Human Ubiquitin D (UBD) 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 Ubiquitin D (UBD) ELISA Kit
Method:	Sandwich
Synonyms:	UB-D; FAT10; UBD-3; Diubiquitin; Ubiquitin-like protein FAT10
Detection range:	3.125-200ng/mL
Target Protein:	UBD
Size:	96T/48T
Quality guarantee period:	for 12 months, 16 months
Catalog number:	DL-UBD-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

1.Overview：

Other names：UB-D; FAT10; UBD-3; Diubiquitin; Ubiquitin-like protein FAT10

Function: Ubiquitin-like protein modifier which can be covalently attached to target protein and subsequently leads to their degradation by the 26S proteasome, in a NUB1L-dependent manner. Probably functions as a survival factor. Conjugation ability activated by UBA6. Promotes the expression of the proteasome subunit beta type-9 (PSMB9/LMP2). Regulates TNF-alpha-induced and LPS-mediated activation of the central mediator of innate immunity NF-kappa-B by promoting TNF-alpha-mediated proteasomal degradation of ubiquitinated-I-kappa-B-alpha. Required for TNF-alpha-induced p65 nuclear translocation in renal tubular epithelial cells (RTECs). May be involved in dendritic cell (DC) maturation, the process by which immature dendritic cells differentiate into fully competent antigen-presenting cells that initiate T cell responses. Mediates mitotic non-disjunction and chromosome instability, in long-term in vitro culture and cancers, by abbreviating mitotic phase and impairing the kinetochore localization of MAD2L1 during the prometaphase stage of the cell cycle. May be involved in the formation of aggresomes when proteasome is saturated or impaired. Mediates apoptosis in a caspase-dependent manner, especially in renal epithelium and tubular cells during renal diseases such as polycystic kidney disease and Human immunodeficiency virus (HIV)-associated nephropathy (HIVAN).

Sequence：

       10         20         30         40         50
 MAPNASCLCV HVRSEEWDLM TFDANPYDSV KKIKEHVRSK TKVPVQDQVL
         60         70         80         90        100
 LLGSKILKPR RSLSSYGIDK EKTIHLTLKV VKPSDEELPL FLVESGDEAK
        110        120        130        140        150
 RHLLQVRRSS SVAQVKAMIE TKTGIIPETQ IVTCNGKRLE DGKMMADYGI
        160
 RKGNLLFLAC YCIGG

2．Features
INTENDED USE
The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of UBD in human serum, plasma or other biological fluids.

DETECTION RANGE
3.12-200ng/mL. The standard curve concentrations used for the ELISA’s were 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.12ng/mL.

SENSITIVITY
The minimum detectable dose of UBD is typically less than 1.34ng/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 UBD.
No significant cross-reactivity or interference between UBD and analogues was observed.

You can reference link of the kit as following
https://dldevelop.com/Research-reagent/dl-ubd-hu.html
https://www.dldevelop.com/uploadfile/data/DL-UBD-Hu.pdf

Introduction

Item	Standard		Test
Description	The kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of UBD 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 UBD 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.