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Multiplex Assay Kit for Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array)

CTLA3; HFSP; HF; H factor; Granzyme 1; Fragmentin-1; Hanukkah factor; Cytotoxic T-Lymphocyte-Associated Serine Esterase 3; CTL tryptase; Cytotoxic T-lymphocyte proteinase 1

Specificity

This assay has high sensitivity and excellent specificity for detection of Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array).
No significant cross-reactivity or interference between Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array) and the recovery rates were calculated by comparing the measured value to the expected amount of Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 97-105 101
EDTA plasma(n=5) 88-99 94
heparin plasma(n=5) 80-98 89

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array) 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 Granzyme A (GZMA) ,etc. by CBA (Cytometric Bead Array) 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) 90-97% 89-103% 84-92% 89-99%
EDTA plasma(n=5) 97-105% 96-105% 81-95% 99-105%
heparin plasma(n=5) 79-102% 80-97% 78-93% 94-102%

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

Assay procedure summary

1. Add 200μL analysis buffer solution to each well of the plate for pre-wetting;
2. Preparation of standards, reagents and samples before the experiment;
3. Add 100μL standard or sample to each well, add 10μL magnetic beads, and incubate 120 minutes protect from light at 25°C on shaker;
4. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent A. Incubate 60 minutes protect from light at 25°C on shaker;
5. Remove liquid on magnetic frame, add 100μL prepared Detection Reagent B, and incubate 30 minutes protect from light at 25°C on shaker;
6. Wash plate once on magnetic frame;
7. Add 100μL sheath fluid, swirl for 10 minutes, read on the machine.

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Magazine Citations
Cell Reports Granzyme A Produces Bioactive IL-1β through a Nonapoptotic Inflammasome-Independent Pathway Pubmed:25437548
Journal of Dermatological Science Relationships among plasma granzyme B level, pruritus and dermatitis in patients with atopicdermatitis. pubmed:27686401
Journal of Maternal-Fetal & Neonatal Medicine Extracellular granzyme A in amniotic fluid is elevated in the presence of sterile intra-amniotic inflammation in preterm prelabor rupture of membranes Pubmed: 32912008
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