Human igm

Alkaline phosphatase-conjugated rabbit antibody to the 39/93 kDa LDB antigens (Strategic Biosciences, Stow, MA, USA) diluted in human serum was used as a calibration control in both TBRF and LD IBs and bands with lower intensity than the calibration control were considered to be negative [34 (link)]. The two proteins used as controls in both IBs were a mixture of human IgM and IgG (Sigma, St. Louis, MO, USA) for detecting the addition of alkaline phosphatase conjugated anti-human antibodies (C1), and Protein L (Sigma, St. Louis, MO, USA) for detecting the addition of human serum (C2), as previously described [34 (link)].
Immune rabbit sera for use as a marker control in TBRF IBs were prepared by immunizing individual rabbits separately with each of the recombinant antigens from the different RFB species used in TBRF IBs (Pacific Immunology, Ramona, CA, USA). The rabbit sera were then pooled and used at a dilution of 10⁻⁴ in a single control IB strip. Alkaline phosphatase-labelled goat anti-rabbit whole IgG (KPL, Gaithersburg, MD, USA) was used as the secondary antibody.

Immulon 4 HBX plates were coated with 50 μl of human IgM (Sigma) at 1.6 μg/ml in 0.1 m carbonate buffer (pH 9.6) overnight at 4 °C. Wells were blocked with PBS containing 1% BSA (w/v) for 2 h at room temperature. Serum was diluted 1/100 in GVBSCaMg or GVBSMgEGTA, added to plates, and incubated for 30 min at 37 °C. IgM-activated C3d deposition was detected as described for C3 activation. To examine the effects on classical pathway activity, ES-62 was added at 0.1 μg/ml with or without CRP for 30 min at 37 °C prior to addition to the IgM-coated plates.

Anti-PC and anti-MDA were extracted as previously described^{15 ,26 (link)}. Briefly, PC-BSA and MDA-HAS, 1 mg/mL was coupled to Hitrap NHS column (GE Healthcare, Sweden). Human IgM (Sigma Aldrich, Israel) was passed through Sepharose column coupled with PC-BSA and MDA–HSA. Unbound IgM considered as non-anti-MDA or non-anti-PC (mentioned as flow through, FT) was collected by washing the columns with binding buffer followed by elution with 0.1 mol/L glycine–HCl, elution buffer. The eluted antibodies were desalted in PD-10 columns (GE Healthcare, UK) and concentrated by a Centriprep centrifugal filter (Millipore, Ireland). After filtration through a 0.22-lm filter (Sarstedt, Germany), extracted antibodies were stored at −20 °C.

As a C1q activator, human IgM (Sigma, St. Louis, MO, USA) was coated on 96-well plates at 2 µg/mL overnight at 4 °C, then blocked with 5% BSA in PBS at 37 °C for 2 h. On each well of the plates, 2 µg of C1q was added that had been pre-incubated with different amounts of rEmCRT (0, 2, 4 µg) or BSA (4 µg, as a comparison) in total volume of 100 µL for 2 h at 37 °C and incubated for 1 h at 37 °C. After washing with PBST, C1q-D diluted at 1:100 in 1 × Veronal buffer (VB, Lonza, Basel, Switzerland) containing 0.05% Tween-20 and 0.1% gelatin was added into each well for 1 h at 37 °C to finish the classical complement activation. NHS at dilution of 1:50 was used as a positive control. After being washed for three times with PBST, each well was added with 100 µL goat anti-human C4 mAb (1:1000 Abcam, Cambridge, UK) or rabbit anti-human C3 polyclonal antibodies (1:100, BOSTER Biological Technology Co., Ltd..., Chengmai, China) to determine C4 and C3 intermediate product of classical complement activation. HRP-conjugated rabbit anti-goat or goat anti-rabbit IgG (1:5000 or 1:1000, Affinity Biosciences, Liyang, China) was used as the secondary antibody and OPD (Beyotime Biotechnology, Shanghai, China) was used as the substrate.

Rosetting assays were performed using cultures with high rosetting rates (60–95%) and frequencies were assessed by counting 200 ethidium bromide‐stained IEs and noting those that had two or more erythrocytes adhering using wet slide preparations and fluorescence microscopy. To determine the role of F_c‐mediated IgM binding in rosetting, synchronous ring stage IEs were incubated overnight (37°C, 5% CO₂) with NHS (10%), IgM‐depleted NHS, IgM‐depleted NHS plus human IgM (Sigma; 4 mg ml⁻¹), Albumax II (10%) or Albumax II (10%) plus IgM (4 mg ml⁻¹). The following day the relative rosetting frequency of triplicate wells was assessed as described above. To examine the involvement of different HB3VAR06 domains in rosetting, synchronous HB3VAR06⁺ IEs were grown from the ring to the late trophozoite stages in 10% NHS in the presence of 1:20 dilutions of domain‐specific antisera followed by assessment of rosetting rates as above.

IgM concentrations in supernatants from all cell cultures were determined by ELISA. A calibration curve using human IgM 1.6–100 ng/mL (Sigma-Aldrich, St. Louis, MO, USA) was used to quantify results. All experiments were performed in duplo (medians were used for end result). Supernatants were diluted, if necessary, to fit within the measurements of the calibration curve. Measurements <1.6 ng/mL were considered negative. IgG3 concentrations were measured in the same way using an ELISA-kit with a calibration curve of 4.4–200 ng/mL (Affymetrix/eBioscience, Santa Clara, CA, USA).