Serological techniques

U .lC rith id ia luciliae staining.

Serum samples from transgenic and negative control littermates were screened for the presence of anti-dsDNA antibodies using a commercially available

Crithidia luciliae dsDNA kit (The Binding Site Ltd.). C. luciliae contains an organelle (the kinetoplast), a giant mitochondrion containing dsDNA but apparently free of histones and other mammalian nuclear antigens (Aarden et al, 1975). Slides coated with C. luciliae were incubated for thirty minutes at room temperature with serum samples diluted 1:10 in PBS. The slides were washed in PBS and then incubated for 30 minutes with FITC-conjugated goat anti mouse IgG. After further washing the slides were mounted in Gelvatol and viewed either using a Zeiss Axiophot Microscope (Carl Zeiss Ltd.) or a confocal laser scanning microscope (Model MRC-1000, Bio Rad). A series of 1 pm optical sections through the specimens was obtained with a confocal microscope and a composite image (Z series) was constructed.

2.7.2 Assay for antibodies against extractable nuclear antigens (ENA)

Serum samples were screened for the presence of antibodies against extractable nuclear antigens (Sm, U1 RNP, SS-A (Ro), SS-B (La), Jol and Scl-70) using a commercially available ENA screening counter current immunoelectrophoresis (CIE) kit (The Binding Site Ltd.). This procedure was performed by Miss Karen Walker, University of Birmingham. Transgenic serum found to be positive on this screen was further characterised using an ENA typing CIE kit specific for the same antigens (The Binding Site Ltd.). For ENA screening, 120 pi of ENA extract (buffered sheep spleen extract preserved in 100 mM PMSF and 10 mM mercaptoethanol) and 20 pi of test serum were applied to the

surface of an agarose gel by means of an application mask (15 to 20 pi of test serum and appropriate positive controls for ENA typing). For both procedures electrophoresis was carried out at 50V for 1 hour 15 minutes on a Beckman Paragon power pack. Gels were dried and then stained in Acid Blue 29 in 5% v/v acetic acid for 2 seconds and destained in 5% v/v acetic acid for 5 minutes. Gels were dried completely by placing in a 45° C incudryer for 15 minutes and then examined for a visible immunoprecipitate.

2.7.3 Antihistone, anti-dsDNA and antinucleosome ELISA 2.7.3.1 Chromogenic substrate solution

ELIS As were developed using the following chromogenic substrate solution: 10 mg of 0-phenylene diamine (Sigma) in 29.5 ml of 0.15 M citrate-phosphate buffer pH 5.0 containing 6% hydrogen peroxide (BDH). 200 pi of chromogenic substrate solution was added to each well of the ELISA plate following the final wash. The chromogenic reaction was stopped by adding 50 pi of 3 M sulphuric acid and the absorbance read at 492 nm in a Titertek Multi skan MCC/340 spectrophotometer.

2.7.3.2 ELISA protocol

The levels of total Ig and IgG antihistone and anti-dsDNA antibodies in serum were measured using a modification of previously described methods (Kotzin et al 1987). Calf thymus histones (Sigma) were diluted in PBS to a concentration of 2.5 pg/ml and 0.2 ml of this antigen solution was added to each well of an Immulon II microtitre plate (Dynatech Laboratories, Inc.). After overnight incubation at 4°C, wells were coated with 0.4 ml gelatin (1 mg/ml in PBS) for at least 24 hours at 4°C. After washing, 0.2 ml of serum

samples diluted 1/100 to 1/1000 in 0.1% Tween, 1 mg/ml gelatin and 0.5% bovine serum albumin in PBS were added and incubated for 1.5 hours at room temperature. After washing, total Ig bound was measured by adding HRP- conjugated rabbit anti-mouse Ig (DAKO) or IgG was measured using HRP- conjugated goat anti-mouse IgG (Sigma). Both secondary antibodies were used at a dilution of 1/4000 in 0.1% Tween in PBS. After 1.5 hours incubation at room temperature the wells were washed and substrate solution added. The optical density (OD) was then read with an automated spectrophotometer at 492 nm.

To measure anti-dsDNA antibody levels, wells were coated with dsDNA (Sigma). To attach dsDNA, microtitre wells were first coated with poly-L- lysine (Sigma) at 5.0 p.g/ml in H2O for 1.5 hours at 37°C. After washing,

dsDNA was added at 5.0 pg/ml in PBS and incubated overnight at 4°C. After washing, serum samples diluted 1/100 to 1/1000 were added as described above.

Levels of IgG antinucleosome antibodies were measured by ELISA as previously described (Amoura et al 1994). These assays were carried out by Dr. Sophie Koutouzov, Hôpital Necker, Paris, France. Briefly, Purified mononucleosomes prepared as previously described (Koutouzov et al 1996) were dissolved in PBS at 5 pg/ml and 100 pi added to Luxlon microtitre plates (CML). Plates were incubated overnight at 4°C. Wells were washed with PBS- 0.1% Tween, pH 7.4 (PBST) and postcoated for 2 hours with 0.1 ml of PBS- 10% FCS, pH 7.4. After washing, sera (1/100) diluted in PBS/T were added and reacted for 2 hours. Bound antibodies were detected with peroxidase- conjugated goat anti-mouse Fc antisera (Sigma). Binding was measured by adding ABTS substrate (Southern Biotechnology), and the OD was read at 405 nm by an automated spectrophotometer (Dynatech Laboratories, Inc.).

A serum sample known to be positive for anti-ssDNA and negative for anti- dsDNA showed no significant binding in the dsDNA ELISA (OD 0.00+/- 0.05). All sera were also tested on uncoated ELISA plate plastic. Non-specific binding to plastic was low (OD on uncoated wells ranged from 0 to 5% of values on antigen coated wells). The antihistone and anti-dsDNA ELISA tests were run in triplicate and anti-nucleosome ELISA in duplicate. In addition, serum samples were checked for non-specific binding to control wells lacking antigen. We have previously shown that this anti-dsDNA ELISA system shows no cross reactivity with ssDNA.

2.8 Characterisation of lymph node and splenic composition in IFN-y