| SKU: | EK0995 |
|---|---|
| Pack Size: | 96wells/kit, with removable strips. |
| Validated Species: | Human |
| Application: | ELISA |
| Sensitivity: | <10pg/ml |
| Assay Range: | 312pg/ml-20,000pg/ml |
| Sample Type: | cell culture supernates, cell lysates, serum and plasma (heparin, EDTA). |
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Overview
| Product Name | Human ALCAM/Cd166 PicoKine™ ELISA Kit |
|---|---|
| SKU/Catalog Number | EK0995 |
| Description | Sandwich High Sensitivity ELISA kit for Quantitative Detection of Human ALCAM. 96wells/kit, with removable strips. |
| Cite This Product | Human ALCAM/Cd166 PicoKine™ ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK0995) |
| Validated Species | Human |
| Application | ELISA *Our Boster Guarantee covers the use of this product in the above tested applications. **For positive and negative control design, consult "Tissue specificity" under Protein Target Info. |
| Immunogen | Expression system for standard: NSO; Immunogen sequence: W28-A526 |
| Cross Reactivity | There is no detectable cross-reactivity with other relevant proteins. |
| Pack Size | 96wells/kit, with removable strips. |
Properties
| Sensitivity | <10pg/ml *Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive. |
|---|---|
| Assay Range | 312pg/ml-20,000pg/ml *This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range. |
| Sample Type | cell culture supernates, cell lysates, serum and plasma (heparin, EDTA). *The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine™ ELISA kit should detect it. **For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide. |
| Capture Antibody | monoclonal antibody from mouse |
| Detection Antibody | polyclonal antibody from goat |
| Normal Levels | Some research articles suggesting the natural levels of ALCAM are listed below: |
| Storage | Store at 4°C for 6 months, at -20°C for 12 months. Avoid multiple freeze-thaw cycles(Shipped with wet ice.) |
Kit Components
| Catalog number | Description | Quantity |
| EK0995-CAP | 96-well plate precoated with anti-Human ALCAM antibody | 1 |
| EK0995-ST | lyophilized recombinant Human ALCAM standard | 20ng/tubex2 |
| EK0995-DA | biotinylated anti-Human ALCAM antibody | 130ul(dilution 1:100) |
| AR1103 | Avidin-Biotin-Peroxidase Complex(ABC) | 130ul(dilution 1:100) |
| AR1106-1 | sample diluent buffer | 30ml |
| AR1106-2 | antibody diluent buffer | 12ml |
| AR1106-3 | ABC diluent buffer | 12ml |
| AR1104 | TMB color developing agent | 10ml |
| AR1105 | TMB stop solution | 10ml |
| PLA-SEA | Adhesive cover | 4 |
Washing buffer Preparation: Disolve AR0030-E to 1000ml distilled water and adjust pH to 7.2~7.6. Finally, adjust the total volume to 1L.
*Additional components can be purchased. If you need extra of the above components please order them together to avoid additional shipping charges.
See Prices For Extra ComponentsMaterial Required But Not Provided
- Microplate reader in standard size.
- Automated plate washer.
- Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
- Clean tubes and Eppendorf tubes.
- 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 H2
- Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na O and adjust pH to 7.2-7.
Protein Target Info (Source: Uniprot.org)
You can check the tissue specificity below for information on selecting positive and negative control.
| Gene Name | ALCAM |
|---|---|
| Protein Name | CD166 antigen |
| Molecular Weight | 65102 MW |
| Protein Function | Cell adhesion molecule that binds to CD6. Involved in neurite extension by neurons via heterophilic and homophilic interactions. May play a role in the binding of T- and B-cells to activated leukocytes, as well as in interactions between cells of the nervous system. |
| Tissue Specificity | Spleen, placenta, liver, and weakly in liver. Expressed by activated T-cells, B-cells, monocytes and thymic epithelial cells. Expressed by neurons in the brain. Restricted expression in tumor cell lines. Preferentially expressed in highly metastasizing melanoma cell lines. . |
| Sequence Similarities | Contains 3 Ig-like C2-type (immunoglobulin-like) domains. |
| Subcellular Localization | Membrane; Single-pass type I membrane protein. |
| Uniprot ID | Q13740 |
| Alternative Names | CD166 antigen;Activated leukocyte cell adhesion molecule;CD166;ALCAM;MEMD; |
| Research Areas | |immunology|adaptive immunity|b cells| immunology|t cells| kits/ lysates/ other|elisa kits|cd markers elisa kits| |
Background for CD166 antigen
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.
Human ALCAM/Cd166 PicoKine™ ELISA Kit Images
Click the images to enlarge.
Human ALCAM PicoKine ELISA Kit standard curve
Intra/Inter Assay Precision
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 16 | 16 | 16 | 24 | 24 | 24 |
| Mean(pg/ml) | 648 | 1967 | 8283 | 688 | 2156 | 8924 |
| Standard deviation | 37.58 | 145.55 | 563.24 | 48.16 | 198.35 | 651.45 |
| CV(%) | 5.8% | 7.4% | 6.8% | 7% | 9.2% | 7.3% |
Reproducibility
To assay reproducibility, three samples with differing target protein concentrations were assayed using four different lots.
| Lots | Lot1 (pg/ml) | Lot2 (pg/ml) | Lot3 (pg/ml) | Lot4 (pg/ml) | Mean (pg/ml) | Standard Deviation | CV (%) |
|---|---|---|---|---|---|---|---|
| Sample 1 | 648 | 630 | 693 | 657 | 657 | 22.94 | 3.4% |
| Sample 2 | 1967 | 2006 | 1987 | 2137 | 2024 | 66.54 | 3.2% |
| Sample 3 | 8283 | 8482 | 8134 | 7570 | 8117 | 339.21 | 4.1% |
Typical Data Obtained from Human ALCAM/Cd166 PicoKine™ ELISA Kit
(TMB reaction incubate at 37°C for 25-30min)
| Concentration(pg/ml) | 0 | 312 | 625 | 1250 | 2500 | 5000 | 10000 | 20000 | ||
| O.D. | 0.033 | 0.109 | 0.137 | 0.215 | 0.385 | 0.688 | 1.138 | 2.063 | ||
*The typical data is obtained from Boster's internal QC result and for reference only. It may differ from the lab test results of the end users. It is more important that the user's lab test results reflect the same linearity demonstrated in the typical data than achieving exactly the same O.D. values.
Customer Q&As
A: None of the components in our ELISA kits are produced in humans or primates.
Q: What is the volume of the recombinant protein control? What is its shelf life?
Keywords: expiration, storage, temperature, size
Keywords: expiration, storage, temperature, size
A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead?
Keywords: protocol, alternative buffer, applications, reagent recipe
A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
Q: Why are my O.D. values different than your values on the datasheet?
Keyword: troubleshooting, reference
A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP?
Keyword: kit components, reagents, protocol
A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine?
Keyword: applications
A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay?
Keyword: protocol, reagents
A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits?
Keyword: technical support, help, customer service
A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
Q: The results of my standards do not look correct, what could be the problem?
Keyword: troubleshoot, protocol
A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
Q: What is the well depth of the 96 well plates for the ELISA kits?
Keyword: well capacity, product size
A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
Q: The kit does not include wash solution, what should I do?
Keyword: wash buffer
A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals?
Keyword: antibody clonality
A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
Q: Do your ELISA kits come with sealants or plate covers?
Keyword: storage, sequential use
A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
Q: What is the concentration of Sodium Azide in your Elisa kits?
Keyword: preservative
A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
Q: Are there positive and negative controls available for my ELISA kit?
Keyword: positive control, negative control
A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
Q: Why do I get positive results for my knockout (KO) model when used as a control?
A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
Q: How long do I soak my plate in the wash buffer?
A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
Q: Can I use Tween in my wash buffer?
A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
Q: What plate type do I use to set up the microplate reader?
A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
Q: What should I use for negative control?
A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
Q: What is the normal level of this protein in my sample of interest?
A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
A: Yes the plate is separable. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below: https://www.bosterbio.com/media/pdf/Boster_Sample_Preparation_Protocols.pdf
Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our techincal support.
Q: Can this ELISA kit react with human, mouse, rat or other species?
A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.
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