At the outset of the 2019 SARS-CoV-2 / COVID-19 epidemic, diagnostic immunoassay manufacturers focused on developing anti-SARS-CoV-2 IgM & IgG tests.
While the epidemic has progressed to become a global pandemic, diagnostic test developers’ efforts have also evolved. Recently, we have noticed an increase in interest for patient material positive for Human anti-SARS-CoV-2 IgA. This article will take a look at the difference between the different immunoglobulin sub-classes and seek to understand test developers’ interest in human IgA antibodies specific to SARS-CoV-2.


SARS-CoV-2 Antibody Tests

In a recent article we have explained the reason for interest in tests to detect SARS-CoV-2 specific antibodies. Previously infected individuals may be expected to express antibodies specific to SARS-CoV-2. Therefore antibody tests have utility for serology studies that seek to understand how many in the population have previously been infected, and may also have future use as companion tests for vaccinated populations to understand if satisfactory immune responses have been generated. Furthermore, one idea early in the pandemic which hasn’t gained much traction was to mitigate some of the economic impact by identifying and liberating from freedom of movement constraints, i.e. “lockdown”, those individuals who have already been infected by the virus and may therefore be immune from future infections.


Types of Immunoglobulin

There are 5 major classes of immunoglobulins. These are IgG, IgM, IgA, IgD, and IgE, each with its own structure based around the classic antibody “Y” shape consisting of Heavy and Light chains.



The most common class of immunoglobulin, present in the largest amounts in blood and tissue fluids, and the most commonly detected type in diagnostic infectious disease tests.


The initial class of Immunoglobulin made by B cells following exposure to an antigen, commonly present as a receptor on the B cell surface. Typically the earliest class of immunoglobulin detectable before levels wane. However, the situation can vary for different infections and different individuals, e.g. in Lyme Disease and Toxoplasmosis which we have covered in previous articles.


The main class of antibody found in many bodily secretions including tears and saliva, respiratory and intestinal secretions. Typically, IgA is not as stable as IgG despite being synthesized in large amounts


IgE is present in low concentrations in the blood. IgE antibodies stimulate a histamine response when binding allergens and play a crucial role in allergy testing; allergen specific IgEs produced in response to exposure to a given allergen can be readily detected in human serum or plasma and are diagnostic of specific allergies.


IgD is present on the surface of most B cells early in their development but only limited amounts are released into circulation


Why Test for IgA?

Whereas early studies of SARS-CoV-serological responses focused on IgG and IgM responses, some papers have suggested COVID-19 IgA may be the most readily detectable of the immunoglobulins in COVID-19 patients and detection of it can serve to increase test sensitivity.

Back in March 2020 Guo et al reported that IgM and IgA appeared earlier than IgG while IgG titres were highest followed by IgA and then IgM.

In a May 2020 article, Jääskeläinen et al. analysed sera from 39 patients and determined that IgA levels were higher than IgG in most cases. In many cases, the IgA level was high enough for the patient to test positive whereas the IgG level was below the threshold for a positive test result. Euroimmun SARS-CoV-2 IgG and IgA kits were used, which are suitable for detection in serum and plasma.

In August 2020 Beavis et al showed 68 out of 82 SARS-CoV-2 PCR positive patients were positive for SARS-CoV-2 IgA whereas 55 out of 82 were positive for SARS-CoV-2 IgG. For patients tested 0, 1 or 2 days after symptom onset the vast majority were negative for IgG whereas most were positive for IgA.



Timelines of IgG and IgA results from SARS-CoV-2 PCR positive patients (from Beavis et al.)


Infantino et al., have recommended the use of IgA tests in order to enhance diagnostic sensitivity of COVID-19 serology tests. They found that IgA levels reached concentrations higher than those observed for IgG and IgM and were often positive in IgM negative patients. Therefore, IgA could shorten the amount of time needed post-infection for virus positive patients to test antibody positive.

SARS-CoV-2 IgG, IgA and IgM in SARS-CoV-2 patients who were initially IgM negative


Saliva testing

Collecting blood and converting to serum or plasma in order to detect antibodies is a fairly straightforward process, but perhaps not as straightforward as collecting saliva. Since IgA tends to be present in saliva, detection of  SARS-CoV-2 specific human IgA could theoretically be performed on saliva. Recent studies have confirmed that SARS-CoV-2 IgA is detectable in the saliva of COVID-19 patients.  Another paper in pre-print suggests that antibody levels in serum and saliva do not correlate particularly well so testing in both matrices would enhance test sensitivity even further.


Closing the serology gap

If the time window between viral infection and antibody detectability is short enough (e.g. 0-2 days post-infection) the utility of SARS-CoV-2 serology tests could greatly increase. With many countries struggling to expand molecular PCR testing capacity to the levels needed, serology tests carried out using different technology and by different laboratories/personnel to PCR tests, would be additive to the existing COVID-19 test capacity. The price per test would also likely be significantly lower. Unfortunately, it would be hard to tell from the test results exactly when the patient had become infected, especially as antibody levels post-infection vary greatly between individuals, so the utility of such a test is still in doubt.

Interestingly, SARS-CoV-2 IgA also has potential as a prognostic marker, being associated with more severe disease.



Data from several sources suggests that the detection of SARS-CoV-2 IgA in addition to the other immunoglobulins (IgG and IgM) represented in SARS-CoV-2 antibody tests  can increase the sensitivity of COVID-19 tests when compared with tests to detect anti-SARS-CoV-2 IgG and/or IgM .

Logical Biological offers serum and plasma samples with measured positive levels of SARS-CoV-2 IgA, IgM and IgG as well as SARS-CoV-2 positive swabs.

What is HAMA?

HAMA is an acronym for Human Anti-Mouse Antibodies. Some humans produce HAMA and have it present in their blood. Unfortunately for them, and the in vitro diagnostics industry, the presence of Human anti-mouse Antibodies (HAMA) in patient samples can lead to false positive and false negative results in immunoassays.

Murine MC HAMA

Why do some Humans produce HAMA?

Some people work directly with mice while others might inadvertently encounter mouse proteins or immunoglobulins by coming into contact with mouse urine or contaminated food. These people could develop an immune response against mouse immunoglobulins (antibodies) they encounter such that their immune system produces HAMA.

In the past, mouse monoclonal antibodies were used as therapeutics and could elicit an immune response resulting in the presence of HAMA in human individuals. However, these days monoclonal antibody-based pharmaceuticals are “humanised” to avoid this problem.

Presence of HAMA in individuals is rare, but still needs to be accounted for in immunoassay design.

Could I interfere with your immunoassay?

How can HAMA impact Immunoassays?

When a patient is tested for a condition, the test performed is commonly an immunoassay and the sample that is tested is serum or plasma derived from the patient’s blood. Immunoassays are typically developed using a “matched pair” of mouse monoclonal antibodies to bind to and detect the marker of interest. The ‘marker’ of interest depends on the condition being diagnosed but could be, for example, Troponin I which is a marker of acute myocardial infarction, or HIV p24 antigen which is a marker for the presence of HIV virus. The patient sample (e.g. serum or plasma) is applied to the immunoassay and if the marker being tested for is present the mouse monoclonal antibodies within the assay bind to it and a signal is generated.

However, where HAMA is present in the patient sample the HAMA can bind to the mouse monoclonal antibodies used as immunoassay components and can either i) block the mouse monoclonal antibodies from binding to the marker of interest resulting in a false negative result, or ii) form a bridge between the pair of mouse monoclonal antibodies, generating false positive signal.  Where a patient receives an incorrect diagnosis due to the presence of HAMA, the consequences can be devastating. At least 34 cases of hCG false-positive tests in the United States between 1999-2004 resulted in the patients receiving chemotherapy or surgery, including 10 hysterectomies, for assumed cancer1.

How can HAMA interference be prevented in immunoassays?

Assay manufacturers can develop their assays in such a way as to minimise the interference from HAMA, for example by adding excess mouse immunoglobulin to their assay buffers. When this is done, the HAMA present in the patient sample can bind the excess mouse immunoglobulin rather than the reagents being used in the assay.

Immunoassay developers will need to access patient material in order to design an assay that is not affected by HAMA, and also to show that the assay works in the presence of HAMA. Logical Biological is able to provide HAMA-positive serum and plasma.

Even if blockers are used to control HAMA, the heterogeneous nature of its presence in patients mean that it is difficult to rule out its influence entirely, unless it is measured. If HAMA is suspected, the clinical laboratory can perform serial dilutions with an appropriate buffer to demonstrate nonparallelism (higher recovery of the signal than expected).

An alternative solution to controlling for HAMA interference is to use monoclonal antibodies from an alternative species in the immunoassay, in place of mouse monoclonal antibodies. Rabbit and Sheep monoclonal antibodies are candidates here. However, this is not a great solution as some individuals in the population will be producing human anti-rabbit and anti-sheep antibodies. Synthetic peptide binders and/or recombinant antibodies can theoretically avoid the problem of HAMA when used as alternatives to antibodies raised in animals.


  1. Human Chorionic Gonadotropin (hCG), By Laurence A. Cole, Stephen A. Butler. Elsevier. 2010.

Irregular antibodies are antibodies found in the blood of transfusion donors that have the potential to cause hemolysis of the recipient blood. Screening using an indirect Coombs Test should be performed to ensure that the donor blood is compatible with the recipient. “Irregular Antibodies” refers to all antibodies, other than those detecting ABO blood group antigens, that can cause incompatibility in blood transfusions and between mother & child.

The ABO Blood Group System

Most people are aware of the ABO blood group system. There are 4 major blood types in humans – A, B, O and AB. Some of the blood groups are incompatible with others; if a blood group is transfused into a patient with an incompatible blood group, hemolysis results. The incompatibility can result in death.

Example of ABO Incompatibility

Terence (recipient) is Blood Group A. He has A antigens on his red blood cells and anti-B antibodies in his plasma.
Theresa (donor) is Blood Group B. She has B antigens on her red blood cells and anti-A antibodies in her plasma.

If Theresa’s Group B blood is given to Terence, Terence’s anti-B antibodies will attack Theresa’s blood and cause it to hemolyse.

Since blood group incompatibility is life-threatening it is essential to confirm donor and recipient compatibility before a transfusion occurs. The ABO and Rhesus blood group systems are the most well-known but there are other less well known factors that can cause hemolysis in transfusion patients and new-borns – these are the Irregular Antibodies  

Anti-Kell – an example of an Irregular Antibody

Anti-Kell antibodies may develop in individuals which lack the Kell antigen upon:

  1. Receipt of a blood transfusion containing Kell antigen
  2. At childbirth following transplacental hemorrhage

In these cases the individual’s immune system will recognise the Kell antigen as a foreign molecule and elicit an immune-response, becoming sensitized to it.

Testing for donor compatibility – the Indirect Coombs Test

The indirect Coombs Test, also known as Indirect Antiglobulin Test, detects irregular antibodies. Again, taking anti-Kell as an example (See Figure 1):

  1. Recipient serum may contain irregular antibodies (e.g. anti-Kell)
  2. Donor blood sample is added to recipient serum
  3. Recipient irregular antibodies, if present, bind to donor red blood cells where the corresponding antigen is present
  4. Anti-human Immunoglobulins (Coombs reagent) is added. The antibodies within Coombs reagent bind the Fc region of any irregular antibodies and, where those irregular antibodies have bound the donor red blood cells, form bridges between immune complexes on red blood cells, resulting in agglutination
Positive Coombs Test result for Irregular Antibodies

There are many other irregular antibodies, such as those listed below, available from Logical Biological.

Irregular Antibody Product Identifier
anti-c H191
anti-Cw H193
anti-D H189
anti-E H190
anti-Fya H195
anti-Jka H194
anti-Kell H192
anti-Kpb H205
anti-Lea H200
anti-Leb (Lewis) H201
anti-Lua H203
anti-Lub H204
anti-M H196
anti-N H197
anti-P1 H202
anti-Public H207
anti-S H198
anti-s H199
auto-Pap H206