Product Name:	Human ITGAL / CD11a ELISA Kit
Product Code:	HUFI01521
Size:	96 Assays
Alias:	ITGAL, Integrin alpha-L, Leukocyte function-associated molecule 1 alpha chain, CD11 antigen-like family member A, Leukocyte adhesion glycoprotein LFA-1 alpha chain, LFA-1A, CD11A, LFA-1, LFA1A
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ITGAL concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-103	95
EDTA plasma(n=5)	90-101	96
UFH plasma(n=5)	85-103	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ITGAL and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-97%	87-105%	85-101%
EDTA plasma(n=5)	89-101%	83-101%	89-99%
UFH plasma(n=5)	80-94%	82-100%	93-99%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P20701

UniProt Protein Function:	ITGAL: Integrin alpha-L/beta-2 is a receptor for ICAM1, ICAM2, ICAM3 and ICAM4. It is involved in a variety of immune phenomena including leukocyte-endothelial cell interaction, cytotoxic T-cell mediated killing, and antibody dependent killing by granulocytes and monocytes. Belongs to the integrin alpha chain family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Membrane protein, integral; Cell adhesion; Cell surface Chromosomal Location of Human Ortholog: 16p11.2 Cellular Component: cell surface; membrane; plasma membrane; immunological synapse; intercellular junction; integrin complex; external side of plasma membrane Molecular Function:protein binding; protein heterodimerization activity; metal ion binding; protein complex binding; ICAM-3 receptor activity; cell adhesion molecule binding Biological Process: integrin-mediated signaling pathway; extracellular matrix organization and biogenesis; regulation of immune response; cell-matrix adhesion; activated T cell proliferation; positive regulation of calcium-mediated signaling; signal transduction; receptor clustering; heterophilic cell adhesion; leukocyte adhesion; T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell; positive regulation of T cell proliferation; inflammatory response; blood coagulation; cell adhesion; cell motility; leukocyte migration
NCBI Summary:	ITGAL encodes the integrin alpha L chain. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. This I-domain containing alpha integrin combines with the beta 2 chain (ITGB2) to form the integrin lymphocyte function-associated antigen-1 (LFA-1), which is expressed on all leukocytes. LFA-1 plays a central role in leukocyte intercellular adhesion through interactions with its ligands, ICAMs 1-3 (intercellular adhesion molecules 1 through 3), and also functions in lymphocyte costimulatory signaling. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P20701
NCBI GenInfo Identifier:	88911345
NCBI Gene ID:	3683
NCBI Accession:	P20701.3
UniProt Secondary Accession:	P20701,O43746, Q45H73, Q96HB1, Q9UBC8,
UniProt Related Accession:	P20701
Molecular Weight:
NCBI Full Name:	Integrin alpha-L
NCBI Synonym Full Names:	integrin, alpha L (antigen CD11A (p180), lymphocyte function-associated antigen 1; alpha polypeptide)
NCBI Official Symbol:	ITGAL
NCBI Official Synonym Symbols:	CD11A; LFA-1; LFA1A
NCBI Protein Information:	integrin alpha-L; LFA-1A; LFA-1 alpha; integrin gene promoter; CD11 antigen-like family member A; lymphocyte function-associated antigen 1; leukocyte adhesion glycoprotein LFA-1 alpha chain; leukocyte function-associated molecule 1 alpha chain; antigen CD11A (p180), lymphocyte function-associated antigen 1, alpha polypeptide
UniProt Protein Name:	Integrin alpha-L
UniProt Synonym Protein Names:	CD11 antigen-like family member A; Leukocyte adhesion glycoprotein LFA-1 alpha chain; LFA-1A; Leukocyte function-associated molecule 1 alpha chain; CD_antigen: CD11a
Protein Family:	Integrin
UniProt Gene Name:	ITGAL
UniProt Entry Name:	ITAL_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.