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ELISA Antibody FAQs
1. Q: What is an ELISA-validated TrueMAB antibody?
A: ELISA validated TrueMABs are monoclonal antibodies made against authentic full length human protein antigens which have been validated specifically in an ELISA application. The formulation of ELISA-validated TrueMab antbodies is also different from standard TrueMab antibodies. Capture antibodies are supplied in a PBS buffer suitable for plate coating or coupling to other solid phases. Detection antibodies are supplied biotinylated in a PBS buffer containing glycerol and BSA.
2. Q: Why does OriGene offer matched capture/detection antibody recommendations?
A: Matched capture and detection antibody recommendations are the result of our in-house validation studies. Since OriGene often offers multiple TrueMAB antibodies per protein target, our validation process selects the best combinations of specific capture and detection antibodies to help our customers better decide which antibodies to use in their ELISA applications. For some targets, multiple pairs are available as different pairs may be optimal for different applications.
3. Q: Can ELISA-validated TrueMABs be used in other applications?
A: Yes it is possible, but those applications have not been tested in-house. OriGene recommends that customers view our TrueMAB collection of antibodies to find other applications for which OriGene’s antibodies have been validated.
4. Q: What protein standards are used?
A: The specific protein standards used in OriGene’s validation process can be found in the ELISA Validated TrueMAB datasheets. Most protein standards are authentic full length human proteins. For some matched ELISA pairs, no protein standard is yet available. Please contact technical support for more information on source and supply of standards for these antibodies.
5. Q: Can OriGene’s ELISA-validated TrueMAB antibodies be used with another company’s protein standard?
A: Yes. However, recognition and binding of the antibody to the native full-length protein is the primary consideration for validation of OriGene TrueMab antibodies. Truncated recombinant proteins or proteins expressed in non-mammalian expression systems may not give similar results.
6. Q: Will trehalose or sodium azide affect ELISA performance?
A: Trehalose is used to help protect antibodies against damage caused by storage at -20ºC and precipitation from freeze-thaw cycles. Sodium azide is used as a preservative to help increase shelf life. Trehalose and sodium azide present in the capture antibodies will not affect antibody ELISA performance in any significant way.
7. Q: Will BSA/Glycerol in the detection antibody affect ELISA performance?
A: BSA is used to stabilize and prevent the adhesion of the biotinylated detection antibody to reaction vessels. Glycerol is used as a cryoprotectant to prevent antibodies from precipitation and damage from freezing and freeze thaw-cycles. For most applications, BSA and glycerol will not affect antibody ELISA performance.
8. Q: When making antibody aliquots what is the smallest recommended volume?
A: To prevent damage caused by repeated freeze-thaw cycles it is recommended that aliquots of the antibody be made into various volumes no less than 20?l. In volumes smaller than 20 ?l, stock concentration can be affected by a variety of factors.
9. Q: When do the antibodies expire?
A: Antibodies are generally stable for years when stored under ideal conditions. OriGene has not tested antibody storage for longer than stated on the Certificate of Analysis.
10. Q: How can the results similar to those found in the datasheets be generated?
A: It is possible to generate similar data to that found in the datasheet by following the procedures found in Origene’s standard ELISA protocol. However, variations in reagents, substrates, buffers, instruments, and specific lab conditions will cause variation in results between labs.
11. Q: Is a full ELISA kit available for selected ELISA-validated capture/detection antibody pairs?
A: Not at present. However, if you are interested in having OriGene create an ELISA kit for you using our ELISA-validated capture and detection antibodies, please email us at customELISA@origene.com with your request and we will contact you shortly.
12. Q: How should I dilute my samples?
A: Each investigator must determine the best dilution for their samples. Make dilutions so that your samples will fall in the linear portion of the standard curve. In the absence of any experience with a particular analyte or sample type, it is recommended to make several dilutions of a small set of samples (eg, 1:10, 1:50, 1:250, and 1:1000) to determine the optimal dilution range for your samples.
13. Q: What should I use to dilute my protein standards?
A: In order to accurately quantitated your samples, protein standards should be diluted in as similar manner to the samples as possible. For example, if the samples are tissue culture supernatants and the samples will be diluted 1:5 before testing, then the protein standards should be diluted in a buffer than contains 20% tissue culture medium. If the samples are serum or plasma and the samples will be diluted 1:10 before testing, then the protein standards should be diluted in a buffer than contains 10% negative serum (fetal bovine serum or newborn calf serum can usually substitute for negative serum.
14. Q: What if my raw OD values are too low or too high?
A: If the raw OD values are too low, then the concentration of the detection antibody and/or the streptavidin-HRP conjugate should be increased. If the raw OD values are too high, then the concentration of the detection antibody and/or the streptavidin-HRP conjugate should be decreased.
15. Q: What can I do to decrease my background?
A: Add additional blocking agents to the Assay Diluent used for dilution of the biotinylated detection antibody and the streptavidin HRP conjugate. Blocking agents commonly used to reduce non-specific background are goat, rabbit or mouse IgG, goat, rabbit or mouse serum, newborn calf serum, gelatin (bovine or fish), non-fat dry milk and casein or casein hydrolysate. High background can also be caused by insufficient washing or cross-contamination of samples and reagents. If possible, use a well-maintained plate washer for all wash steps. For manual washing, use a multichannel pipette to add wash buffer, and use a multichannel vacuum manifold to remove the wash buffer. Avoid using the “squirt and dump” method for washing. For additional background reduction, modify the formulation or pH of the Wash Buffer, or use wash buffer that has been heated to 37ºC of 42ºC. Alternatively, after several washed to remove the bulk of the material, add a smaller amount of wash buffer to each well and then incubate the plate for 15 minutes to one hour with shaking.
16. Q: The sample quantitation is incorrect or not consistent. How can I improve this?
A: Make sure your samples are diluted so that the signal obtained is in the linear region of the standard curve. Samples that give signals close to the low or high ends of the standard curve may be inaccurate and difficult to reproduce.
17. Q: What type of analysis should be used for quantitation of samples?
A: The 5 Parameter Logistic or 5PL nonlinear regression model is an asymmetric function that is ideal for analyzing ELISA data. The 5PL model differs from the 4-PL or 4 Parameter Logistic model in that it is an asymmetric function which is a usually a better fit for immunoassay data. In addition, weighting the data by “1/Y2” or “1/Y” will often give better fits for ELISA data.
18. Q: What software package should be used for analysis of the standard curve and quantitation of samples?
A: Any software package that can model a 5 Parameter Logistic curve can be used. Generally, Microsoft Excel is not well suited for this type of analysis although an Excel add-on is available that may work (www.xlstat.com). Some of the software choices available are SigmaPlot (Systat), Prism (GraphPad), ReaderFit (Hitachi) or others. Hitachi US (MiraiBio) offers a free on-line version of ReaderFit that is limited to 32 data points (www.ReaderFit.com).