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Analysis of Renal Function & Injury---Cys-C

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Cystatin-C(CYSC) is a non-glycosylated, low molecular weight, cation protein that is regularly synthesized by most nucleated cells. Cystatin-C is an endogenous protein that is known to be affected little by age, race, and muscle mass. It is not only used as a biomarker of renal function but also has positive as­sociations with obesity, insulin resistance, hypertension, and cardiovascular mortality. Higher levels of serum cystatin-C were associated with increased prevalence of diabetic conditions.


Under normal circumstances, cystatin C in serum is 0.47-1.09 mg/L. When renal function is impaired, cystatin C concentration in blood will change with GFR. In renal failure, GFR decrease while cystain C concentration in blood can increase more than 10 times. Under normal glomerular filtration rate and renal tubules dysfunction can hinder cystatin C absorption and its rapid decomposition in the renal tubules, increasing its concentration in urine over 100 times.


Anti-Cystatin-C (CYSC) monoclonal antibody

A new generation of anti-CYSC monoclonal antibodies, which was recently produced by CUSAg, makes possible the development of LETIA assay. Anti-CYSC monoclonal antibodies can be used for a broad range CYSC assay, which is a simple, rapid, sensitive and homogeneous assay that can be performed using automated systems for high-throughput immunoassays.


Calibration curve

The human CYSC reacts with the anti-human CYSC antibody-coated latex, resulting in agglutination and increase in turbidity. Turbidity changes are then measured using a spectrometer to quantitatively measure the CYSC concentration in the sample.Fig.1 shows that the absorbance was increasing gradually along with the CYSC concentration.

Fig.1 Calibration curve for CYSC LETIA assay

 

Accuracy 

Two levels of CYSC serum were analyzed in replicates of three at two separated timeon the CUSAg LETIA assay. Data from this study are summarized in the below table.1, the relative bias was <5%.

Table.1 Accuracy of CYSC reagent


 

Precision

Two members of serum samples based panel were assayed, using a single lot of reagents, in replicates of ten on theCUSAgLETIA assay(from 0.47 to 1.09 mg/L as normal). As shown in table.2, the system shows excellent precision with CV≤5%.

Table.2 Precision profile of CYSC reagent


Repeated freeze-thaw cycles

Our CUSAg antibodies (CSB-DA004AmN①and CSB-DA004AmN②) were dealt with subsequent freeze-thaw cycles, the number of which is 1 time and 5 times from -20°Cto 20°C, and after that wetoke the anti-CYSC antibodies into the reagents onLETIA assay. As shown in Fig.2, the antibodies were stable after 5 times repeated freeze-thaw cycles.

Fig.2 Effect of repeated freeze-thaw on CYSC antibodies

 

Clinical Comparison

Anti-CYSC monoclonal antibodies were also evaluated in medium-scale clinical trials with random blood samples fromdonations (n=50). Fig.4 shows that the correlation coefficient (r) is as high as 0.99 between in-house CYSC assay and commercialLETIA assay. These results show good agreement between the two systems.

Fig.3 Clinicalcomparison ofin-house CYSC reagent and commercial kit

 

Protein activity determination

The active concentration of CYSC protein was determined by using an outsourcing kit. Fig.4 shows thatThe theoretical value and test value of protein activity concentration.

 

Fig.4 The theoretical value and test value of protein activity concentration


References

1. Elsvier Taiwan LLC: Cystatin C as a potential biomarker for neonatal infants. 2016; 1875-9572

2. Hossein-Emad Momtaz, Arash Dehghan, Mohamad Karimian: Correlation of cystatin C and creatinine based estimates of renal function in children with hydroephrosis. 2016 February; 5(1); 25-28

3. Wei Li, Nargis Sultana, Nabeel Siraj, Liam J Ward, Monika Pawlik, Efrat Levy, Stefan Jovinge, Eva Bengtsson, Xi-Ming Yuan: Autophagy dysfunction and regulatory cystatin C in macrophage death of atherosclerosis. 2016; pp. 1-9

4. Monika Pergande and Klaus Jung: Sandwich enzyme immunoassay of cystatin C in serum with commercially available antibodies. 1993; 39/9; 1885-1890 


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