RIA and Elisa

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RIA and Elisa

Pipette filler on tableYou should always consult with the Analytical and Assay Core prior to starting a series of experiments.

  • To ensure that you have the latest version of these instructions (revisions may be in progress - but not yet posted - which could affect your experiments).
  • To ensure that the Analytical and Assay Core has a sufficient stock of critical reagents, such as antibody, to be able to process ALL of your samples from that set of experiments. This is necessary to allow your samples to be compared to each other without the intrusion of errors due to different cross-reactivities, etc.
  • To ensure that other critical reagents and supplies are on hand so that the Analytical and Assay Core can supply you with any special buffers or additives you might need when you need them.
  • To ensure that the Analytical and Assay Core has all supplies on hand necessary to process your samples when you submit them.
  • To ensure that the Analytical and Assay Core schedules its workload in order to free enough time to be able to process your time-critical samples.
  • To ensure that the Analytical and Assay Core understands what you are trying to do, so that they may offer advice that may help you accomplish your goal. Considerations include:
    • The organization of samples within and between assays to minimize intrinsic error;
    • The sample volume recommended to maximize sensitivity to the analyte concentrations you expect to find;
    • The use of other assays necessary to correct the requested assay for cross-reactivity; The use of other assays to complement the findings from the assay you requested.

Other reasons for consultation

  • EDTA anticoagulants. There are 3 EDTAs normally employed as anticoagulants: Na2 EDTA, K2 EDTA, and K3 EDTA. Na2 and K2 EDTA are the anticoagulants of choice for samples requiring EDTA as the anticoagulant. (Any EDTA can adversely affect some assays, however. The choice of which, if any, anticoagulant (EDTA, heparin, etc.) always depends upon the analyte, the antibody, and the assay system. If EDTA is an acceptable anticoagulant, however, Na2 and K2 EDTA are almost certain to be acceptable EDTAs. K3 EDTA can adversely affect some antibodies or assays that Na2 and K2 EDTA would not affect. K3 EDTA must be tested with each such antibody and assay system to determine if it is an acceptable anticoagulant. Furthermore, since K3 EDTA is a liquid, it could have a dilutional effect on small sample volumes. (E.g., a 0.5 mL blood sample having a hematocrit of 40 is collected in a 5 mL Vacutainer containing the usual 20 mLs of K3 EDTA. The plasma volume = (0.5 mL * ((100-40)/100)) = 0.3 mLs. The dilution = 0.3 mLs/(0.3 mLs + 0.02 mLs K3 EDTA) = 0.3/0.32 = 0.9375. I.e., use of K3 EDTA would cause the sample's analyte concentration to be diluted by » 6%.)
  • Sample type. Samples are routinely performed on plasma, plasma extracts, serum and cell culture media as indicated herein. Should you need assays on other biological fluids, such as urine samples, not specified herein, or on extracts, consult the lab prior to sample collection.
  • Sample storage. Generally, you may store, for subsequent assay, plasma, serum, urine or culture medium samples in plastic or siliconized (silanized) glass tubes. Unsiliconized glass tubes should be avoided since they may adsorb some analytes and lower their concentrations in the sample. Do NOT store blood samples without first removing the RBCs etc. If platelets bind or metabolize the analyte(s) of interest, centrifugation at ³ 2500g for 30 minutes is necessary to remove platelets prior to storage.
  • Sample containers. Submit samples in 12x75mm tubes if possible. If not possible, or if another tube size is more convenient, consult the Analytical and Assay Core before sample collection to be sure that the lab's automated equipment can accommodate that tube size for that assay.

Arranging your samples for assay

  • Determine which samples should be assayed together in the same assay in order to minimize the effects of random error on your results. (Between-assay inaccuracies and CVs are always as large or, usually, larger than within-assay inaccuracies and CVs. Therefore, samples that you wish to compare closely with each other should be assayed in the same assay. Example: control, experimental and recovery samples from the same animal should be assayed in the same assay. An alternate, but incorrect, method would be to assay all controls from all animals in one assay, and all experimental samples from all animals in another assay. Sounds good, but this latter method would allow a between-assay error to sum with control vs. experimental differences and perhaps make a non-significant difference seem significant, or vice-versa.
  • Fill out your sample worksheet
  • Arrange your samples in a sample rack in the same order in which they are listed on the sample worksheet. Place samples in rack in the following order: left to right, then front to back so that the first sample is located in the front, left hand corner.
  • Double-check that the sample ID on the tube matches that on the sample worksheet.

New assays

If you require an assay of some analyte not listed above, consult the lab staff.

Materials and methods

Consult the Analytical and Assay Core for technical details and published references when writing "Material and Methods" sections. Since these assays are frequently modified, you should seek the methods used to obtain the results for each paper.

Funding acknowledgment

NOTE: The below information is for departmental members only.

From the Bimolecular Resource Facilities webpage: Personnel in core facilities provide essential services for their users and it is important to recognize their contributions to the scientific advancement of the projects. The type of recognition that is most appropriate may be different for individual projects, depending on the contribution that core facility personnel provides.

Thus, it is important that the Analytical and Assay Core Laboratory is acknowledged in publications in order to document the value of our core facility.

The appropriate acknowledgment text can include any of the following:

  • The authors gratefully acknowledge the Analytical and Assay Core Laboratory.
  • You can also list the core services provided such as:
    • The authors gratefully acknowledge the Analytical and Assay Core Laboratory, which provided analysis of ________________.
  • Remember to include NIH HL51971 under sources of funding.