Human ALCAM ELISA Kit

CD166 antigen is a 100-105 kD typeI transmembrane glycoprotein that is a member of the immunoglobulin superfamily of proteins. In humans it is encoded by the ALCAM gene. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. It is also called CD166 (cluster of differentiation 166), MEMD, SC-1/DM-GRASP/BEN in the chicken, and KG-CAM in the rat. It is expressed on activated T cells, activated monocytes, epithelial cells, fibroblasts, neurons, melanoma cells, and also in sweat and sebaceous glands. CD166 protein expression is reported to be upregulated in a cell line deriving from a metastasizing melanoma. CD166 plays an important role in mediating adhesion interactions between thymic epithelial cells and CD6+ cells during intrathymic T cell development. Recently, CD166 has also been used as a potential cancer stem cell marker.

Sku #Product NameProduct SizePriceQTY
2904000015 Human ALCAM ELISA Kit 1 plate 1 plate $429.00 USD
 

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For quantitative detection of human ALCAM in cell culture supernates, serum and plasma(heparin , EDTA).

Typical Data Obtained from Human ALCAM

(TMB reaction incubate at 37°C for 25 min)
Concentration(pg/ml)
0.0
312
625
1250
2500
5000
10,000
20,000
O.D
0.033
0.109
0.137
0.215
0.385
0.688
1.138
2.063

Typical Human ALCAM ELISA Kit Standard Curve

This standard curve was generated at Eton for demonstration purpose only. A standard curve must be run with each assay.

Range 312pg/ml-20,000pg/ml

Sensitivity < 10pg/ml
Specificity Natural and recombinant human ALCAM
Cross-reactivity No detectable cross-reactivity with other relevant proteins

Storage

Store at 4°C for 6 months, at -20°C for12 months. Avoid multiple freeze-thaw cycles (Shipped with wet ice.)
Precision

Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plateto assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separateassays to assess inter-assay precision.
Intra-Assay Precision
Inter-Assay Precision
Sample
1
2
3
1
2
3
n
16
16
16
24
24
24
Mean(ng/ml)
3.07
6.42
13.26
3.58
7.04
14.98
Standard deviation
0.150
0.327
0.756
0.218
0.444
0.959
CV(%)
4.9
5.1
5.7
6.1
6.3
6.4

Principle

Eton’s human ALCAM ELISA Kit was based on standard sandwich enzyme-linked immune-sorbent assay technology. A monoclonal antibody from mouse specific for ALCAM has been precoated onto 96-well plates. Standards(NSO, W28-A526) and test samples are added to the wells; a biotinylated detection polyclonal antibody from goat specific for ALCAM is added subsequently and then followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex was added and unbound conjugates were washed away with PBS or TBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the human ALCAM amount of sample captured in plate.

Kit Components

Description
Quantity
96-well plate precoated with anti- human ALCAM antibody
1
Lyophilized recombinant human ALCAM standard
20ng/tube×2
Biotinylated anti- human ALCAM antibody
130μl(dilution 1:100)
Avidin-Biotin-Peroxidase Complex (ABC)
130μl(dilution 1:100)
Sample diluent buffer
30 ml
Antibody diluent buffer
12ml
ABC diluent buffer
12ml
TMB color developing agent
10ml
TMB stop solution
10ml

Material Required But Not Provided

1. Microplate reader in standard size.
2. Automated plate washer.
3. Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
4. Clean tubes and Eppendorf tubes.
5. Washing buffer (neutral PBS or TBS).
Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g Nacl; 450μl of purified acetic acid or 700μl of concentrated hydrochloric acid to 1000ml H2O and adjust pH to 7.2-7.6. Finally, adjust the total volume to 1L.
Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na2HPO4 and 0.2g NaH2PO4 to 1000ml distilled water and adjust pH to 7.2-7.6. Finally, adjust the total volume to 1L.

Notice for Application of Kit

1. To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experiment using standards and a small number of samples is recommended.
2. The TMB Color Developing agent is colorless and transparent before using, contact us freely if it is not the case.
3. Before using the Kit, spin tubes and bring down all components to the bottom of tubes.
4. Duplicate well assay is recommended for both standard and sample testing.
5. Don’t let 96-well plate dry, for dry plate will inactivate active components on plate.
6. Don’t reuse tips and tubes to avoid cross contamination.
7. Avoid using the reagents from different batches together.

8. In order to avoid marginal effect of plate incubation due to temperature difference (reaction may be stronger in the marginal wells), it is suggested that the diluted ABC and TMB solution will be pre-warmed in 37°C for 30 min before using.



Preparation
1. Sample Preparation and Storage
Store samples to be assayed within 24 hours at 2-8°C. For long-term storage, aliquot and freeze samples at -20°C. Avoid repeated freeze-thaw cycles.
Cell culture supernates: Remove particulates by centrifugation, assay immediately or aliquot and storesamples at
-20°C.
Serum: Allow the serum to clot in a serum separator tube (about 4 hours) at room temperature.Centrifuge at approximately 1000 X g for 15 min. Analyze the serum immediately or aliquot and store frozen at -20°C.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 15 min at 1000 x gwithin 30 min of collection. Analyze immediately or aliquot and store frozen at -20°C.
2. Sample Dilution Guideline
The user needs to estimate the concentration of the target protein in the sample and select a proper dilution factor so that the diluted target protein concentration falls near the middle of the linear regime in the standard curve. Dilute the sample using the provided diluent buffer. The following is a guideline for sample dilution. Several trials may be necessary in practice. The sample must be well mixed with the diluents buffer.
High target protein concentration (200-2000ng/ml). The working dilution is 1:100. i.e. Add 3μlsampleinto 297 μl sample diluent buffer.
Medium target protein concentration (20-200ng/ml). The working dilution is 1:10. i.e. Add 25μlsample into 225 μl sample diluent buffer.
Low target protein concentration (312-20,000pg/ml). The working dilution is 1:2. i.e. Add 100μlsample to 100 μl sample diluent buffer.
Very Low target protein concentration (≤312pg/ml). No dilution necessary, or the working dilution is1:2.
3. Reagent Preparation and Storage
A. Reconstitution of the human ALCAM standardALCAM standard solution should be prepared no more than 2 hours prior to the experiment. Two tubes of ALCAM standard (20ng per tube) are included in each kit. Use one tube for each experiment.
a. 20,000pg/ml of human ALCAM standard solution: Add 1 ml sample diluent buffer into one tube, keep the tube at room temperature for 10 min and mix thoroughly.
b. 10,000pg/ml312pg/ml of human ALCAM standard solutions: Label 6 Eppendorf tubes with
c. 10,000pg/ml, 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312pg/ml, respectively. Aliquot 0.3ml of the sample diluent buffer into each tube. Add 0.3 ml of the above 20,000pg/ml ALCAM standard solution into 1st tube and mix. Transfer 0.3 ml from 1st tube to 2nd tube and mix. Transfer 0.3 ml from 2nd tube to 3rd tube and mix, and so on.
Note: The standard solutions are best used within 2 hours. The 20ng/ml standard solution should be storedat 4°C for up to 12 hours, or at -20°C for up to 48 hours. Avoid repeated freeze-thaw cycles.
B. Preparation of biotinylated anti-human ALCAM antibody working solution: The solution should be prepared no more than 2 hours prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume)
b. Biotinylated anti-human ALCAM antibody should be diluted in 1:100 with the antibody diluent buffer and mixed thoroughly. (i.e. Add 1μl Biotinylated anti-human ALCAM antibody to 99μl antibody diluent buffer.)
C. Preparation of Avidin-Biotin-Peroxidase Complex (ABC) working solution: The solution should be prepared no more than 1 hour prior to the experiment.
a. The total volume should be: 0.1ml/well x (the number of wells). (Allowing 0.1-0.2 ml more than total volume)
b. Avidin- Biotin-Peroxidase Complex (ABC) should be diluted in 1:100 with the ABC dilution buffer and mixed thoroughly. (i.e. Add 1μl ABC to 99μl ABC diluent buffer.)
Assay Procedure
The ABC working solution and TMB color developing agent must be kept warm at 37°C for 30 min before use. When diluting samples and reagents, they must be mixed completely and evenly. Standard ALCAM detection curve should be prepared for each experiment. The user will decide sample dilution fold by crude estimation of ALCAM amount in samples.
1. Aliquot 0.1ml per well of the 20,000pg/ml, 10,000pg/ml, 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312pg/ml human ALCAM standard solutions into the precoated 96-well plate. Add 0.1ml of the sample diluent buffer into the control well (Zero well). Add 0.1ml of each properly diluted sample of human cell culture supernates, serum or plasma (heparin, EDTA) to each empty well. See “Sample Dilution Guideline” above for details. We recommend that each human ALCAM standard solution and eachsample is measured in duplicate.
2. Seal the plate with the cover and incubate at 37°C for 90 min.
3. Remove the cover, discard plate content, and blot the plate onto paper towels or other absorbent material. Do NOT let the wells completely dry at any time.
4. Add 0.1ml of biotinylated anti-human ALCAM antibody working solution into each well and incubate the plate at 37°C for 60 min.
5. Wash plate 3 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (Plate Washing Method: Discard the solution in the plate without touching the side walls. Blot the plate onto paper towels or other absorbent material. Soak each well with at least 0.3 ml PBS or TBS buffer for 1~2 minutes. Repeat this process two additional times for a total of THREE washes. Note: For automated washing, aspirate all wells and wash THREE times with PBS or TBS buffer, overfilling wells with PBS or TBS buffer. Blot the plate onto paper towels or other absorbent material.)
6. Add 0.1ml of prepared ABC working solution into each well and incubate the plate at 37°C for 30 min.
7. Wash plate 5 times with 0.01M TBS or 0.01M PBS, and each time let washing buffer stay in the wells for 1-2 min. Discard the washing buffer and blot the plate onto paper towels or other absorbent material. (See Step 5 for plate washing method).
8. Add 90μl of prepared TMB color developing agent into each well and incubate plate at 37°C in dark for 25-30 min (Note: For reference only, the optimal incubation time should be determined by end user. And the shades of blue can be seen in the wells with the four most concentrated human ALCAM standard solutions; the other wells show no obvious color).
9. Add 0.1ml of prepared TMB stop solution into each well. The color changes into yellow immediately.
10. Read the O.D. absorbance at 450nm in a microplate reader within 30 min after adding the stop solution.
For calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of Zero well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The human ALCAM concentration of the samples can be interpolated from the standard curve.
Note: if the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation toobtain the concentration before dilution.
Summary
1. Add samples and standards and incubate the plate at 37°C for 90 min. Do not wash.
2. Add biotinylated antibodies and incubate the plate at 37°C for 60 min. Wash plate 3 times with 0.01M TBS.
3. Add ABC working solution and incubate the plate at 37°C for 30 min. Wash plate 5 times with 0.01M TBS.
4. Add TMB color developing agent and incubate the plate at 37°C in dark for 25-30 min.
5. Add TMB stop solution and read.

1. Bowen MA, Patel DD, Li X, Modrell B, Malacko AR, Wang WC, Marquardt H, Neubauer M, Pesando JM, Francke U, et al. (Jun 1995). "Cloning, mapping, and characterization of activated leukocyte-cell adhesion molecule (ALCAM), a CD6 ligand". J Exp Med 181 (6): 2213–20.

2. "Entrez Gene: ALCAM activated leukocyte cell adhesion molecule".


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