A1, Dec. 9, 2021
Int. Cl. G01N 33/543 (2006.01); G01N 33/545 (2006.01); G01N 33/68 (2006.01)
CPC G01N 33/54346 (2013.01) [G01N 33/545 (2013.01); G01N 33/68 (2013.01); G01N 2333/4727 (2013.01)] 10 Claims
 
1. A method for measuring the presence of calprotectin in a biological sample of a patient by a particle-enhanced turbidimetric immunoassay using a test kit comprising:
a first aqueous reagent-component comprising:
20 to 1000 mmol/L organic buffer with a pH in the range from 5.0 to 6.0;
50 to 300 mmol/L salt of sodium, potassium or lithium;
0.1 to 1.5% protease-treated serum albumin;
0.01 to 0.1% (w/v) sodium dodecyl sulfate, and
optionally beta-aldoses, triose, tetroses, pentoses, hexoses, glucan, dextran and/or sugar to achieve an osmolality of at least 200 mosmol/kg of H2O; and
a second reagent-component comprising:
0.01 to 0.5% (w/v) latex particles of 150 to 350 nm diameter carrying immobilized monoclonal antibodies which bind either one of S100A8 and S100A9 proteins or calprotectin (S100A8/A9);
the method comprising the steps of:
a) collecting a predetermined amount of said biological sample;
b) solubilizing and extracting said biological sample in the first aqueous reagent-component having i) a pH between 5.0 and 6.0, ii) an osmolality of at least 150 mosmol/kg of H2O, iii) wherein the organic buffer is co-coordinating and sequestering calcium and zinc ions, and optionally, homogenizing and extracting the matrix of said biological sample followed by a removal of any particulate material to obtain a sample solution with a solubilized presence of essentially heterodimeric calprotectin (S100A8/A91);
c) mixing a defined amount of said sample solution of step (b) with an amount of the second reagent-component comprising latex nanoparticles having immobilized two or more monoclonal antibodies or fragments thereof which specifically bind either one of S100A8 and S100A9 or calprotectin (S100A8/A9) to obtain a particle-bound antibody-antigen reaction with calprotectin (S100A8/A9) being present in a defined molecular state;
d) incubating the mixture of step c) for an interval of time; and
e) acquiring an optical property of the mixture and determining a signal indicative of the content of calprotectin (S100A8/A9) based on the optical property of the mixture;
f) relating said content to a calibrated control and assessing the clinical condition of said patient based on the measured presence of calprotectin (S100A8/A9) in said biological sample.