Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human CRY alphaB in samples. No significant cross-reactivity or interference between Human CRY alphaB and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human CRY alphaB. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CRY alphaB and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human CRY alphaB, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human CRY alphaB. The concentration of Human CRY alphaB in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	CRYAB: a major structural protein of the eye lens. A member of the small heat shock protein (sHSP also known as the HSP20) family. Alpha-B is expressed in the lens as well as other tissues. Elevated expression of alpha-B crystallin occurs in many neurological diseases; a missense mutation cosegregated in a family with a desmin-related myopathy.
UniProt Protein Details:	Protein type:Heat shock protein; Chaperone Chromosomal Location of Human Ortholog: 11q22. 3-q23. 1 Cellular Component: actin filament bundle; cell surface; cytoplasm; cytosol; Golgi apparatus; microtubule cytoskeleton; mitochondrion; myelin sheath; nucleoplasm; nucleus; plasma membrane; Z disc Molecular Function:identical protein binding; metal ion binding; microtubule binding; protein binding; protein homodimerization activity; structural constituent of eye lens; unfolded protein binding Biological Process: aging; lens development in camera-type eye; microtubule polymerization or depolymerization; muscle contraction; muscle development; negative regulation of apoptosis; negative regulation of caspase activity; negative regulation of cell growth; negative regulation of intracellular transport; protein folding; protein homooligomerization; response to estradiol stimulus; response to hydrogen peroxide; response to hypoxia; stress-activated MAPK cascade; tubulin folding Disease: Cardiomyopathy, Dilated, 1ii; Cataract 16, Multiple Types; Myopathy, Myofibrillar, 2; Myopathy, Myofibrillar, Fatal Infantile Hypertonic, Alpha-b Crystallin-related
NCBI Summary:	Mammalian lens crystallins are divided into alpha, beta, and gamma families. Alpha crystallins are composed of two gene products: alpha-A and alpha-B, for acidic and basic, respectively. Alpha crystallins can be induced by heat shock and are members of the small heat shock protein (HSP20) family. They act as molecular chaperones although they do not renature proteins and release them in the fashion of a true chaperone; instead they hold them in large soluble aggregates. Post-translational modifications decrease the ability to chaperone. These heterogeneous aggregates consist of 30-40 subunits; the alpha-A and alpha-B subunits have a 3:1 ratio, respectively. Two additional functions of alpha crystallins are an autokinase activity and participation in the intracellular architecture. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. Alpha-A and alpha-B gene products are differentially expressed; alpha-A is preferentially restricted to the lens and alpha-B is expressed widely in many tissues and organs. Elevated expression of alpha-B crystallin occurs in many neurological diseases; a missense mutation cosegregated in a family with a desmin-related myopathy. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2014]
UniProt Code:	P02511
NCBI GenInfo Identifier:	117385
NCBI Gene ID:	1410
NCBI Accession:	P02511. 2
UniProt Secondary Accession:	P02511,O43416, Q9UC37, Q9UC38, Q9UC39, Q9UC40, Q9UC41 B0YIX0,
UniProt Related Accession:	P02511
Molecular Weight:	20,159 Da
NCBI Full Name:	Alpha-crystallin B chain
NCBI Synonym Full Names:	crystallin alpha B
NCBI Official Symbol:	CRYAB
NCBI Official Synonym Symbols:	MFM2; CRYA2; CTPP2; HSPB5; CMD1II; CTRCT16; HEL-S-101
NCBI Protein Information:	alpha-crystallin B chain
UniProt Protein Name:	Alpha-crystallin B chain
UniProt Synonym Protein Names:	Alpha(B)-crystallin; Heat shock protein beta-5; HspB5; Renal carcinoma antigen NY-REN-27; Rosenthal fiber component
Protein Family:	Alpha-crystallin
UniProt Gene Name:	CRYAB
UniProt Entry Name:	CRYAB_HUMAN

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
2000	51337 60105	55721	55693
1000	23668 24280	23974	23946
500	11561 10503	11032	11004
250	5208 5380	5294	5266
125	2665 2551	2608	2580
62.5	1315 1307	1311	1283
31.25	653 695	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CRY alphaB were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CRY alphaB were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	109.23	188.73	884.48	102.93	178.03	883.63
Standard deviation	9.95	18.00	59.61	12.69	14.62	90.13
C V (%)	9.11	9.54	6.74	12.33	8.21	10.20

Recovery

The recovery of Human CRY alphaB spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	94-111	102
EDTA plasma (n=5)	97-110	103
Cell culture media (n=5)	89-100	95

Linearity

Samples were spiked with high concentrations of Human CRY alphaB and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	100-115	89-102	90-102
1:2	Average (%)	106	96	96
1:4	Range (%)	89-100	97-109	85-97
1:4	Average (%)	94	103	92
1:8	Range (%)	96-111	101-117	102-116
1:8	Average (%)	102	107	110
1:16	Range (%)	97-112	83-98	98-112
1:16	Average (%)	104	90	106

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.