Comparison of the Calibration Standards of Three Commercially Available Multiplex Kits for Human Cytokine Measurement to WHO Standards Reveals Striking Differences

Serum parameters as indicators for the efficacy of therapeutic drugs are currently in the focus of intensive research. The induction of certain cytokines (or cytokine patterns) is known to be related to the status of the immune response e.g. in regulating the TH1/TH2 balance. Regarding their potential value as surrogate parameters in clinical trials and subsequently for the assignment of treatment efficacy, the accurate and reliable determination of cytokines in patient serum is mandatory. Because serum samples are precious and limited, test methods—like the xMAP multiplex technology—that allow for the simultaneous determination of a variety of cytokines from only a small sample aliquot, can offer great advantages

Dual specificity antibodies using a double-stranded oligonucleotide bridge

AbstractThe covalent conjugation of oligonucleotides to antibody Fab’ fragments was optimized by using oligonucleotides modified with a hexaethylene linker arm bearing three amino groups. One oligonucleotide was coupled to antibody of one specificity and a complementary oligonucleotide to antibody of a second specificity. The antibodies were then allowed to hybridize by base pairing of the complementary nucleotide sequences and the generation of bispecific antibody was analyzed on SDS-PAGE and confirmed using BIAcore analysis. The strategy of complementary oligonucleotide-linked bispecific molecules is not limited to antibodies but is applicable to linking any two molecules of different characteristics

The Human Chorionic Gonadotropin-β Arginine 68 to Glutamic Acid Substitution Fixes the Conformation of the C-Terminal Peptide

Wild-type human chorionic gonadotropin (hCG) has been used as a contraceptive vaccine. However, extensive sequence homology with LH elicits production of cross-reactive antibodies. Substitution of arginine68 of the β-subunit (hCGβ) with glutamic acid (R68E) profoundly reduces the cross-reactivity while refocusing the immune response to the hCG β -specific C-terminal peptide (CTP). To investigate the molecular basis for this change in epitope usage, we immunized mice with a plasmid encoding a truncated hCG β-R68E chain lacking the CTP. The animals produced LH-cross-reactive antibodies, suggesting that the refocused immunogenicity of R68E is a consequence of epitope masking by a novel disposition of the CTP in the mutant rather than a structural change in the cross-reactive epitope region. This explanation was strongly supported by surface plasmon resonance analysis using a panel of anti-hCGβ-specific and anti-hCGβ/LH cross-reactive monoclonal antibodies (mAbs). Whereas the binding of the LH cross-reactive mAbs to hCGβ-R68E was eliminated, mAbs reacting with hCGβ-specific epitopes bound to hCGβ and hCGβ-R68E with identical affinities. In a separate series of experiments, we observed that LH cross-reactive epitopes were silent after immunization with a plasmid encoding a membrane form of hCGβ-R68E, as previously observed with the soluble mutant protein itself. In contrast, the plasmid encoding the soluble secreted form of hCGβ-R68E evoked LH cross-reactive antibodies, albeit of relatively low titer, suggesting that the handling and processing of the proteins produced by the two constructs differed

Inhibition of HIV virus by neutralizing Vhh attached to dual functional liposomes encapsulating dapivirine

Although highly active antiretroviral therapy (HAART) has greatly improved the life expectancy of HIV/AIDS patients, the treatment is not curative. It is a global challenge which fosters an urgent need to develop an effective drug or neutralizing antibody delivery approach for the prevention and treatment of this disease. Due to the low density of envelope spikes with restricted mobility present on the surface of HIV virus, which limit the antibody potency and allow virus mutation and escape from the immune system, it is important for a neutralizing antibody to form bivalent or multivalent bonds with the virus. Liposome constructs could fulfil this need due to the flexible mobility of the membrane with its attached antibodies and the capacity for drug encapsulation. In this study, we evaluated the neutralization activity of a range of liposome formulations in different sizes coated with anti-gp120 llama antibody fragments (Vhhs) conjugated via either non-covalent metal chelation or a covalent linkage. The non-covalent construct demonstrated identical binding affinity to HIV-1 envelope glycoprotein gp120 and neutralizing ability for HIV virus as free Vhh. Although covalently linked Vhh showed significant binding affinity to gp120, it unexpectedly had a lower neutralization potency. This may be due to the comparability in size of the viral and liposome particles restricting the number which can be bound to the liposome surface so involving only a fraction of the antibodies, whereas non-covalently attached antibodies dissociate from the surface after acting with gp120 and free the remainder to bind further viruses. Covalently conjugated Vhh might also trigger the cellular uptake of a liposome-virion complex. To explore the possible ability of the antibody-coated liposomes to have a further function, we encapsulated the hydrophobic antiviral drug dapivirine into both of the non-covalently and covalently conjugated liposome formulations, both of which revealed high efficacy in reducing viral replication in vitro. Thus, dual function liposomes may lead to a novel strategy for the prophylaxis of HIV/AIDS by combining the neutralizing activity of Vhh with antiviral effects of high drug concentrations

POKOK-POKOK ILMU KEKEBALAN

ix,362 hlm ; 14 x 21 c