Immunoassay Quality

Linearity of Dilution

Linearity of dilution (natural linearity, spiked linearity) is another important fundamental of immunoassay accuracy and specificity. No matter what dilution samples are run at, you should always derive the same concentration once back-calculated, giving you consistency in results. If the sample matrix is not properly controlled for, sample values often increase as you perform higher dilutions. This is illustrated in the image above. As you dilute the sample, the interfering factors also dilute out, making the target analyte more accessible, and the concentration of the target analyte increases. When your sample has high endogenous levels, natural linearity can be performed by running a 5-fold serial dilution. If endogenous levels are lower, you can spike recombinant protein into your sample and dilute it out, which is commonly referred to as spiked linearity. In R&D Systems assays, all sample types are optimized for linear dilutions through the standard curve range.

Performing Linearity of Dilution

FIGURE 3. Human Luminex Performance Panel Linearity Competitor Comparison. Serial dilutions of serum and plasma samples in the Human XL Cytokine Luminex Performance Panel (Catalog #FCSTM18) and two competitor panels shown with average percent linearity for all dilutions. R&D Systems meets the acceptable range (70%-130%) for Luminex Performance assays. In contrast, the competitor panels did not, showing matrix effects.

FIGURE 4. Human TNF-alpha QuicKit Spiked Linearity Competitor Comparison. TNF-alpha is spiked at high concentration in various sample matrices, then diluted with the appropriate calibrator diluent and run on the Human TNF-alpha Quantikine QuicKit ELISA (Catalog #QK210). R&D Systems ELISA meets acceptable range (80%-120%). The competitors do not, showing matrix effects.

Specificity

One of the most important features of an immunoassay is that you are measuring the correct intended analyte, so you can have confidence in results and move your research forward. Immunoassays are, by nature, subject to several potential interferences from components in complex samples like serum or plasma. The nature of these interferences can be both positive and negative as shown in this graphic, but all result in inaccurate values. The burden is on the assay manufacturer to recognize these and correct them, but not all manufacturers are equal. Removing false positives and false negatives are key to obtaining the correct specificity.

FIGURE 5. Removal of False Positives. To demonstrate the false positive phenomenon, we acquired samples from patients with known values of rheumatoid factor (RF) or HAMA (human anti-mouse antibodies) both of which are known to be potential interferants in immunoassays. One portion of each sample was treated with a commercial quenching reagent (HBT) designed to block those interferences and the other portion was left untreated (NT). All samples were run in both R&D Systems and in a competitors Luminex assay according to the manufacturers protocol and the data collected. The chart on the above left is a composite graph showing all samples for IFNg and IL-1b. This shows that the competitor’s assay is prone to false positive interference, but the R&D Systems assay is not. The chart on the right is a dramatic example of the effect it can have on data where the blue bars represent false positive values in the competitor’s assay, but not in the R&D Systems assay.

FIGURE 6. The importance of blocking reagents when detecting human GDNF. Blocking reagents are commonly used in ELISA kits to reduce interference from proteins in samples which may produce false positive results. In this experiment, we ran our human GDNF Quantikine ELISA (Catalog #DGD00) with our Quantikine diluent which contains blockers and with the Quantikine diluent with blockers removed. The absence of our proprietary blocking reagents resulted in false positives in 9 of 11 samples using the Quantikine ELISA (8A). Conversely, the same samples were tested on a competitor Human GDNF ELISA with their provided diluent and with their diluent plus our proprietary blocking reagents. There were 9 of 11 false positives using the Competitor A ELISA as provided, there were no false positive results in Competitor A’s ELISA when our blocking reagents were added to the diluent (8B). Note that Competitor A lacked the sensitivity needed to detect GDNF in positive control supernates from U-118MG and U-87MG cell cultures.

Consistency

R&D Systems assays ensure lot-to-lot consistency, enabling long term research studies. Extensive quality control testing is done on each kit component and each complete kit to make sure standard curves, background, and sample values remain consistent.

In particular, master calibration is used to prevent variability over time due to differences in protein immunoreactivity. Each new standard lot is calibrated against a master calibrator which is manufactured during assay development. This, combined with our stringent manufacturing and quality control measures ensure reliable and reproducible results between kit lots and prevent drift in sample values.

FIGURE 7 Consistent Control Values in Quantikine® ELISAs. High (blue line), medium (green line), and low (dark blue line) controls are assayed with every manufactured lot of the Human IL-6 Quantikine® ELISA Kit (Catalog #D6050). Controls for the Human IL-6 Quantikine® ELISA Kit fall within acceptable ranges (gray bars) and remain consistent from lot to lot.

FIGURE 8 Consistent Control Values in Luminex Panel. High (blue line), medium (dark blue line), and low (green line) controls are assayed with every manufactured lot of the Human IL-6 Performance Luminex Assay (Catalog# LUXLM206). Controls for the Human IL-6 Luminex Assay remain consistent from lot to lot over the course of 6 years.