Anti human igg antibodies

Nanoparticle suspensions (50 and 60 nm diameter) in PBS and all chemicals used in the assay preparation were purchased from Sigma Aldrich, Gillingham, UK; including 4-nitrobenzenethiol (NBT), anti-human IgG antibodies dissolved in PBS, human IgG, BSA (bovine serum albumin), casein, and Tween-20. The only exceptions were anti-MPT64 monoclonal antibodies and MPT64 protein (antigen), which were purchased from BBI Solutions, Portland, OR, USA, and Enogen, Cambridge, UK respectively. Microscope slides coated with gold film (100 nm thick layer, 99.9% purity) over a Cr (2–3 nm) layer were purchased from EFM Co., Salt Lake, UT, USA.

The ELISA assay for the binding of antibodies to collagen type I and II was performed using commercially available kits (CHONDREX, Inc., Redmond, WA, USA).
In the ELISA assay, for the detection of anti-fibronectin antibodies, plates (Immulon 2HB Thermo Scientific, Illkirch, France) were coated with 10 μg/ml of human fibronectin (SIGMA, St. Louis, MO, USA), and the tested antibodies were diluted in PBS 1% BSA and incubated overnight at 4°C. Plates were washed 3 times with PBS 1% Tween and one time with PBS alone. Alkaline phosphatase-labelled anti-human IgG antibodies were purchased from Sigma. IgG antibodies to Klebsiella pneumoniae were detected by ELISA using a bacterial extract adsorbed on the solid phase as described in detail elsewhere [23 (link)]. For the binding to recombinant Klebsiella pneumoniae, proline dipeptidase (MyBioSource, San Diego, CA, USA) plates were coated with 20 microgram/ml of recombinant protein in PBS. The test was then performed as described above. The binding to the other two Klebsiella pneumoniae-derived proteins was assessed using the two synthetic peptides (LFI and SET peptide) using DELFIA as described above.

Protein fractions separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were transferred to a nitrocellulose membrane according to the method described by Blake [38 (link)]. The membrane was blocked with Tris Buffered Saline with Tween 20 (TBST) 0.5% milk at room temperature for 2 hours, washed 5 times with TBST for 5 minutes, and cut into strips that were incubated for 1 hour with anti-His antibody (Sigma-Aldrich) at 1:10,000 or with a positive control, which consisted of a pool of hyperimmune sera from P. vivax-immune primates at 1:50. Strips were then incubated for 1 h with either alkaline phosphatase-conjugated anti-mouse IgG or anti-human IgG antibodies respectively (Sigma-Aldrich) at a dilution of 1:10,000 and were then washed as before. Finally, formation of immune complexes in the membrane was assessed visually by adding TMB or NBT-BCIP substrates (Sigma Aldrich) accordingly.