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Method for determination of members of the S100 family of calcium binding proteins by immunoturbidimetry

A technology of immunoturbidimetry and calprotectin, applied in the field of turbidimetry and detection system

Pending Publication Date: 2020-05-01
IMMUNDIAGNOSTIK AG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While monoclonal antibodies are easier to standardize and allow quality control to prescribed standards throughout the product lifecycle, there are combined issues of obtaining agglutination reactivity, measurement sensitivity, and specificity of granulocyte-secreted calprotectin

Method used

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  • Method for determination of members of the S100 family of calcium binding proteins by immunoturbidimetry
  • Method for determination of members of the S100 family of calcium binding proteins by immunoturbidimetry
  • Method for determination of members of the S100 family of calcium binding proteins by immunoturbidimetry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Example 1 Preparation of anti-calprotectin immune particles

[0098] Latex particles from well-known manufacturers such as MERCK, Bangs Laboratories were used. The latex is carboxylated polystyrene or chloromethyl latex. The latex particles had the following parameters: surface charge density 62 μC / cm2; surface charge density 163 μEq / g pol; solids content 9.0%; stabilized with 0.05% sodium azide. Immune particles were prepared by covalently linking purified murine monoclonal antibodies (mAB 1062, 1067, 1069, 1089, and incorporated in the first reaction component) against purification from granulocytes with intact lysosomal membranes Human Calprotectin. The size of the carboxylated polystyrene particles is preferably uniform (175 nm). Alternatively, nanoparticles of two different sizes (160-175nm and 250-275nm) were coated with a single monoclonal antibody (see Image 6 ). Latex particles of a single size of 175 nm can also be coated with two different monoclonal ...

Embodiment 2

[0100] Example 2 Extraction of stool samples

[0101] The fecal sample extraction method is as follows. 15 mg of feces were diluted 1:100 in 1.5 ml of buffer. Fill empty sample tubes with 1.5 ml assay buffer, 50 mM maleate (pH 5.0), 150 mM NaCl, sodium azide, 0.1% sodium dodecyl sulfate, bovine serum albumin, with or without IgM antiserum was added. For comparison, the ready-to-use IDK was also used at room temperature Extraction buffer (Cat. No. K 6967) and Bühlmann fCal Turbotm extraction buffer were used to extract samples. Fecal samples were collected and stored at 2-8°C for 48h. Long-term (up to 12 months) storage at -20°C is recommended. At the beginning of the turbidimetric test, thaw the frozen sample slowly, preferably at 2-8°C. In some cases, inhomogeneous samples were mechanically homogenized. For sample collection, use Fecal Sample Application System (SAS) (Cat. No. K6998SAS). The tip of the SAS test strip has a notch to hold a certain amount of raw ma...

Embodiment 3

[0102] Example 3 Calprotectin immunoturbidimetric analysis

[0103] Samples extracted with any of the above extraction buffers were used for turbidimetric assays using an apparatus Roche Hitachi 912 according to the manufacturer's instructions. 10 μl of extraction samples were added to 200 μl of assay buffer and 50 μl of storage buffer with sodium citrate (pH 5.0), bovine serum albumin, Tween 20, sucrose, sodium azide. The automated analyzer gently mixes the immunoparticles with the extracted sample while incubating at 37°C for 5 minutes. While gently shaking, add the second reaction component containing the particles bearing the immobilized monoclonal antibody. At 37°C, the agglutination time was 5 minutes, during which time the absorbance was measured. Repeat the measurement. Absorbance values ​​were obtained by reading at a wavelength of 570 nm.

[0104] Commercially purified calprotectin was diluted in pH=5.4 citrate buffer, ranging from 0 to 2000 μg / g. (6 calibrati...

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Abstract

A method for measuring the presence of calprotectin (S100A8 / A) heterodimer in a biological sample using a particle-enhanced turbidimetric immunoassay (PETIA) based on monoclonal antibodies. The methodcan be adapted on automated standard analyzers and provides a reliable clinical measurement of calprotectin in faecal samples and extracts. The method is comparable to commerical two-site sandwich ELISA. The disclosed method counters spontaneous agglutination caused by calcium ions and low-molecular weight calcium-binding S100 proteins as observed with conventional PETIAs. The method can be usedfor measuring the presence of human calprotectin in stool, urine, serum, plasma, synovial liquid and other body liquids. Metrological traceability and high commutability with conventional immunoassays(ELISA) has been shown despite of different measurement principles used.

Description

technical field [0001] The present application relates to turbidimetry and detection systems for the quantitative determination of Calbindin S100 family members in body fluids and feces. The test kit contains antibodies, buffers, and kits of parts for diagnosing acute and chronic inflammatory diseases and conditions. Background technique [0002] The term "calprotectin" includes two granulocyte proteins with a relative molecular weight of 36500 Daltons and an isoelectric point of pH 6.3 and 6.5 (US 4,833,074; Fagerhol et al., Scand.J.Haematol.24,393- 398 (1980). Fagerhol et al., Bull. Europ. Physiopath. Resp. 16 (suppl), 273-281 (1980). Alternative names include low molecular weight S100A8 and S100A9 calcium-binding proteins, myeloid-associated protein 8 (MRP8), Myeloid-related protein 14 (MRP14), metastasis suppressor-related protein 8 / 14 (MRP8-MRP14), S100a / b, calgranulin A / B, cystic fibrosis antigen (CFA), human leukocyte protein, leukocyte L1 protein complex, 60BB anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/54313G01N33/54346G01N2333/4727G01N33/545G01N33/68
Inventor F-P·安布鲁斯特M·格里姆勒P·舒T·贝克尔F·瓦尔策
Owner IMMUNDIAGNOSTIK AG
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