Human Klotho (KL) ELISA Kit

two product lines: Traditional ELISA Kit and Ready-to-Use ELISA Kit.

	Traditional ELISA Kit	Ready-to-Use ELISA KIT
Product name:	Human Klotho (KL) ELISA Kit
Method:	Sandwich
Synonyms:
Detection range:	0.156-10ng/mL
Target Protein:	KL
Size:	96T/48T
Quality guarantee period:	for 12 months, 16 months
Catalog number:	DL-KL-Hu (traditional)	(ready-to-use)
Assay length	1-4.5Hours	1-3.5Hours
Advantages:	Competitive price. High sensitivity. High stability. 12 months shelf life.	Pre-diluted Detection Reagent A and B Reduction in the number of steps when conducting the test All the reagents can be stored at -20℃ Faster reaction compare to other brands 16 months shelf life
Instruction Manual

Overview

Function: May have weak glycosidase activity towards glucuronylated steroids. However, it lacks essential active site Glu residues at positions 239 and 872, suggesting it may be inactive as a glycosidase in vivo. May be involved in the regulation of calcium and phosphorus homeostasis by inhibiting the synthesis of active vitamin D (By similarity). Essential factor for the specific interaction between FGF23 and FGFR1 (By similarity).By similarity The Klotho peptide generated by cleavage of the membrane-bound isoform may be an anti-aging circulating hormone which would extend life span by inhibiting insulin/IGF1 signaling.By similarity
Sequence:
MPASAPPRRP RPPPPSLSLL LVLLGLGGRR LRAEPGDGAQ TWARFSRPPA PEAAGLFQGT FPDGFLWAVG SAAYQTEGGW QQHGKGASIW DTFTHHPLAP PGDSRNASLP LGAPSPLQPA TGDVASDSYN NVFRDTEALR ELGVTHYRFS ISWARVLPNG SAGVPNREGL RYYRRLLERL RELGVQPVVT LYHWDLPQRL QDAYGGWANR ALADHFRDYA ELCFRHFGGQ VKYWITIDNP YVVAWHGYAT GRLAPGIRGS PRLGYLVAHN LLLAHAKVWH LYNTSFRPTQ GGQVSIALSS HWINPRRMTD HSIKECQKSL DFVLGWFAKP VFIDGDYPES MKNNLSSILP DFTESEKKFI KGTADFFALC FGPTLSFQLL DPHMKFRQLE SPNLRQLLSW IDLEFNHPQI FIVENGWFVS GTTKRDDAKY MYYLKKFIME TLKAIKLDGV DVIGYTAWSL MDGFEWHRGY SIRRGLFYVD FLSQDKMLLP KSSALFYQKL IEKNGFPPLP ENQPLEGTFP CDFAWGVVDN YIQVDTTLSQ FTDLNVYLWD VHHSKRLIKV DGVVTKKRKS YCVDFAAIQP QIALLQEMHV THFRFSLDWA LILPLGNQSQ VNHTILQYYR CMASELVRVN ITPVVALWQP MAPNQGLPRL LARQGAWENP YTALAFAEYA RLCFQELGHH VKLWITMNEP YTRNMTYSAG HNLLKAHALA WHVYNEKFRH AQNGKISIAL QADWIEPACP FSQKDKEVAE RVLEFDIGWL AEPIFGSGDY PWVMRDWLNQ RNNFLLPYFT EDEKKLIQGT FDFLALSHYT TILVDSEKED PIKYNDYLEV QEMTDITWLN SPSQVAVVPW GLRKVLNWLK FKYGDLPMYI ISNGIDDGLH AEDDQLRVYY MQNYINEALK AHILDGINLC GYFAYSFNDR TAPRFGLYRY AADQFEPKAS MKHYRKIIDS NGFPGPETLE RFCPEEFTVC TECSFFHTRK SLLAFIAFLF FASIISLSLI FYYSKKGRRS YK

Features

DL-KL-Hu Human Klotho (KL) ELISA Kit
INTENDED USE
The kit is a sandwich enzyme immunoassay for in vitro quantitative measurement of (KL) in human serum, plasma and other biological fluids.
DETECTION RANGE
0.156-10ng/mL. The standard curve concentrations used for the ELISA’s were 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.312ng/mL, 0.156ng/mL.

SENSITIVITY

The minimum detectable dose of (KL) is typically less than 0.055ng/mL.
The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.

SPECIFICITY

This assay has high sensitivity and excellent specificity for detection of (KL).
No significant cross-reactivity or interference between KL and analogues was observed.
Note:
Limited by current skills and knowledge, it is impossible for us to complete the cross-reactivity detection between (KL) and all the analogues, therefore, cross reaction may still exist.

Introduction

Item	Standard		Test
Description	The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of KL in human serum, plasma and other biological fluids.		Conform
Identification	Colorimetric		Positive
Composition	Traditional ELISA Kit	Ready-to-Use ELISA KIT	Conform
	Pre-coated, ready to use 96-well strip plate 1	Pre-coated, ready to use 96-well strip plate 1
	Plate sealer for 96 wells 2	Plate sealer for 96 wells 2
	Standard 2	Standard 2
	Diluents buffer 1×45mL	Standard Diluent 1×20mL
	Detection Reagent A 1×120μL	Detection Solution A 1×12mL
	Detection Reagent B 1×120μL	Detection Solution B 1×12mL
	TMB Substrate 1×9mL	TMB Substrate 1×9mL
	Stop Solution 1×6mL	Stop Solution 1×6mL
	Wash Buffer (30 × concentrate) 1×20mL	Wash Buffer (30 × concentrate) 1×20mL
	Instruction manual 1	Instruction manual 1

Test principle

The microtiter plate provided in this kit has been pre-coated with an antibody specific to the index. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to the index. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain the index, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of the index in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Recovery

Matrices listed below were spiked with certain level of recombinant KL and the recovery rates were calculated by comparing the measured value to the expected amount of the index in samples.

Matrix	Recovery range (%)	Average(%)
serum(n=5)	81-93	86
EDTA plasma(n=5)	80-97	88
heparin plasma(n=5)	90-101	95

Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of the index 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)	82-96%	83-98%	81-99%	93-101%
EDTA plasma(n=5)	88-101%	86-95%	90-102%	80-93%
heparin plasma(n=5)	80-91%	82-90%	95-104%	79-95%

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level the index were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level the index were tested on 3 different plates, 8 replicates in each plate.
CV(%) = SD/meanX100
Intra-Assay: CV<10%
Inter-Assay: CV<12%

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 conditions.
Note:
To minimize unnecessary influences on the performance, operation procedures and lab conditions, especially room temperature, air humidity and incubator temperatures should be strictly regulated. It is also strongly suggested that the whole assay is performed by the same experimenter from the beginning to the end.

Assay procedure summary

1. Prepare all reagents, samples and standards;
2. Add 100µL standard or sample to each well. Incubate 2 hours at 37℃;
3. Aspirate and add 100µL prepared Detection Reagent A. Incubate 1 hour at 37℃;
4. Aspirate and wash 3 times;
5. Add 100µL prepared Detection Reagent B. Incubate 1 hour at 37℃;
6. Aspirate and wash 5 times;
7. Add 90µL Substrate Solution. Incubate 15-25 minutes at 37℃;
8. Add 50µL Stop Solution. Read at 450nm immediately.