WesternBlots - ImmunoBlots                             


Epstein-Barr-Virus IgM/IgA-Immunoblot

Immunoblot for the detection of IgG-antibodies against Epstein-Barr-Virus in serum


Epstein-Barr-virus is a member of the Herpes-virus family. It is morphologically very similar but antigenically distinct from other Herpes viruses.

EBV is the main etiological agent of infectious mononucleosis (IM). IM may range from an asymptomatic mild disease, most frequently in early childhood, to a full blown symptomatic IM, most frequently in early adult age.

The virus is mostly acquired via saliva (kissing disease) and infects the epithelia of the oropharynx first. From there B-lymphocytes are infected and transformed into highly proliferating lymphoblasts. They form the typical mononuclear cells which can easily be detected by blood count. On the other hand, the proliferation of white blood cells in not typical of IM alone. This can be found in leukaemia, CMV-infection etc. Therefore, IM must be clearly differentiated from these diseases by other means.

Because virus isolation is very difficult and slow, serology is widely accepted as the best method of identification. The Paul-Bunnel method for the detection of heterophile antibodies is  acceptable for adults, although false positive results have been reported, but not for children due to missing up to 50% of all primary infections.

Therefore, test systems were established to detect antigen-specific antibodies against EBV. First the virus capsid antigen (VCA) was used for screening purposes. But with VCA alone, seropositivity can only be confirmed, because VCA-IgM can be absent in some cases of primary and in most cases of reactivated disease.

Reactivation of the disease can occur because the virus persists in the B-lymphocytes lifelong. Reactivation of EBV is not as common as in cases of CMV, but is a problem in immunocompromised patients, too, and must be differentiated from primary infection.

The best marker for active disease are antibodies against early antigen (EA). In most cases EA-D (diffuse) is of importance, because EA-R (restricted) antibodies can only be found in young children. EA-antibodies are present in nearly all cases of active disease, but a differentiation of primary and recurrent disease is not possible. For that, the detection of antibodies against the nuclear antigen 1(EBNA-1) is helpful. EBNA-1-antibodies are present 8 10 weeks after infection at the earliest and persist lifelong. Therefore active primary infections should be negative for EBNA-1, but reactivations are positive.

In conclusion, 4 (four !!!) different Elisa tests are needed for correct diagnosis and staging of an EBV-infection. This immunoblot system offers the possibility of looking for all the discussed antibodies within one highly specific test system. This could be especially worthwhile in patients

who are suspected or at risk of reactivation like AIDS patients, cancer patients undergoing chemo- and radiotherapy, transplantation patients etc.


  1. Sumaya, C.V. Serological testing for Epstein Barr Virus - Developemens in interpretation. J. infect. dis. 151, 984-987 (1985)
  2. Henle, W., Henle, G., Lannette, E.T. Epstein Barr Virus. Scientific American, 241, 40-52, (1979)
  3. Lannette, E.T. Epstein Barr Virus. In Manual of clinical Microbiology, 5 th ed. Eds.: Balows, A., Hausler, Jr., W.J., Hermann, K.L., Isenberg, H.D., Shadomy, H.J. pp 847-852, ASM, Washington D.C. (1991)
  4. Schillinger, M., Kampmann, M., Henninger, K., Murray, G., Hanselmann, I., Bauer, G. Variability of humoral immune response to acute Epstein Barr Virus (EBV) infection:  Evaluation of the significance of serological markers. Med. Microbiol. Lett. 2, 296-303, (1993)

Epstein-Barr-Virus IgG-Immunoblot

Immunoblot for the detection of IgG-antibodies against Epstein-Barr-Virus in serum

Analysis of band pattern and interpretation of the results

Cut off - Standard setting: IgM/IgA - 10 %, IgG - 20 %

The following antigens can be identified by this immunoblot:


Membrane-antigen (MA)

The role of antibodies against membrane-antigens is not yet known. They seem to appear in every stage of disease, but are rarely present later than 6 months after infection.


Viruscapsid-antigen (VCA)

VCA-IgG is the classical marker for seropositivity. Antibodies against VCA persist lifelong. and are present very soon after infection. A differentiation between primary and recurrent disease is not possible, because VCA-antibodies do not differ in titer nor can a difference in band pattern be observed. On the nitrocellulose-strips up to seven different bands associated with VCA can be detected. The high molecular weight bands (intact protein) are often very faint, because antigenic determinants are lost in the electrophoretic process. Therefore the strips should be carefully inspected, to avoid overlooking these faint bands. The low molecular weight bands are clearly visible in most cases, but in some cases they are totally missing. In conclusion, a sample is positive for VCA when 1 of 7 seven bands can be detected. VCA-IgM is the classic marker of fresh infection (1 to 3 weeks), and it is seldom seen in case of reactivation.


Early-antigen diffuse (EA-D) and Early-antigen restricted (EA-R)

EA-IgG-bands are positive when there is active disease, this is true for primary infection and for reactivation, whether VCA antibodies can be detected or not. The bands vanish when the disease become inactive, and in healthy, seropositive individuals normally no EA-band can be detected. Even 1 (one !!!) labelled EA-band (R or D) is sufficient to report a positive result for EA-antibodies.

In most cases the EA-D band is positive. This is normally the earliest band which can be detected in primary infection (3 - 6 weeks after start of infection). Then, the band is often broad and very strongly coloured in the middle and diffuse at the rims, whereas in cases of reactivation the band appears more distinctly and clearer.

The EA-R band appears 3 6 months after start of infection.

Simultaneous detection of an EA-band and EBNA-1-band (with at least 1 VCA band) is nearly a proof for a reactivated disease.

Only in very rare cases can an EA-band persist over a long time period, but then EA-antibodies are of IgG-type only, and no IgM can be detected. Therefore, in cases of no clinical evidence of disease and simultaneous detection of EBNA-1 and EA-antibodies, the EBV-IgM-blot should be negative and a reactivation can be excluded.


Epstein-Barr-Virus-Specific Nuclear Antigen (EBNA)

At least 6 different EBNA proteins are known so far, two of which, the EBNA-1 and EBNA-2, are well described at literature. About the other EBNA proteins and their possible role in the serodiagnosis of EBV infections, detailed information is not available from literature. IgG antibodies against EBNA 1 are found several weeks after onset of symptoms at the earliest after the original infection. Therefore when EBNA 1-antibodies can be detected, a primary infection can be excluded with high probability. Antibodies against EBNA-2 appear earlier, but can usually be detected later than antibodies against VCA and EA antigens. If the EBNA-1 band is stronger than the EBNA-2 band, it points to a late stage of the disease (2 years after start of infection). The clinical significance of the EBNA for the IgM and IgA response is not known at the movement.

 Summery of interpretation 

Stage of disease


EA (R/D)



Primary infection





Healthy seropositives 6 8 weeks after infection





Healthy seropositives 3 months after infection








(reaction zone till 1 cm)



Chronic Active EBV infection

IgA +

IgA + (EA-D)



Malignant Tumors


very strongly +


D and R




Primary infection

VCA-IgM/ IgA positive

Early stage of infection, usually 1 to 3 weeks after start of infection.

VCA-IgG positive

Early stage of infection.

Isolated appearance could persist long-life

EA-D-IgG positive

3 6 weeks after start of infection

EA-R IgG positive

3th 6th month of infection

EBNA-2 IgG positive

6th 12th month of infection

EBNA-1 IgG positive

If this band is stronger than the EBNA-2, it points to a late stage (2 years after start of infection).

Chronic active EBV infection:

VCA IgA positive

EA-D IgA in some cases positive

Malign tumors:

VCA IgG very strongly positive

EA-D and EA-R positive


bullet The interpretation given here is limited to classical primary infection and reactivation. For  special forms of diseases caused by EBV, such as chronic active EBV- infection or Burkitt lymphoma or nasopharyngeal carcinoma etc. there may be other band patterns as described.
bulletA negative result does not preclude the possibility of a recent infection. If clinical sign can be found, an IgM test is recommended. If both tests are negative, an additional sample should be taken after 6 to 8 days from this patient to exclude a delayed antibody response.




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