Comparison of different methods for the detection of the fine specificity of anti-Ro/SSA response

Background

the determination of the fine specificity of anti-Ro/SSA response is useful in the classification of the risk for the occurrence of congenital complete heart block (CCHB) in newborn of anti-Ro/SSA mothers.

Aim of the study

to evaluate different methods for the detection of anti-52 and 60 kD Ro/SSA antibodies.

Patients and methods

132 sera (82 from anti-Ro/SSA patients by counterimmunoelectrophoresis (CIE), 30 from anti-ENA positive/anti-Ro/SSA negative and 20 from ANA and anti-ENA negative) were tested by ELISA with recombinant 52 and 60 kD Ro protein (Pharmacia Upjohn, Germany) and immunoblotting (IB) with human spleen extract (HSE) as substrate to the aim of determining the fine specificity of anti-Ro response. In addition, 21 sera from mothers of CCHB children were tested by CIE, two ELISAs with recombinant proteins (Pharmacia and Euro-diagnostica, The Netherlands), two IBs with HSE and with HEp-2 extract as substrates (MarDx, USA).

Results

the total agreement between ELISA (Pharmacia) and IB (HSE) was 76% for anti-Ro 60 kD and 44% for anti-Ro 52 kD. The ELISA was more sensitive than IB both for anti-Ro 60 kD (91% vs 84%) and for anti-Ro 52 kD detection (82% vs 51%). Seven sera positive by CIE were negative by IB (non blotters): six of these sera were positive for anti-60 kD and 2 for anti-52 kD by ELISA. The mean antibody titre for anti-60 kD was significantly lower (P < 0.00005) than that of sera detected by IB.

A correlation ranging from 78 to 100% was detected between the different methods testing the sera from CCHB mothers. The agreement between the IB methods for anti-Ro 60 kD and for anti-52 kD was 79% and 68.5% respectively while between the ELISAs was 44% and 67% respectively. The best agreement obtained comparing IB and ELISA for anti-Ro 60 and 52 kD was 78% between IB with HSE and ELISA (Euro-diagnostica).

Conclusions

ELISA seems to be the most sensitive method to detect the fine specificity of anti-Ro/SSA response. The majority of IB negative/CIE positive sera (non blotters) were positive for anti-60 kD by ELISA at low titer. IB with HSE as substrate performed slightly better (p not significant) than IB with HEp-2 cells extract in CCHB mothers.