9. IgG Avidity Tests
 

Rubella reinfection can occur, especially in those whose immunity were induced by vaccination rather than by natural infection. However, reinfection by rubella during the first trimester of pregnancy is thought to pose minimal risks to the fetus. Cases of CRS arising from rubella reinfection have rarely been reported and termination of pregnancy is not recommended. Therefore, it is important to distinguish reinfection from primary infection by rubella during the first trimester of pregnancy. In the absence of reliable confirmatory tests, needless abortions may result.

Rubella specific IgM can be detected following both primary and reinfection. Although in the latter case, it is likely to be more transient and of a lower level One solution for the differentiation of primary from reinfection could be the measurement of the antigen-binding avidity of specific IgG. The avidity of IgG is low after primary antigenic challenge but matures slowly within weeks and months.

Methods

1. Semi-quantitative test ;- This is based on the recognition of a characteristic pattern in the radial haemolysis test. The zone of haemolysis was assessed; Haemolytic zones with "soft" diffuse outer margins are produced by antibodies of low avidity. Haemolytic zones with a discrete outer margins (designated "ordinary" ) are produced by antibodies of high avidity. Zones that were neither diffuse nor discrete are classified as equivocal.
2. Quantitative test "Avidity ELISA" ;-
3.  
   1. Elution principle - in this test a mild protein denaturing agent such as urea or diethylamine (DEA), is added to the antibody - antigen mixture. Antibodies of low avidity are more likely to dissociate from the antigen-antibody complexes than those of higher avidity. The rest of the test is as for a normal ELISA. The results obtained in the presence and absence of the denaturing agent is compared and a ratio is derived. Low avidity antibodies will have a much higher ratio than high avidity antibodies.
   2. The sera to be tested is diluted at various concentrations in the presence and absence of DEA and assayed for IgG1 and IgG3. The optical densities obtained were plotted. The highest OD (V) was noted and halved (V/2) and the distance between the OD curves at V/2 was measured as the DEA shift value.

Hedman et al (1989) measured the IgG avidity from 64 sera. According to their avidity IgG ELISA, 29 had low avidity Ab, 29 had high avidity Ab and 6 were borderline. Comparison with known clinical records showed that all patients with low avidity Ab had recent primary infection. Those with high avidity had previously been immune. Amongst this group were 4 that had originated from confirmed reinfections. Of the equivocal sera, 5 out of 6 were obtained within 2 months of primary infection.

Thomas and Morgan-Capner (1988) measured the avidities of Rubella specific IgG1 and IgG3. IgG3 is rarely demonstrated in reinfection. They tested sera from 24 patients who were immunized or infected in the distant past, 66 who recent rubella primary infection, 11 from those with symptomatic reinfection and 64 from those with asymptomatic reinfection. For IgG1 the DEA shift value was< 0.6 for cases of rubella in the distant past, compared with 0.8 for the first month after primary infection. The maximum DEA shift value for the sera from cases of reinfection was 0.65. No serum from cases of rubella in the distant past contained sufficient specific IgG3 to estimate avidity. The sera collected within one month of the onset of rubella gave DEA shift values of 0.7 compared to sera from reinfection. In general, the elution-principle test is more sensitive for past infection but less sensitive for recent infection. Whereas the dilution-principle test is more sensitive for recent infection.

Molecular Methods

Virological Methods Slideset