For some ELISA based antibody tests (e.g. h-tTg antibody test), labs report units as RU/mL or U/mL. Also different labs have different cut off (normal range) values.

I understand that different kit manufacturers recommend different ranges because the units are not standardized (e.g. IU/mL) for enzyme activity concerned. But I didn't understand the difference between RU, U and AU

Question Is there a difference between RU/mL, AU/mL and U/mL? Are the figures comparable across reports from different labs (which may be using different kits)?

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    $\begingroup$ No actually. U is definitely not IU. I am referring to ELISA tests where there is no international standards yet. For example, IgA tTg antibody test. I know AU is Arbitrary Units. RU is either Relative or Reference Unit. U is simply unit. Doubts is what's the difference between these. $\endgroup$ – Dayne Jul 16 '19 at 4:11
  • $\begingroup$ Thanks for informative comment. I am not from biology background so your comment helped. Now what I meant by standardization is that what conjugate to use and incubation times are not standardized for some tests. Because of this, extinction coefficients are not known. So the lab depends on calibrators given in the kit. Calibrators have specified units and using that curve is fitted. Based on fitted curve sample's concentration is derived. Now, from what i read, manufacturers give their own units. But some give RU some U some AU. For tests where standard reaction conditions are defined IU. $\endgroup$ – Dayne Jul 16 '19 at 11:53

The ELISA kits are calibrated to meet the requirements of internationally certified laboratories such as Center for Biologics Evaluation and Research (CBER) associated with FDA. In most cases, the presence and specific concentration of an antigen is the target for the measurements. Such measurements take place through the absorbance measurement. According to the Beer-Lambert law, concentration of a compound in a solution is proportional to the quantity of light absorbed at its specific wavelength and at a constant optical path-length. Following absorbance measurement, a simple calculation leads to the concentration of the analyte via equation below:

A = εlc

where A is absorbance of the solution at the particular wavelength, ε is the molar absorptivity, l is the path length or deepness of the solution (which for common spectrophotometers is 1 cm or less if a well plate reader is employed), and c is the concentration of the substrate in the reaction solution (M or mol/dm3). Majority of ELISA microplate readers correspond to this method for the calculation of the analyte concentration. Introducing standard solutions with known analyte concentrations can be the basis of such calculations for the instrument. Measurement unit are typically expressed in gram per liter (g/l), mol per liter (mol/l) and copies per milliliter (copies/ml) for nucleotides, plaque forming units per milliliter (pfu/ml) for infectious viruses, colony forming units per milliliter (cfu/ml) for cells and phages, as well as unit per milliliter (u/ml) for specific enzymes. These measurement units can be also reported over time (i.e. ng/ml/h). Relative units (RU/ml) or equivalent units (Eq/ml) are normally used if no international standard references exist for the conversion.

Source: Enzyme-linked Immunosorbent Assay (ELISA): From A to Z (SpringerBriefs in Applied Sciences and Technology) by Samira Hosseini, Patricia Vázquez-Villegas, et al. | Dec 31, 2017; ISBN-13: 978-9811067655; ISBN-10: 9811067651. Page 65.

Contrary, Units Per Millilitre (U/mL) are normally used when there are international standard references, agreed upon by scientists and doctors.

Please find the following information on Arbitrary Units (AU):

The WHO Expert Committee on Biological Standardization (ECBS) is the scientific body that establishes WHO biological reference standards which serve to define an internationally agreed unit to allow comparison of biological measurements worldwide. WHO International Standards (IS) for biological substances are recognized as the highest order of reference materials for biological substances and they are assigned potencies in International Units (IU). International Standards are used to quantify the amount of biological activity present in a sample in terms of the IU, allowing comparisons between assays from different laboratories. Calibration of assays using System of Units (SI)units is not appropriate given the variability of the specific activity of a biological material. Use of an arbitrary unit makes it possible to better define parameters such as the analytical sensitivity of tests or clinical parameters such as protective levels of antibody.

Usually, arbitrary units are defined within a particular study. For example:

A human serum pool positive for anti-dsDNA IgG antibodies (DNAPo) was prepared by mixing equal parts of serum from 10 patients with SLE who had a positive CLIFT test ≥ 1:640. The DNAPo was arbitrarily designed as having 100 arbitrary antibody units (AU) per ml. Artificial serum standards containing different AU/ml were prepared by diluting the DNAPo with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBS-T). A human serum pool negative for anti-dsDNA IgG antibodies (DNANe) was prepared by mixing equal parts of serum from 10 healthy persons who had a negative CLIFT test and no clinical evidence of SLE. The DNAPo and DNANe pools were used as positive and negative controls in the ELISA and CLIFT reactions.

Thus, the U/mL figures are comparable, while RU/ml or AU/ml are not.

  • $\begingroup$ Thanks for the answer. I thought when there are international standard references, IU/mL is used. In any case what does AU mean? $\endgroup$ – Dayne Jun 14 '20 at 3:49
  • $\begingroup$ @Dayne, thank you! I have updated my answer on AU (arbitrary units). $\endgroup$ – Maxim Masiutin Jun 14 '20 at 19:53

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