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Meaning of units in ELISA based tests?

Meaning of units in ELISA based tests?


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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)?


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.


ELISA: Types of ELISA

The ELISA Basics Guide has the right amount of detail to help you plan your experiment and achieve a successful ELISA.


ELISA Tests

ELISA is an abbreviation for "enzyme-linked immunosorbent assay." In 1974, P. Perlmann and E. Engvall developed the test as a substitute for certain radioimmunoassay tests, and eventually, it replaced the western blot test for HIV confirmation. The ELISA test is versatile and medical professionals can perform it easily as compared to other more complicated tests many variations are available commercially.

What is an ELISA test?

An ELISA test uses components of the immune system (such as IgG or IgM antibodies) and chemicals for the detection of immune responses in the body (for example, to infectious microbes). The ELISA test involves an enzyme (a protein that catalyzes a biochemical reaction). It also involves an antibody or antigen (immunologic molecules) that may form an antigen-antibody reaction to provide a positive result or, if they do not react, a negative result. Examples of the uses of an ELISA test include diagnosing infections such as HIV (human immunodeficiency virus) and some allergic diseases like food allergies and experimental investigations to identify compounds (antigens from a cell lysate in a wide array of organisms). ELISA tests are also known as an immunosorbent assay or an enzyme immunoassay when an enzyme is bound to another substance as an indicator (can cause a color change, for example).

The test is based on a microtiter plate that has a solid phase substrate (target protein, antigen) at a known concentration fixed to the plate that when exposed to an antibody that has an indicator attached (dye for color change or enzyme-labeled antibody) that can produce a color change. Depending on a standard curve for absorption of enzyme-labeled antibody versus antigen level as related to the dye color change, tests may provide semi-quotative, quantitative, and/or identification of many diverse substances. This type of test is termed a direct ELISA.

There are other types of ELISA tests. Indirect ELISA uses a secondary antibody to attach to the substrate, and the sandwich ELISA that uses the antibody as the substrate fixed to the microtiter plate. For examples and additional details, see http://ruo.mbl.co.jp/bio/e/support/method/elisa.html.

Types of ELISA Tests

HIV Test

Antibody testing is usually done on a blood sample, often using an enzyme-linked assay called an ELISA or EIA. In this test, a person's serum is allowed to react with virus proteins that have been produced in the laboratory. If the person has been infected with HIV, the antibodies in the serum will bind to the HIV proteins, and the extent of this binding can be measured. Negative EIA results are usually available in a day or so.

What is the use of an ELISA test?

ELISA tests primarily detect proteins (as opposed to small molecules and ions such as glucose and potassium). Medical professionals frequently use ELISA tests as blood tests to detect antigens that may be present in the blood. The substances detected by ELISA tests can include hormones, an allergen, viral antigens (dengue fever, for example), bacterial antigens (TB, for example), and antibodies that the body has made in response to infection (antibodies to hepatitis B, for example) or vaccination. They can also identify an infectious disease agent in patients.

What is an ELISA kit?

An ELISA kit is a commercially available ELISA test that usually contains pre-coated polystyrene plates, detection antibodies, and usually all of the chemicals needed to perform an ELISA test. However, people can purchase special kits with substances designated by the customer.

SLIDESHOW

How does ELISA testing work?

There are variations of the ELISA test (see below), but the most utilized type consists of an antibody attached to a solid surface (polystyrene plate). This antibody has affinity for (will latch on to) the substance of interest, such as a hormone, bacteria, or another antibody. For example, human chorionic gonadotropin hormone (HCG), the commonly measured protein that indicates pregnancy, can be detected by ELISA. A mixture of purified HCG linked to an enzyme and the test sample (blood or urine) are added to the test system. If no HCG is present in the test sample, then only the linked enzyme will bind to the solid surface. The more substance of interest that is present in the test sample, the less linked enzyme will bind to the solid surface. The more of the substance of interest is present it will cause a reaction and show up on the test plate in some way, such as a color change of the solution (or like a pregnancy test "two pink lines" or a "+" mark).


Principle of ELISA Test

  1. An enzyme: horse radish peroxidase, alkaline phosphatase which is labelled or linked, to a specific antibody.
  2. A specific substrate:
  • o-Phenylenediamine dihydrochloride for peroxidase
  • P Nitrophenyl Phosphate (PNPP)- for Alkaline Phosphatase

Substrate is added after the antigen-antibody reaction. The enzyme catalyses (usually hydrolyses) the substrate to give a color end point (yellow compound in case of alkaline phosphatase). The intensity of the color is proportional to the amount of antibody or antigen present in the test sample, which can be quantified using ELISA reader.


ELISA advantages and disadvantages

Advantages

  • High sensitivity and specificity: it is common for ELISAs to detect antigens at the picogram level in a very specific manner due to the use of antibodies.
  • High throughput: commercial ELISA kits are normally available in a 96-well plate format. But the assay can be easily adapted to 384-well plates.
  • Easy to perform: protocols are easy to follow and involve little hands-on time.
  • Quantitative: it can determine the concentration of antigen in a sample.
  • Possibility to test various sample types: serum, plasma, cellular and tissue extracts, urine, and saliva among others.

Disadvantages

  • Temporary readouts: detection is based on enzyme/substrate reactions and therefore readout must be obtained in a short time span.
  • Limited antigen information: information limited to the amount or presence of the antigen in the sample.

Cross-Reactivity Concerns

COVID-19 serology assays are designed to be specific for SARS-CoV-2, but how do we know they will not cross react with other coronaviruses? Cross-reactivity with the common coronaviruses (cCoV's) would be especially detrimental since 60-75% of children have antibodies to one or more cCoV&rsquos, and 90% of adults over 50 years of age have antibodies to all 4 cCoV's. Fortunately, there is not a lot of sequence identity shared between SARS-CoV-2 and cCoV&rsquos (approximately 21-34% AA homology), but the FDA does require laboratories to include a note on all positive serology reports stating, &ldquoFalse positive results due to antibodies to cCoV&rsquos may occur.&rdquo

What about the closer relatives of SARS-CoV-2? SARS-CoV-2 and SARS-CoV share 90% amino acid identity for their respective N proteins and 77% for S proteins. SARS-CoV-2 and MERS-CoV share 49% amino acid identity for N proteins and 33% for S proteins. These data highlight the value of using S protein antigens to increase the specificity of serology tests.


Objective

To investigate the diagnostic value of serological testing and dynamic variance of serum antibody in coronavirus disease 2019 (COVID-19).

Methods

This study retrospectively included 43 patients with a laboratory-confirmed infection and 33 patients with a suspected infection, in whom the disease was eventually excluded. The IgM/IgG titer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was measured by chemiluminescence immunoassay analysis.

Results

Compared to molecular detection, the sensitivities of serum IgM and IgG antibodies to diagnose COVID-19 were 48.1% and 88.9%, and the specificities were 100% and 90.9%, respectively.In the COVID-19 group, the IgM-positive rate increased slightly at first and then decreased over time in contrast, the IgG-positive rate increased to 100% and was higher than IgM at all times. The IgM-positive rate and titer were not significantly different before and after conversion to virus-negative. The IgG-positive rate was up to 90% and not significantly different before and after conversion to virus-negative. However, the median IgG titer after conversion to virus-negative was double that before, and the difference was significant.

Conclusions

Viral serological testing is an effective means of diagnosis for SARS-CoV-2 infection. The positive rate and titer variance of IgG are higher than those of IgM in COVID-19.


Understanding Rheumatoid Arthritis Lab Tests and Results

Rheumatoid arthritis (RA) is a chronic autoimmune disease that primarily affects the joints, but can affect other parts of the body. Diagnosing and managing RA involves clinical evaluation by a rheumatologist, as well as several different laboratory tests that require blood work. The results of these tests may be used in two ways:

The doctor and healthcare team use the results of these tests to guide treatment options for each patient. In turn, understanding how the results of blood tests used to monitor RA and its treatment can help patients better able to manage their RA.

Rheumatoid Factor

Rheumatoid factor (RF) was the first autoantibody to be discovered in people with RA. (Autoantibodies develop in response to the body’s own tissue, and are characteristic of autoimmune diseases, such as RA.) Despite the name, however, RF is not specific to RA, and there are many factors that can impact RF lab results. About 20% of those with confirmed RA will not have an abnormal RF test, while 5% of people who do not have RA will have an abnormal RF test. Negative levels do not exclude the disease, and positive levels do not guarantee the diagnosis.

The normal range of RF is from 0-20 IU/ml. RF above 20 IU/ml is not considered enough to diagnose RA, as there other reasons the RF level may be elevated. Some conditions and medical procedures that can raise RF levels include: other autoimmune diseases, certain chronic infections, diabetes, bacterial endocarditis, cancer, normal aging, vaccinations and transfusions. It’s important to note that once the RF level is elevated, it will often remain so even if the disease goes into remission.

Anticyclic Citrullinated Peptide

Another test which is ordered when rheumatoid arthritis is suspected is the anticyclic citrullinated peptide (anti-CCP). The normal level of anti-CCP is less than 20 Units. (At Hospital for Special Surgery, anti-CCP is reported in Units. Some labs report this same result using a different measurement notation, that is, as less than 20 EU/ml.) A level above 20 suggests the possibility of RA. As with rheumatoid factor, some people with positive anti-CCP antibody will not have RA, but this test is somewhat more specific for RA than the rheumatoid factor. The higher the levels of anti-CCP antibody, the more likely it is to suggest RA.

This test is 97% specific for RA if it is present. Once a patient develops a positive anti-CCP, it will usually remain positive, despite remission.

About 20% of RA patients are seronegative, meaning that their RF and anti-CCP lab results both continue to come back negative. In these cases, the physician makes the diagnosis based on physical examination and imaging.

RF and anti-CCP are not used to monitor disease activity, because they both tend to remain positive despite remission. Once the diagnosis of RA has been made and confirmed, these tests are not repeated.

Sedimentation Rate

Sedimentation rate (also known as erythrocyte sedimentation rate or ESR), is a crude measure of inflammation. It is calculated by measuring the rate at which red blood cells sediment in a test tube in one hour. Normal levels for men range from 0-15 mm/hr to 0-20mm/hr and for women 0-20 mm/hr/ to 0-30mm/hr, depending on age – higher for people over the age of 50). The ESR rate is not specific for RA, and there are many factors that can interfere with the results, such as bad processing, an infection, and aging in patients over the age of 50.

C-reactive Protein

C-reactive protein is another measure of clinical inflammation. The normal measurement is less than 1.0 in many labs. This test, however, can be influenced by factors such as obesity and infection and is not specific to RA.

Both ESR and C-reactive protein are non-RA-specific measures of inflammation. Both tests are used to test disease activity when they are high, this suggests that the disease is very active (assuming no other causes for high results, such as infection, are present). The healthcare team orders these labs regularly to monitor the patient’s disease and check how his or her medications are working.

Complete Blood Count

A complete blood count (CBC) test looks at red and white blood cell counts. Below are the normal measurements in our lab – other labs may well have their own set of normal values.

The CBC tests help to inform the healthcare team about side effects of treatment and any secondary consequences of RA, such as anemia. If the patient’s hemoglobin levels indicate anemia, this will be further investigated looking for its cause.

Complete Metabolic Panel

A complete metabolic panel is used to monitor kidney and liver function, in order to assess whether changes to medication must be made or whether they are working well. A complete metabolic panel measures sodium (Na), potassium (K), chloride, glucose, creatinine (a measure of kidney function), and AST and ALT (markers of liver function).

Both the CBC and the complete metabolic panel are used to monitor disease activity as well as side effects and efficacy of medication.

With these laboratory tests as a guide, the healthcare provider may need to make adjustments to the patient’s medications and RA treatment. Laboratory tests provide important information in the diagnosis, management and treatment of rheumatoid arthritis. By becoming informed about the normal values for tests, as well as their own numbers, patients with RA can better communicate with the health care team and gain a better understanding about some of the information that is used in developing and monitoring their treatment plans.

Learn more about the HSS Early RA Support and Education Program, a free support and education group, developed specially for people recently diagnosed with RA and early RA.

Summary by Lysa Petrsoric, MPH, MSW, April 20, 2015
Edited by Nancy Novick.

Authors

Monica Richey, MSN, ANP-BC/GNP
Mary Kirkland Center for Lupus Care, Hospital for Special Surgery


What is Indirect ELISA?

ELISA can be performed using two types of antibodies namely primary antibody and secondary antibody. Indirect ELISA tool uses both types of antibodies to amplify the signals for better detection. Indirect ELISA technique is performed as follows.

  1. Plates are incubated with antigens and washed to block nonspecific binding.
  2. Then primary antibodies are added and washed.
  3. Enzyme-linked secondary antibody are added and washed.
  4. A substrate is added and allowed to react with enzymes.
  5. Signals are detected, and the presence or absence of the specific antigen in the sample is identified.

In indirect ELISA test, several secondary antibodies can bind to a single primary antibody. Secondary antibodies are linked with easily assayed enzymes. Therefore, one binding can make a strong signal due to more than one interactions. Hence, indirect ELISA is more sensitive than direct ELISA. However, indirect ELISA can make nonspecific signals due to cross reactions of the secondary antibodies.

Figure 02: Indirect ELISA Test


References

Aoyagi K, Ashihara Y, Kasahara Y. Immunoassay and immunochemistry. In: McPherson RA, Pincus MR, eds. Henry's Clinical Diagnosis and Management by Laboratory Methods. 23rd ed. St Louis, MO: Elsevier 2017:chap 44.

Murray PR. The clinician and the microbiology laboratory. In: Bennett JE, Dolin R, Blaser MJ, eds. Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases, Updated Edition. 8th ed. Philadelphia, PA: Elsevier Saunders 2015:chap 16.



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