An ELISA method to estimate the mono ADP-ribosyltransferase activities: e.g in pertussis toxin and vaccines.

ADP-ribosyltransferase activities have been observed in many prokaryotic and eukaryotic species and viruses and are involved in many cellular processes, including cell signalling, DNA repair, gene regulation and apoptosis. In a number of bacterial toxins, mono ADP-ribosyltransferase is the main cause of host cell cytotoxicity. Several approaches have been used to analyse this biological system from measuring its enzyme products to its functions. By using a mono ADP-ribose binding protein we have now developed an ELISA method to estimate native pertussis toxin mono ADP-ribosyltransferase activity and its residual activities in pertussis vaccines as an example. This new approach is easy to perform and adaptable in most laboratories. In theory, this assay system is also very versatile and could measure the enzyme activity in other bacteria such as Cholera, Clostridium, E. coli, Diphtheria, Pertussis, Pseudomonas, Salmonella and Staphylococcus by just switching to their respective peptide substrates. Furthermore, this mono ADP-ribose binding protein could also be used for staining mono ADP-ribosyl products resolved on gels or membranes.

Development of an ELISA-Based Potency Assay for Inactivated Influenza Vaccines Using Cross-Reactive Nanobodies.

Inactivated vaccines are the main influenza vaccines used today; these are usually presented as split (detergent-disrupted) or subunit vaccines, while whole-virus-inactivated influenza vaccines are rare. The single radial immune diffusion (SRD) assay has been used as the gold standard potency assay for inactivated influenza vaccines for decades; however, more recently, various alternative potency assays have been proposed. A new potency test should be able to measure the amount of functional antigen in the vaccine, which in the case of influenza vaccines is the haemagglutinin (HA) protein. Potency tests should also be able to detect the loss of potency caused by changes to the structural and functional integrity of HA. To detect such changes, most alternative potency tests proposed to date use antibodies that react with native HA. Due to the frequent changes in influenza vaccine composition, antibodies may need to be updated in line with changes in vaccine viruses. We have developed two ELISA-based potency assays for group 1 influenza A viruses using cross-reactive nanobodies. The nanobodies detect influenza viruses of subtype H1N1 spanning more than three decades, as well as H5N1 viruses, in ELISA. We found that the new ELISA potency assays are sensitive to the nature of the reference antigen (standard) used to quantify vaccine antigens; using standards matched in their presentation to the vaccine type improved correspondence between the ELISA and SRD assays.

The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency.

The response to the 2009 A(H1N1) influenza pandemic has highlighted the need for additional strategies for intervention which preclude the prior availability of the influenza strain. Here, 18 single domain VHH antibodies against the 2009 A(H1N1) hemagglutinin (HA) have been isolated from a immune alpaca phage displayed library. These antibodies have been grouped as having either (i) non-neutralising, (ii) H1N1 restricted neutralising or (iii) broad cross-subtype neutralising activity. The ability to neutralise different viral subtypes, including highly pathogenic avian influenza (H5N1), correlated with the absence of hemagglutination inhibition activity, loss of binding to HA at acid pH and the absence of binding to the head domain containing the receptor binding site. This data supports their binding to epitopes in the HA stem region and a mechanism of action other than blocking viral attachment to cell surface receptors. After conversion of cross-neutralising antibodies R1a-B6 and R1a-A5 into a bivalent format, no significant enhancement in neutralisation activity was seen against A(H1N1) and A(H5N1) viruses. However, bivalent R1a-B6 showed an 18 fold enhancement in potency against A(H9N2) virus and, surprisingly, gained the ability to neutralise an A(H2N2) virus. This demonstrates that cross-neutralising antibodies, which make lower affinity interactions with the membrane proximal stem region of more divergent HA sub-types, can be optimised by bivalency so increasing their breadth of anti-viral activity. The broad neutralising activity and favourable characteristics, such as high stability, simple engineering into bivalent molecules and low cost production make these single domain antibodies attractive candidates for diagnostics and immunotherapy of pandemic influenza.