Systemic lupus erythematosus and rheumatoid arthritis

 
 
11. Genotyping and serological analyses 
DNA from the patients and controls was purified from ethylenediamine 
tetraacetic acid-treated blood using a standard salting-out method.
190
The 6 
polymorphisms genotyped for these projects were 
PvuII
T/C (rs2234693), 
XbaI
A/G (rs9340799), PD-1.3 G/A (rs11568821), rs3811021, 
PTPN22
1858C/T 
(rs2476601), and 
PTPN22
-1123G/C (rs2488457). A mixture of the TaqMan 
assay and TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, 
CA, USA) were added to 96-well microtitre plates containing 10 ng of air-dried 
genomic DNA. PCR reactions were performed according to the manufacturer’s 
instructions. The genotypes of the patients and controls were determined using 
an ABI PRISM 7900HT Sequence Detector System and the SDS 2.1 software 
(Applied Biosystems). 
In paper IV and V, 
HLA-DRB1
genotyping was performed using PCR sequence-
specific primers from DR low-resolution kit and DRB1*04 subtyping kit 
(Olerup SSP AB, Saltsjöbaden, Sweden) with a method previously described.
191
The shared epitope (HLA-SE) alleles were defined as DRB1*0401 and 
DRB1*0404. ACPAs were analysed using the Diastat kit (anti-CCP2) from 
Axis-Shield Diagnostics (Dundee, UK) (cut-off value = 5 units/ml). In paper 
IV, RFs of IgM, IgG and IgA isotypes were determined using in-house enzyme-
linked immunoassays as previously described.
83
In paper V, IgM isotype 
rheumatoid factor (IgM-RF) was determined using the agglutination test 
(Waaler-Rose). Number of individuals analysed and positive for 
HLA-SE
, 
ACPAs, and RFs are shown in Table 6. 
- 38 -

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Table 6.
Frequencies of 
HLA-SE
, ACPA, and RFs in paper IV and V. Presented as number of 
individuals positive/analysed and percentage positive within parentheses.