Anti human igg antibody

Wild type (WT) and mutant AP33 MAbs were produced by transient transfection of HEK cells with expression vectors encoding the appropriate antibody heavy and light chain combinations as previously described^{29 (link)}. The mutagenesis was carried out on a human-mouse chimera comprising the variable region of AP33 combined with a human constant region, to enable testing of interaction with murine Ab2s. To measure the concentrations of WT and mutant AP33 MAbs, serially diluted IgG-containing media were added, with an appropriate IgG standard alongside, to microtitre plates coated with anti-human IgG antibody (Sigma I-9010; 1:10,000). Captured chimeric MAbs were detected with anti-human IgG HRP (Sigma A-0170; 1:10,000), followed by TMB. Equal amounts of WT and mutant AP33 MAbs were then captured onto microtitre plates coated with anti-human IgG antibody. Ab2 MAbs were added at 1 µg/ml and incubated for 1 h at RT. Bound Ab2 MAbs were detected with anti-mouse IgG HRP (Sigma A4416; 1:5000) followed by TMB. Absorbance was measured at 450 nm.

Serum samples were immediately prepared and stored at −80 °C until use. Plasma samples were diluted 1:1 with PBS before freezing at −80 °C. Recombinant His-YB-1 (2.5 µg), Flag-YB-1 (1.5 µg) and GST-YB-1 proteins (FL 0.5µg; ∆1 0.5 µg; ∆2 0.5 µg; ∆3 0.5 µg; ∆4 1 µg; ∆5 1 µg; ∆8 1 µg; ∆9 1.5 µg) were used as antigens. After electrophoresis, proteins were blotted onto nitrocellulose membranes and blocked with 2.5% dry milk in TBST for 1 h at room temperature. Blots were probed with human serum samples diluted 1:200 in TTBS and incubated overnight at 4 °C. Bound antibodies were detected with secondary anti-human-IgG antibody (Sigma-Aldrich; 1:5000) and visualized with peroxidase-conjugated anti-mouse IgG antibody (GE Healthcare, Buckinghamshire, UK; 1:2000) using the ECL-system (Thermo Fisher Scientific) on an Intas gel imaging system (Intas, Göttingen, Germany). Densitometry readings were performed with LabImage (Kapelan, Leipzig, Germany).

For the ELISA assays, each well of 96-well Nunc Immobilizer^TM microtitration plates (Thermo scientific) was coated with 1 μg of one of the peptides diluted in Bicarbonate/carbonate coating buffer (100 mM) pH 9.6. The plates were then washed 3 times with PBS-Tween 0.2% (PBS-T) and blocked with skim milk at 2% in PBS-T for 1 h at 37 °C. They were subsequently washed 3 times with PBS-T and incubated for 45 min at 37 °C with 100 μl of a 1:200 solution of primary antibody diluted in PBS. Thereafter, the plates were washed 3× with PBS-T and then incubated for 30 min at 37 °C with a 1:1000 solution of secondary anti-human IgG antibody (Sigma-Aldrich) and/or anti-mouse IgG and IgM (Sigma-Aldrich), all horseradish peroxidase-labeled in 100 μl of PBS. After incubation with the secondary antibodies, the plates were washed 4× and O-phenyl-diaminobenzidine plus 30% H₂O₂ (1 μl/ml) (Sigma-Aldrich) was added to 0.05 M phosphate-citrate buffer, pH 5.0 as a peroxidase substrate before further incubation for 15 min at 27 °C. The reaction was stopped with a solution of 0.1 M 2 N H₂SO_4, and absorption measured at 492 nm in an ELISA Multiskan spectrum reader (Thermo). Sera from healthy individuals living in the same areas of Chile and Panama or mouse preinfected sera were used to calculate the cut-off value using the formula: median + 3 × SD of the negative samples.

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.