Human alpha-La (Alpha-Lactalbumin) CLIA Kit (HUES00580)

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HUES00580
€599

Description

ELISA Kit Technical ManualMSDS

Human alpha-La (Alpha-Lactalbumin) CLIA Kit

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 alpha-La . 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 alpha-La 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 alpha-La, 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 alpha-La. The concentration of Human alpha-La in the samples can be calculated by comparing the RLU of the samples to the standard curve.

Assay typeSandwich
Format96T
Assay time4.5h
ReactivityHuman
Detection methodChemiluminescence
Detection range0.16-10 ng/mL
Sensitivity0.09 ng/mL
Sample volume100uL
Sample typeBreast milk
RepeatabilityCV < 15%

Specificity

This kit recognizes Human alpha-La in samples. No significant cross-reactivity or interference between Human alpha-La and analogues was observed.

Typical Data

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
(ng/mL)
RLU Average Corrected
1031660
33282
3247132447
514857
16083
1547015446
2.57686
7320
75037479
1.253456
3850
36533629
0.631804
1718
17611737
0.31831
817
824800
0.16338
376
357333
023
25
24--

Precision

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

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

  Intra-assay Precision Inter-assay Precision
Sample123123
n202020202020
Mean (ng/mL)0.551.564.950.591.655.40
Standard deviation0.050.140.410.080.130.57
C V (%)9.098.978.2813.567.8810.56

Recovery

The recovery of Human alpha-La 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)93-10799
EDTA plasma (n=5)90-10496
Cell culture media (n=5)102-117108

Linearity

Samples were spiked with high concentrations of Human alpha-La 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)
1:2Range (%)90-102101-114103-118
Average (%)95108110
1:4Range (%)100-11591-10694-107
Average (%)10797101
1:8Range (%)102-11388-98103-121
Average (%)10793110
1:16Range (%)87-97101-113100-116
Average (%)92107108

Kit Components & Storage

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 X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent A1 vial, 5 mL4'C (shading light)
Substrate Reagent B1 vial, 5 mL4'C (shading light)
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human alpha-La (Alpha-Lactalbumin) CLIA Kit (HUES00580) Assay procedure

    1. 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.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids.) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. 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.
    7. 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.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. 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.
    11. Determine the RLU value of each well immediately.

Human alpha-La (Alpha-Lactalbumin) CLIA Kit (HUES00580) Protein Information

UniProt Protein Function:LALBA: Regulatory subunit of lactose synthase, changes the substrate specificity of galactosyltransferase in the mammary gland making glucose a good acceptor substrate for this enzyme. This enables LS to synthesize lactose, the major carbohydrate component of milk. In other tissues, galactosyltransferase transfers galactose onto the N-acetylglucosamine of the oligosaccharide chains in glycoproteins. Belongs to the glycosyl hydrolase 22 family.
UniProt Protein Details:

Protein type:Secreted; Secreted, signal peptide; Transferase; Apoptosis

Chromosomal Location of Human Ortholog: 12q13

Cellular Component: extracellular space; Golgi lumen; Golgi membrane

Biological Process: apoptosis; cell-cell signaling; defense response to bacterium; lactose biosynthetic process; signal transduction

NCBI Summary:This gene encodes alpha-lactalbumin, a principal protein of milk. Alpha-lactalbumin forms the regulatory subunit of the lactose synthase (LS) heterodimer and beta 1,4-galactosyltransferase (beta4Gal-T1) forms the catalytic component. Together, these proteins enable LS to produce lactose by transfering galactose moieties to glucose. As a monomer, alpha-lactalbumin strongly binds calcium and zinc ions and may possess bactericidal or antitumor activity. A folding variant of alpha-lactalbumin, called HAMLET, likely induces apoptosis in tumor and immature cells. [provided by RefSeq, Jul 2008]
UniProt Code:P00709
NCBI GenInfo Identifier:126001
NCBI Gene ID:3906
NCBI Accession:P00709.1
UniProt Secondary Accession:P00709,Q6FGX0, Q9UDK4,
UniProt Related Accession:P00709
Molecular Weight:16,225 Da
NCBI Full Name:Alpha-lactalbumin
NCBI Synonym Full Names:lactalbumin alpha
NCBI Official Symbol:LALBA  
NCBI Official Synonym Symbols:LYZG  
NCBI Protein Information:alpha-lactalbumin
UniProt Protein Name:Alpha-lactalbumin
UniProt Synonym Protein Names:Lactose synthase B protein; Lysozyme-like protein 7
Protein Family:Alpha-lactalbumin
UniProt Gene Name:LALBA  
UniProt Entry Name:LALBA_HUMAN
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Additional Information

Reactivity:
Human
ELISA Type:
CLIA
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