RayBiotech has a vast library of array-validated antibody sandwich pairs, however not all of them have been developed in the 96-well plate ELISA format. Thus, ELISA kits that are listed as “custom” require a 5-7 week development phase, which RayBiotech begins after receiving an order for the kit. The development only needs to occur once, after which the final kit will have passed RayBiotech’s ELISA quality control tests. At this point, the kit will be added to our stock kit inventory.
On each 96 well plate, an 8-point standard curve is run with duplicate wells. The remaining wells may then be used for samples (duplicates are strongly recommended). If the entire plate is used in 1 experiment, the remaining 80 wells will accommodate 40 samples in duplicate. If not run all at once, a new standard curve must be included with every run, reducing the number of potential samples by 8 (to account for the new standard curve points).
RayBio Sandwich ELISA kits do not contain biological reference samples. The recombinant standard may serve as a positive control or may be used for spike recovery studies.
The kit may have diminished activity after an extended period at room temperature, but it is most likely still usable, especially if left out for only a small amount of time. The most temperature-sensitive components in the kit are the standards, HRP-streptavidin, and detection antibody.
At RayBiotech we take our product quality very seriously. Every kit we produce is subjected to a battery of rigorous product-specific quality control tests and manufactured in compliance with GMP and ISO 13485 standards. Our Quality Statement can be viewed here.
Our sandwich ELISA kits are evaluated for intra-assay reproducibility by running 2-3 positive control samples in duplicate on a single plate (maximum tolerance = 10% CV). Inter-assay reproducibility is evaluated by at least 2 independent experiments with 2-3 positive control samples and a full standard curve (maximum tolerance = 12% CV). Lot-to-lot consistency is tested by comparing calculated concentrations of 2-3 positive control samples with calibration curves of current lot and previous lot (maximum tolerance = 20% CV).
Recovery is determined by spiking various levels of target protein into biological samples. Dilution linearity is tested by performing 2-fold and 4-fold dilutions of biological samples. Recoveries typically range from 80-130% (maximum tolerance 70-150%). Tested biological sample types include serum, EDTA plasma, citrate plasma, heparin plasma (normal healthy donors) and cell culture medium (DMEM or 1640). Recommended dilution ranges for serum and plasma are determined from this testing series. For lysate-specific sandwich ELISAs (catalog numbers ending in “-CL”), sample types include cell lysates and tissue homogenates of various origins.
Our tips for sample preparation can be found here:Tips on Sample Preparation
The ELISA kits with catalog numbers ending in “-CL” are optimized and validated for use with cell and tissue lysate samples. All other ELISA and EIA kits (catalog numbers without “-CL”) have not been validated by RayBiotech with cell or tissue lysates, however RayBio kits can generally accommodate many different samples types. All RayBio Phosphorylation ELISA kits are validated with cell lysates.
When testing lysate samples, RayBiotech recommends diluting the samples at least 5-fold with the appropriate diluent buffer indicated in the protocol. This dilution minimizes interference caused by the detergents present in the lysis buffer. The recommended total protein concentration of your lysate is at least 1 mg/ml. The original lysate should then be diluted (5-fold or more is preferred) to a working concentration range of 50-500 µg/ml. Please note that these concentration ranges represent a starting point and may need to be further optimized by the researcher.
RayBiotech carries a lysis buffer (cat# EL-Lysis) which may be purchased separately. If the researcher prefers to prepare their own lysis buffer, they should review the guidelines stated at this link:
In brief, a lysis buffer used with RayBio ELISAs must meet the following specifications:
Buffers optimized for immunoprecipitation, such as RIPA buffer, generally meet these specifications.
If you’re unsure where to start, the technical support team can assist you in locating a publication referencing a RayBio kit with a sample type similar to yours.
If your standard curve OD signals are low (highest standard < 0.7), please consider the following tips and best practices:
When troubleshooting low OD, RayBiotech recommends preparing a fresh stock of standard (each kit comes with an extra vial of Item C) and retest using the tips above.
Low overall signals arise from a problem in the detection process – i.e., the Detection Antibody, HRP-Streptavidin or TMB One-Step Substrate). Please consider the following tips and best practices:
Common causes of high background include:
Matrix effects are a common cause of non-linear dilution responses. This can occur when proteins or other components within the sample affect the immunoreactivity of the target molecule. These matrix components can also affect the ability of the antibody to recognize its target within the sample. Auto-antibodies, binding proteins, hemolysis, or certain disease states (e.g. highly lipemic samples) can contribute to this phenomenon. If matrix effects are suspected, centrifuge the sample and dilute further. When testing serum or plasma, a minimum 2-fold dilution is always recommended to avoid matrix effects.
Common causes of high well-to-well variability include: