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Multiplex Assay Kit for Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay)

HSPB1; HSP-B1; CMT2F; HSP28; Hsp25; Heat Shock 27kDa Protein 1; 28 kDa heat shock protein; Heat shock protein beta-1; Estrogen-regulated 24 kDa protein;

(Note: Up to 8-plex in one testing reaction)

Specificity

This assay has high sensitivity and excellent specificity for detection of Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay).
No significant cross-reactivity or interference between Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay) and the recovery rates were calculated by comparing the measured value to the expected amount of Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 93-104 98
EDTA plasma(n=5) 92-99 96
heparin plasma(n=5) 93-101 97

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay) were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Heat Shock Protein 27 (Hsp27) ,etc. by FLIA (Flow Luminescence Immunoassay) and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 79-91% 97-105% 81-99% 79-101%
EDTA plasma(n=5) 87-101% 92-99% 78-97% 80-93%
heparin plasma(n=5) 96-103% 82-90% 78-97% 79-97%

Stability

The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end.

Reagents and materials provided

Reagents Quantity Reagents Quantity
96-well plate 1 Plate sealer for 96 wells 4
Pre-Mixed Standard 2 Standard Diluent 1×20mL
Pre-Mixed Magnetic beads (22#:Hsp27) 1 Analysis buffer 1×20mL
Pre-Mixed Detection Reagent A 1×120μL Assay Diluent A 1×12mL
Detection Reagent B (PE-SA) 1×120μL Assay Diluent B 1×12mL
Sheath Fluid 1×10mL Wash Buffer (30 × concentrate) 1×20mL
Instruction manual 1

Assay procedure summary

1. Preparation of standards, reagents and samples before the experiment;
2. Add 100μL standard or sample to each well,
    add 10μL magnetic beads, and incubate 90min at 37°C on shaker;
3. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60min at 37°C on shaker;
4. Wash plate on magnetic frame for three times;
5. Add 100μL prepared Detection Reagent B, and incubate 30 min at 37°C on shaker;
6. Wash plate on magnetic frame for three times;
7. Add 100μL sheath solution, swirl for 2 minutes, read on the machine.

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Magazine Citations
Animal Science Journal The effect of heat stress on gene expression and synthesis of heat‐shock and milk proteins in bovine mammary epithelial cells PubMed: 26467738
Nature Communications HSP27 is a partner of JAK2-STAT5 and a potential therapeutic target in myelofibrosis Pubmed:29650953
Advances in Experimental Medicine and Biology Molecules of Damage-Associated Patterns in Bronchoalveolar Lavage Fluid and Serum in Chronic Obstructive Pulmonary Disease Pubmed:29429028
Diabetes Research and Clinical Practice The beneficial effects of 15 units of high-intensity circuit training in women is modified by age, baseline insulin resistance and physical capacity Pubmed: 31102684
Journal of Immunology Research Damage-Associated Molecular Patterns and Myeloid-Derived Suppressor Cells in Bronchoalveolar Lavage Fluid in Chronic Obstructive Pulmonary Disease Patients
Journal of Clinical Medicine Damage-Associated Molecular Patterns and Th-Cell-Related Cytokines Released after Progressive Effort Pubmed: 32210109
Medicine Regulatory T cells, damage-associated molecular patterns, and myeloid-derived suppressor cells in bronchoalveolar lavage fluid interlinked with chronic obstructive … Pubmed:35687771
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