A first WHO reference reagent for the detection of anti-human platelet antigen-15b.

Background And Objectives: Alloantibodies to human platelet antigen-15b (anti-HPA-15b) have been detected in mothers with foetal-neonatal alloimmune thrombocytopenia and in multiply transfused patients. Assays used to detect this antibody, which aids in disease diagnosis, can be unreliable and vary in sensitivity. The objective was to generate a stable, lyophilized anti-HPA-15b preparation and evaluate its suitability as a World Health Organization (WHO) reference reagent for use in the quality control of platelet alloantibody detection assays.

The third international standard for anti-D immunoglobulin: international collaborative study to evaluate candidate preparations.

Background And Objectives: The purpose of the study was to evaluate a lyophilized anti-D immunoglobulin preparation to serve as a replacement WHO International Standard for the calibration of potency assays of anti-D immunoglobulin products. Such products are used to prevent haemolytic disease of the foetus and newborn due to maternal alloanti-D.

Materials And Methods: The candidate 3rd International Standard for anti-D immunoglobulin (16/332) was evaluated and calibrated against the 2nd International Standard for anti-D immunoglobulin (01/572), along with a coded duplicate, a second candidate preparation (16/278) and a comparability sample (16/272) in an international collaborative study.

An autologous endothelial cell:peripheral blood mononuclear cell assay that detects cytokine storm responses to biologics.

There is an urgent unmet need for human tissue bioassays to predict cytokine storm responses to biologics. Current bioassays that detect cytokine storm responses in vitro rely on endothelial cells, usually from umbilical veins or cell lines, cocultured with freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy adult volunteers. These assays therefore comprise cells from 2 separate donors and carry the disadvantage of mismatched tissues and lack the advantage of personalized medicine.

Antibody C region influences TGN1412-like functional activity in vitro.

The unexpected outcome of the clinical trial of the superagonistic CD28 mAb TGN1412 (IgG4κ) continues to stimulate interest. We show that TGN1412 binds similarly to human and cynomolgus macaque FcγR, eliminating the possibility that differences in Fc-mediated interactions with FcγR contributed to the failure of preclinical testing in macaques to predict toxicity in humans. The influence of the Fc domain and C region structure on the in vitro functional activity of TGN1412 was investigated using F(ab')(2) and Fab fragments derived from TGN1412 recovered from the trial and recombinant TGN1412 subclass variants and mutants.

Endothelial cells co-stimulate peripheral blood mononuclear cell responses to monoclonal antibody TGN1412 in culture.

The failure of preclinical testing to predict the severity of the cytokine storm experienced by the recipients of the superagonistic anti-CD28 monoclonal antibody (mAb) TGN1412 during its Phase 1 clinical trial prompted the development of new in vitro experimental approaches for mimicking in vivo cytokine release and lymphoproliferation. Peripheral blood mononuclear cells (PBMC) presented to TGN1412 immobilised on plastic has previously been shown to stimulate a pro-inflammatory cytokine response. The aim of the present study was to investigate a 'co-culture' model for the detection of TGN1412-like immunomodulatory activity in which TGN1412 was presented to PBMC in the presence of monolayers of endothelium-derived cells and other cell types, followed by measurement of cytokine levels in the culture supernatants and proliferation of PBMC.

A WHO reference reagent for the Serum Transferrin Receptor (sTfR): international collaborative study to evaluate a recombinant soluble transferrin receptor preparation.

Background: The usefulness of serum transferrin receptor (sTfR) as a marker of iron deficiency is limited by lack of standardization of commercial immunoassays for sTfR. An international collaborative study was performed to evaluate a lyophilized preparation of recombinant soluble transferrin receptor (rsTfR) for its suitability to serve as a World Health Organization (WHO) Reference Reagent to standardize immunoassays for sTfR.

Methods: The concentration of pure rsTfR was determined from the A(280 nm) using the adjusted theoretical extinction coefficient and molecular weight calculated from its sequence, before dilution and lyophilization in a sTfR-depleted serum matrix.

Improved in vitro methods to predict the in vivo toxicity in man of therapeutic monoclonal antibodies including TGN1412.

TGN1412 is a "superagonistic" CD28 monoclonal antibody (IgG4) that caused serious adverse events at its first time in human clinical trial. In the present study, different in vitro methods for detecting and quantifying unwanted pro-inflammatory activity of therapeutic monoclonal antibodies (mAbs) such as TGN1412 are described. The antibody of interest is immobilised by wet-coating or air-drying onto polypropylene or polystyrene 96-well plates prior to the addition of human peripheral blood mononuclear cells (PBMCs).