Materials
Oligonucleotide primers were synthesised by Thermobiosciences (Germany) and used without further purification. Restriction enzymes NcoI, XhoI and BglII and T4 DNA ligase were purchased from Promega, (Southampton, UK). Plasmids pET-26b(+) and BL21(DE3) E. coli cells were purchased from Novagen (Nottingham, UK). DH5α E. coli cells were supplied by Gibco (Paisley, UK). Automated sequencing was carried out by MWG-Biotech (Ebensburg, Germany).
Monoclonal antibodies
Three human monoclonal antibodies (mAb) were produced in Chinese Hamster Ovary (CHO) cells, an in-vitro expression system, which has been described in detail in previous papers [25, 26]. Affinity purification of the antibodies from these cells was carried out by Chemicon Europe Ltd, Southampton, UK. Murine monoclonal anti-human DI antibodies mAb-16 and 6C4C10 were kind gifts from Dr M Linnik and Dr M Iverson, La Jolla Pharmaceuticals (LJP), California, USA.
Production of a construct containing a synthetic gene encoding the DI sequence
A synthetic gene was designed to encode for DI of human β2GPI, an N-terminal OmpA leader sequence and BglII/NcoI flanking restriction sites. This gene was designed by the computer programme Juniper, which designed six 60 mer overlapping oligonucleotide primers based on the published amino acid sequence of DI [6]. The gene encoding for DI was synthesised using these primers by recursive PCR [15] (figure 1). Twenty pmol of each outer primer and two pmol of each internal primer were used in a reaction containing 2 U of Vent DNA polymerase (New England Biolabs (NEB), Hertfordshire, UK) and 25 mM of 2'-deoxynucleoside 5'-triphosphates (dNTPs') in 100 μl of the 10× supplied buffer and ddH20. PCR was performed under the following conditions: 95°C for 8 min, 30 cycles of 94°C for 2 min, 57°C for 1 min, 72°C for 1 min with a final extension step of 72°C for 10 min. This gene was then ligated into the expression plasmid and sequenced to exclude PCR errors.
The pET system originally described by Studier and Moffat [27] was used to express recombinant DI. A variety of constructs were used to optimise expression. The final cloned expression construct encoded for a pelB leader sequence followed by DI and a C-terminal his6-tag cloned into pET-26b(+). Both the leader and the his6-tag are present within the pET-26b(+) plasmid so that the OmpA leader sequence that had originally been created by recursive PCR (figure 1) was redundant and removed at this stage. The target gene was under the control of the high stringency phage T7lac promoter [20] and a strong T7 translation initiation site.
Induction experiments
The final recombinant expression vector pET26b(+), containing the DI gene and a kanamycin resistance gene, was transferred to the expression strain BL21(DE3). Single colonies were picked from the transformants, and 5-ml cultures prepared in 50-ml falcons (overnight with shaking, 30°C). Fresh 500-ml cultures in 2.5-litre flasks were then set up using the overnight culture to inoculate the medium to an optical density (OD)600 of 0.1. Cultures were induced with a range of IPTG (0.1 mM, 0.4 mM or 1 mM) at an OD600 of 0.6 and allowed to grow for a further 4 hours (30°C, shaking). 'Terrific' broth containing 60 μg/ml of kanamycin was used to prepare the overnight cultures with the addition of 1% glucose for the expression cultures. OD600 was recorded at periodic intervals before and after induction as a measure of bacterial growth in the culture.
Preparation of periplasmic fraction and purification
The method for periplasmic extraction of protein from E. coli is based on previously published methods by our group for the expression of periplasmic Fabs of human autoantibodies in W3110 E. coli [28]. Briefly, cultures were spun (4000 g, 20 min, 4°C) following 4 hours induction and supernatant was saved (at 4°C) for the detection of any leaked DI protein from the cells. The cell pellet was then exposed to osmotic shock by suspension in ice-cold water (30 ml of dH20 per litre of culture), stirred for 30 min at 4°C, and spun (8000 g, 20 min, 4°C). This supernatant constitutes the periplasmic fraction containing recombinant DI, an aliquot of which was stored at -20°C for subsequent SDS-PAGE and Western blot analysis.
For purification periplasmic extract was loaded on a column containing nickel charged resin, Novagen (Nottingham, UK). DI bound to the column by virtue of the C-terminal his6-tag and was eluted with 300 mM imidazole, (2 M NaCl, 80 mM Tris-HCl, pH 7.9). Recombinant his6-tagged DI was then dialysed extensively against PBS-10% glycerol overnight at 4°C using dialysis visking tubing, MWCO-3500, Medicell (London, UK). The purity of the eluted DI was assessed on SDS-PAGE 15% gels.
Functional assessment
Affinity purification of IgG aPL
Polyclonal IgG was purified from 21 patients satisfying the preliminary diagnostic criteria for APS [1]. IgG was also purified from 9 normal controls and 14 patients with SLE (but no APS) as disease controls. 19/21 patients with APS, 13/14 patients with SLE and 7/10 healthy controls were female. The mean ages of the subjects in the three groups were comparable (APS – 44.0 years, SD 7.0; SLE 37.2 years, SD 10.0; healthy controls 33.2 years, SD 9.0). Ethical approval for the study was granted by University College London Hospital Research Ethics Committee. Protein G beads (Amersham, Bucks, UK) were prepared by washing with PBS. 1 ml serum was mixed with 2 mls 0.02 M sodium phosphate (pH 7) binding buffer and incubated at room temperature (RT) for two hours with 0.5 ml prepared Protein G beads. This mixture was then spun (200 g, 5 min, 4°C) and the beads washed a further 3 times with binding buffer. Elution of IgG from the beads was performed by mixing the beads with 2 mls 0.1 M glycine (pH 2.7) for 1 min. This was spun (200 g, 5 min, 4°C) and the supernatant stored as the IgG fraction at -20°C. The amount of IgG was quantified using a direct IgG ELISA described in a previous paper [25].
Direct binding ELISA of aPL binding to purified recombinant his6-tagged DI
Nickel chelate-coated microwell plates, VH Bio (Gateshead, UK) were coated with 50 μl of recombinant purified his6-tagged DI and diluted to a concentration of 50 μg/ml using PBS. One half of the plate (the test wells) was coated with DI and the other half with PBS (the control wells). Plates were incubated at RT for 2 hours and were then washed three times with PBS, blocked with 100 μl of 0.25% gelatin, Sigma (Poole, UK) in PBS and incubated for a further 1 hour at RT. After washing the plates three times with PBS, 50 μl of a monoclonal human IgG aPL (IS4VH/IS4VL, IS4VH/B3VL or IS4VH/UK-4VL) derived from CHO cell culture supernatant [24] in sample, enzyme and conjugate dilution (SEC) buffer (100 mM Tris-HCl (pH 7), 100 mM NaCl, 0.02% Tween-20 and 0.2% BSA) was added at concentrations ranging from 9 ng/ml to 80 ng/ml. Serial dilutions of mAb were loaded such that for each dilution loaded in a test well, there was a corresponding control well loaded with the same dilution. For polyclonal IgG aPL purified from APS patients, SLE and normal controls, 50 μl of 20 μg/ml of IgG in SEC buffer was added to each well. Binding of human antibodies to the plate was detected by adding goat anti-human IgG alkaline phosphatase conjugate, Sigma (Poole, UK) diluted 1:2000 in SEC. After incubation at 37°C for 1 hour, bound antibody was detected by addition of alkaline phosphatase chromogenic substrate. The OD was measured in a Genios microplate autoreader (Tecan, Reading, UK). A net OD was calculated for each well to take into account background (OD test well – OD control well). Results of polyclonal IgG were expressed as a percentage binding of a standard APS patient sample known to bind DI, whole β2GPI and CL.
Competitive inhibition ELISA
A direct binding ELISA carried out as above was used to determine the concentration of native affinity purified antibody IS4VH/IS4VL required to achieve ~50% maximum binding. This concentration was 200 ng/ml. DI and β2GPI as test inhibitors were diluted in PBS at concentrations ranging from 0 (i.e. no inhibitor) to 30 μM. Affinity-purified IS4VH/IS4VL was then added to each concentration of inhibitor to achieve 200 ng/ml final concentration of antibody for each sample. The samples were incubated at RT for 2 hours and then tested for binding to DI on an ELISA plate as described earlier for the direct ELISA above. The per cent inhibition for each concentration of inhibitor was determined from the following formula:
% inhibition=(A0-A/A0) × 100, where A is the OD from the well containing the inhibitor (corrected for background) and A0 is the OD from the well containing no inhibitor (corrected for background).