ELISA Sample Preparation FAQs
Krishgen’s ELISA are available under two product types:
GENLISATM branded products are ELISA for cytokine and biomarker testing in various species including humans, rat, mouse and other species. These ELISA have been validated for use with serum, plasma and cell culture supernatant samples.
KRIBIOLISATM branded products are specialised for biopharma related targets. These ELISA have been validated for human serum and plasma samples. For use with cell culture samples, or samples from other species, speak to our team for a custom assay validation.
Typically, it is recommended to follow the below protocols for sample prep:
Cell Culture Supernatant:
If necessary, centrifuge to remove debris prior to analysis. Samples can be stored at temperature < -20°C. Avoid repeated freeze/thaw cycles.
Serum:
Use a serum separator tube and allow clotting for 30 minutes, then centrifuge for 10 minutes at 1000 x g. Remove serum layer and assay immediately or store serum samples at temperature < -20°C. Avoid repeated freeze/thaw cycles.
Plasma:
Collect blood sample in a citrate, heparin or EDTA containing tube. Centrifuge for 10 minutes at 1000 x g within 30 minutes of collection. Assay immediately or store plasma samples at temperature < -20°C. Avoid repeated freeze/thaw cycles.
To ensure correct preparation of samples, the Krishgen team has put together a list of frequently asked questions for use in Krishgen ELISA:
Q: How should I handle and store my ELISA samples before testing?
A: Samples should be stored according to the specific requirements of the analyte being measured. Generally, samples should be handled carefully, avoiding freeze-thaw cycles, and stored at the recommended temperature to maintain sample integrity.
Q: Can I use hemolyzed samples for ELISA testing?
A: Hemolysis can release interfering substances that may affect the assay results. It is recommended to use non-hemolyzed samples for ELISA testing to ensure accurate and reliable results.
Q: Should I filter my samples before using them for ELISA?
A: Filtration can be beneficial when dealing with particulate matter in the sample, but it depends on the specific requirements of the assay and the sample matrix. It is best to check the assay protocol or consult the datasheet for sample preparation recommendations.
Q: Can I dilute my samples for ELISA analysis?
A: Dilution of samples is often necessary to achieve the desired concentration range within the assay’s dynamic range. However, the dilution factor should be carefully chosen to ensure that the target analyte falls within the detectable range of the assay.
Q: What type of buffers should I use for ELISA sample preparation?
A: The choice of buffer depends on the assay requirements and the sample matrix. Typically, using buffers with appropriate pH, ionic strength, and composition that mimic the sample matrix can help maintain the integrity and stability of the target analyte and minimize assay interference.
Q: Is it necessary to pre-treat my samples before ELISA analysis?
A: Pre-treatment may be necessary in certain cases to remove interfering substances or modify sample properties. Techniques such as filtration, centrifugation, or solid-phase extraction can help eliminate unwanted matrix components and improve assay specificity.
Q: Can I use frozen samples for ELISA testing?
A: Freezing samples can be a suitable option for long-term storage, but repeated freeze-thaw cycles should be avoided as they may affect the stability and integrity of the analyte. It is best to follow Krishgen’s recommended guidelines mentioned in the datasheet for sample handling and storage conditions.
Q: How do I handle lipemic samples in ELISA analysis?
A: Lipemic samples with high lipid content can interfere with the assay results. In such cases, it is recommended to clarify the sample by centrifugation or other separation techniques to remove lipids before performing the ELISA. Lipemic samples require internal validation, as Krishgen does not validate for such samples.
Q: What is the minimum sample volume required for ELISA testing?
A: The minimum sample volume can vary depending on the specific assay. Typically for Krishgen ELISA, it will be 100 ul. In some assays, it is as low as 50 ul.
Q: Are there any specific guidelines for sample preparation when performing ELISA in different sample matrices (e.g., serum, plasma, tissue homogenate)?
A: Yes, different sample matrices may have specific sample preparation guidelines. It is recommended to consult the assay protocol for sample preparation instructions tailored to the specific matrix being used.
Q: Can I use proteinase inhibitors in my ELISA sample preparation?
A: Yes, in some cases, the addition of proteinase inhibitors can help prevent proteolysis and preserve the integrity of the target analyte. However, the addition of inhibitors should be validated carefully, as it may interfere with the assay.
Q: Should I remove red blood cells from my ELISA samples?
A: Yes, it is generally recommended to remove red blood cells from samples to minimize hemolysis and reduce sample background. Centrifugation followed by careful pipetting can be an effective approach for separating the plasma or serum from red blood cells.
Q: Can I use EDTA, citrate, or heparin as anticoagulants for ELISA testing?
A: This depends on the type of ELISA. These anticoagulants can be used for ELISA sample preparation. However, the use of anticoagulants can sometimes affect the assay results, and it is important to consult the manufacturer’s guidelines for specific anticoagulant recommendations.
Q: How do I handle samples with high heterophilic antibody content in ELISA analysis?
A: High levels of heterophilic antibodies in the sample can cause false-positive results in ELISA analysis. Pre-treatment using blocking agents or heterophilic antibody-blocking tubes can help reduce assay interference caused by heterophilic antibodies.
Q: Can I use samples that have been previously treated with chemical agents for ELISA testing?
A: The use of chemical agents may interfere with the assay, and it is best to avoid using samples that have been previously treated with chemical agents. If it is necessary to use treated samples, it is recommended to consult
Please note that these answers serve as general guidelines, and it’s important to refer to the specific assay protocols mentioned in the latest product specific IFU for accurate and detailed sample preparation guidelines.
