| Test principle: |
This assay employs a two-site sandwich ELISA to quantitate RCAN2 in samples. An antibody specific for RCAN2 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anyRCAN2 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for RCAN2 is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of RCAN2 bound in the initial step. The color development is stopped and the intensity of the color is measured.
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| Product Overview: |
Using a differential display of mRNA by PCR, Miyazaki et al. (1996) cloned a cDNA fragment of a thyroid hormone-responsive gene, which they called ZAKI4 (RCAN2), from cultured human skin fibroblasts. Using 5-prime and 3-prime RACE, they isolated a full-length cDNA, which gives rise to 2 mRNA species, both of which encode a protein of 192 amino acids. Sequence analysis of the ZAKI4 protein suggested that the 2 mRNAs arise by alternative polyadenylation. Northern blot analysis revealed the presence of differentially expressed transcripts of 3.4 and 1.4 kb. The larger transcript is expressed in skin fibroblasts and skeletal muscle, whereas the smaller transcript is found in heart, brain, and liver but not in placenta, lung, kidney, or pancreas.
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| Components: |
Reagents |
Quantity |
Reagents |
Quantity |
Assay plate (96 Wells) |
1 |
Instruction manual |
1 |
Standard (lyophilized) |
2 |
Sample Diluent |
1 x 20 mL |
Biotin-Conjugate (concentrate 100 x) |
1 x 120 μL |
Biotin-Conjugate Diluent |
1 x 20 mL |
Streptavidin-HRP (concentrate 100 x) |
1 x 120 μL |
Streptavidin-HRP Diluent |
1 x 20 mL |
Wash Buffer (concentrate 25 x) |
1 x 20 mL |
Substrate Solution |
1 x 12 mL |
Stop Solution |
1 x 10 mL |
Adhesive Films |
4 |
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| Specificity: |
This assay has high sensitivity and excellent specificity for detection of Human RCAN2. No significant cross-reactivity or interference between Human RCAN2 and analogues was observed.
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| Recovery: |
Matrices listed below were spiked with certain level of recombinant Human RCAN2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human RCAN2 in samples.
Sample Type |
Number |
Recovery range (%) |
Average(%) |
Serum |
10 |
90-101 |
96 |
EDTA plasma |
10 |
89-97 |
93 |
Heparin plasma |
10 |
91-99 |
95 |
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| Precision: |
Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.
CV (%) = SD/meanX100
Intra-Assay: CV<8%
Inter-Assay: CV<12%
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| Linearity: |
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human RCAN2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample Type |
1：2 |
1：4 |
1：8 |
1：16 |
Serum |
78-89% |
81-99% |
92-103% |
95-105% |
EDTA plasma |
91-101% |
90-98% |
93-101% |
91-98% |
Heparin plasma |
92-103% |
93-102% |
92-99% |
91-101% |
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| Stability: |
The stability of ELISA 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.
The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
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| Sample collection and storage: |
S e r u m : U s e a s e r u m s e p a r a t o r t u b e ( S S T ) a n d a l l o w s a m p l e s t o c l o t f o r t w o h o u r s a t r o o m t e m p e r a t u r e o r o v e r n i g h t a t 2 - 8° C b e f o r e c e n t r i f u g a t i o n f o r 1 5 m i n u t e s a t 1 0 0 0 × g . R e m o v e s e r u m a n d a s s a y i m m e d i a t e l y o r a l i q u o t a n d s t o r e s a m p l e s a t ≤ - 2 0° C . A v o i d r e p e a t e d f r e e z e - t h a w c y c l e s .
P l a s m a : C o l l e c t p l a s m a u s i n g E D T A , o r h e p a r i n a s a n a n t i c o a g u l a n t . C e n t r i f u g e f o r 1 5 m i n u t e s a t 1 0 0 0 × g a t 2 - 8° C w i t h i n 3 0 m i n u t e s o f c o l l e c t i o n . A s s a y i m m e d i a t e l y o r a l i q u o t a n d s t o r e s a m p l e s a t ≤ - 2 0° C . A v o i d r e p e a t e d f r e e z e - t h a w c y c l e s .
O t h e r b i o l o g i c a l f l u i d s : C e n t r i f u g e s a m p l e s f o r 2 0 m i n u t e s a t 1 0 0 0 × g . R e m o v e p a r t i c u l a t e s a n d a s s a y i m m e d i a t e l y o r s t o r e s a m p l e s i n a l i q u o t a t - 2 0° C o r - 8 0° C . A v o i d r e p e a t e d f r e e z e / t h a w c y c l e s .
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| Kits storage instructions: |
Store at 2-8°C. Please refer to Instruction Manual.
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Human Calcipressin-2 (RCAN2) ELISA Kit
