ELISA Kit for Cathepsin D (CTSD)

CPSD; CLN10; Lysosomal Aspartyl Protease; Ceroid-Lipofuscinosis,Neuronal 10

Specificity

This assay has high sensitivity and excellent specificity for detection of Cathepsin D (CTSD).
No significant cross-reactivity or interference between Cathepsin D (CTSD) and analogues was observed.

Recovery

Matrices listed below were spiked with certain level of recombinant Cathepsin D (CTSD) and the recovery rates were calculated by comparing the measured value to the expected amount of Cathepsin D (CTSD) in samples.

Matrix Recovery range (%) Average(%)
serum(n=5) 78-90 81
EDTA plasma(n=5) 78-93 83
heparin plasma(n=5) 78-104 81

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level Cathepsin D (CTSD) were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level Cathepsin D (CTSD) 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 Cathepsin D (CTSD) 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) 78-97% 90-98% 95-102% 78-96%
EDTA plasma(n=5) 82-101% 88-96% 92-101% 96-104%
heparin plasma(n=5) 89-98% 96-104% 89-98% 78-94%

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
Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4
Standard 2 Standard Diluent 1×20mL
Detection Reagent A 1×120µL Assay Diluent A 1×12mL
Detection Reagent B 1×120µL Assay Diluent B 1×12mL
TMB Substrate 1×9mL Stop Solution 1×6mL
Wash Buffer (30 × concentrate) 1×20mL Instruction manual 1

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 50µL standard or sample to each well.
    And then add 50µL prepared Detection Reagent A immediately.
    Shake and mix. Incubate 1 hour at 37°C;
3. Aspirate and wash 3 times;
4. Add 100µL prepared Detection Reagent B. Incubate 30 minutes at 37°C;
5. Aspirate and wash 5 times;
6. Add 90µL Substrate Solution. Incubate 10-20 minutes at 37°C;
7. Add 50µL Stop Solution. Read at 450 nm immediately.

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Magazine Citations
Molecular Immunology Cathepsin D is released after severe tissue trauma in vivo and is capable of generating C5a in vitro ScienceDirect: S0161589011008297
21 Plasma Cathepsin D Levels: A Novel Tool to Predict Pediatric Hepatic Inflammation PubMed: 25732418
Scientific Reportes Plasma cathepsin D correlates with histological classifications of fatty liver disease in adults and responds to intervention pubmed:27922112
Biomedicine & pharmacotherapy Berberine ameliorates intrahippocampal kainate-induced status epilepticus and consequent epileptogenic process in the rat: Underlying mechanisms pubmed:28061403
JGH OPEN Limited applicability of cathepsin D for the diagnosis and monitoring of non‐alcoholic steatohepatitis
Diabetologia Plasma cathepsin D activity is negatively associated with hepatic insulin sensitivity in overweight and obese humans Pubmed: 31690989
Antioxidants Iron and Advanced Glycation End Products: Emerging Role of Iron in Androgen Deficiency in Obesity Pubmed: 32235809
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