May 22, 2022

A competitive hemagglutination inhibition assay for dissecting functional antibody activity to influenza virus

  • Influenza remains one of the most contagious infectious diseases. Approximately, 25-50 million people suffer from influenza-like illness in the United States annually, leading to almost 1 million hospitalizations. Globally, the World Health Organization (WHO) estimates 250,000-500,000 mortalities associated with secondary respiratory complications due to influenza virus infection every year. Currently, seasonal vaccination represents the best countermeasure to prevent influenza virus spread and transmission in the general population. However, presently licensed influenza vaccines are about 60% effective on average, and their effectiveness varies from season to season and among age groups, as well as between different influenza subtypes within a single season.
  • The hemagglutination inhibition (HAI) assay represents the gold standard method for measuring the functional antibody response elicited following standard-of-care vaccination, along with evaluating the efficacy of under-development influenza vaccines in both animal models and clinical trial settings. However, using the classical HAI approach it is not possible to dissect the complexities of variable epitope recognition within a polyclonal antibody response. In this communication, we describe a straightforward competitive HAI-based method using a combination of influenza virus and recombinant hemagglutinin (HA) proteins to dissect the HAI functional activity of HA-specific antibody populations in a single assay format. Importance: The hemagglutination inhibition (HAI) assay is a well-established and reproducible method that quantifies functional antibody activity against influenza viruses and in particular the capability of an antibody formulation to inhibit the binding of HA to sialic acid.
  • However, the HAI assay does not provide with full insights on the breadth and epitope recognition of the antibody formulation, especially in the context of polyclonal sera where multiple antibody specificities contribute to the overall observed functional activity. In this report we introduce the use of Y98F point-mutated recombinant HA (HAΔSA) proteins, which lack sialic acid binding activity, in the context of the HAI assay as a mean to absorb out certain HA-directed (i.e., strain-specific or cross-reactive) antibody populations. This modification to the classical HAI assay, referred to as the competitive HAI assay, represents a new tool to dissect the magnitude and breadth of polyclonal antibodies elicited through vaccination or natural infection

Influence of adjuvants on the amount, specificity and functional activity of antibody response to human influenza vaccine in mice

It’s been almost a century since immunologists started using adjuvants as tools to develop more effective vaccines. Despite the rising number of adjuvanted vaccines in the last decades, we still lack knowledge of the adjuvants’ effects on antibody response. This study was aimed to test the effect of immunizing mice with the human Inactivated Influenza vaccine (IIV), either alone or combined with different widely used adjuvants on the specific antibody response induced. Differential levels of IgM and IgG subclasses were found with the different adjuvants tested. Higher levels of antibodies did not always correspond with a higher efficacy to interfere with the virus infectivity. Differences in neutralization properties are possibly mediated by the specificity of the repertoire of antibodies induced.
The repertoire was studied using a phage display 7-mer peptide library to screen for epitopes/mimotopes recognized by serum pools from vaccinated mice. The selected phage clones included peptides that corresponded to conformational mimotopes since they have no homology with lineal sequences of the Influenza strains’ proteins. Five peptides were identified as recognized by sera from mice immunized with the IIV vaccine alone, including peptides from the hemagglutinin stalk domain, and by sera from mice immunized with the vaccine plus the different adjuvants employed. Adjuvants elicited a more diverse repertoire of epitope-recognizing antibodies that recognized epitopes of the HA recombinant globular head. Mimotopes were theoretically located at the neutralizing antigenic sites of the globular head of Influenza A H1N1pdm09, Influenza A H3N2, and Influenza B hemagglutinin. This study illustrates how different adjuvants can modify the extent and quality of humoral immunity against the IIV vaccine and the effectiveness of vaccination.

Partial Degradation of Recombinant Antibody Functional Activity During Infant Gastrointestinal Digestion: Implications for Oral Antibody Supplementation

Oral administration of engineered immunoglobulins has the potential to prevent enteric pathogen-induced diarrhea in infants. To prevent infection, these antibodies need to survive functionally intact in the proteolytic environment of the gastrointestinal tract. This research examined both ex vivo and in vivo the functional survival across infant digestion of palivizumab, a model FDA-approved recombinant antibody against respiratory syncytial virus (RSV) F protein. Palivizumab-fortified feed (formula or human milk), infant gastric, and intestinal samples were incubated to simulate in vivo digestion (ex vivo digestion). Palivizumab-fortified human milk was also fed to infants, followed by collection of gastric and intestinal samples (in vivo digestion). Palivizumab was purified from the samples of digestate using protein G spin columns followed by filtration through molecular weight cut-off membranes (30 kDa).
Palivizumab functional survival across ex vivo and in vivo digestion was determined via an anti-idiotype ELISA and an RSV plaque reduction neutralization test. Palivizumab concentration and RSV neutralization capacity both decreased when incubated in intestinal samples (ex vivo study). The concentration and neutralization activity of orally-supplemented palivizumab also decreased across infant digestion (in vivo study). These results indicate that if recombinant IgGs were selected for oral supplementation to prevent enteric infections, appropriate dosing would need to account for degradation occurring in the digestive system. Other antibody formats, structural changes, or encapsulation could enhance survival in the infant gastrointestinal tract.

Mapping of Recognition Sites of Monoclonal Antibodies Responsible for the Inhibition of Pneumolysin Functional Activity

The pathogenicity of many bacteria, including Streptococcus pneumoniae, depends on pore-forming toxins (PFTs) that cause host cell lysis by forming large pores in cholesterol-containing cell membranes. Therefore, PFTs-neutralising antibodies may provide useful tools for reducing S. pneumoniae pathogenic effects. This study aimed at the development and characterisation of monoclonal antibodies (MAbs) with neutralising activity to S. pneumoniae PFT pneumolysin (PLY). Five out of 10 produced MAbs were able to neutralise the cytolytic activity of PLY on a lung epithelial cell line. Epitope mapping with a series of recombinant overlapping PLY fragments revealed that neutralising MAbs are directed against PLY loops L1 and L3 within domain 4. The epitopes of MAbs 3A9, 6E5 and 12F11 located at L1 loop (aa 454-471) were crucial for PLY binding to the immobilised cholesterol.
In contrast, the MAb 12D10 recognising L3 (aa 403-423) and the MAb 3F3 against the conformational epitope did not interfere with PLY-cholesterol interaction. Due to conformation-dependent binding, the approach to use overlapping peptides for fine epitope mapping of the neutralising MAbs was unsuccessful. Therefore, the epitopes recognised by the MAbs were analysed using computational methods. This study provides new data on PLY sites involved in functional activity.

Triple Functional Domain Protein (TRIO) Antibody

20-abx147592 Abbexa
  • 425.00 EUR
  • 342.00 EUR
  • 100 ug
  • 50 ug

MAT Activity Assay

IK00401 Arthus 96 tests 844 EUR

SOD Activity Kit

SKT-214-192 Stressmarq 2 plates of 96 wells 494 EUR

Catalase Activity Kit

SKT-215-192 Stressmarq 2 plates of 96 wells 454 EUR

Mouse Triple functional domain protein, Trio ELISA KIT

ELI-16599m Lifescience Market 96 Tests 865 EUR

Human Triple functional domain protein, TRIO ELISA KIT

ELI-17095h Lifescience Market 96 Tests 824 EUR

Recombinant Flavin Containing Monooxygenase 2, Non Functional (FMO2)

4-RPC482Hu01 Cloud-Clone
  • 440.48 EUR
  • 221.00 EUR
  • 1376.80 EUR
  • 525.60 EUR
  • 951.20 EUR
  • 358.00 EUR
  • 3292.00 EUR
  • 100 ug
  • 10ug
  • 1 mg
  • 200 ug
  • 500 ug
  • 50ug
  • 5 mg

Recombinant Flavin Containing Monooxygenase 2, Non Functional (FMO2)

4-RPC482Ra01 Cloud-Clone
  • 476.32 EUR
  • 230.00 EUR
  • 1511.20 EUR
  • 570.40 EUR
  • 1040.80 EUR
  • 382.00 EUR
  • 3628.00 EUR
  • 100 ug
  • 10ug
  • 1 mg
  • 200 ug
  • 500 ug
  • 50ug
  • 5 mg

Flavin Containing Monooxygenase 2, Non Functional (FMO2) Antibody

20-abx131038 Abbexa
  • 453.00 EUR
  • 133.00 EUR
  • 1302.00 EUR
  • 620.00 EUR
  • 342.00 EUR
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Flavin Containing Monooxygenase 2, Non Functional (FMO2) Antibody

20-abx132204 Abbexa
  • 425.00 EUR
  • 133.00 EUR
  • 1205.00 EUR
  • 578.00 EUR
  • 328.00 EUR
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Flavin Containing Monooxygenase 2, Non Functional (FMO2) Antibody

20-abx112558 Abbexa
  • 732.00 EUR
  • 398.00 EUR
  • 150 ul
  • 50 ul

Mouse Triple Functional Domain Protein (TRIO) ELISA Kit

abx390791-96tests Abbexa 96 tests 911 EUR

Human Triple Functional Domain Protein (TRIO) ELISA Kit

abx385496-96tests Abbexa 96 tests 911 EUR

Human Plasma Renin Activity(Plasma Renin Activity) ELISA Kit

QY-E05530 Qayee Biotechnology 96T 361 EUR

Sperm Antibodies ELISA kit

55R-IB79154 Fitzgerald 96 wells 440 EUR

Sperm Antibodies ELISA kit

55R-IB79155 Fitzgerald 96 wells 482 EUR

Sperm Antibodies ELISA kit

55R-IB79156 Fitzgerald 96 wells 440 EUR

Bovine mycobacterium bovis antibodies

QY-E60132 Qayee Biotechnology 96T 426 EUR

Rat Flavin Containing Monooxygenase 2, Non Functional (FMO2) Protein

20-abx168990 Abbexa
  • 662.00 EUR
  • 272.00 EUR
  • 2040.00 EUR
  • 787.00 EUR
  • 481.00 EUR
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Monoclonal Functional BAFF-R (mouse) Antibody, mAb , Clone: 9B9

APR16043G Leading Biology 0.1mg 528 EUR

Monoclonal Functional Rab1-GTP Antibody, mAb (recombinant), Clone: ROF7

APR16044G Leading Biology 0.1mg 528 EUR

Monoclonal Functional Rab6-GTP Antibody, mAb (recombinant), Clone: AA2

APR16045G Leading Biology 0.1mg 528 EUR

Human Flavin Containing Monooxygenase 2, Non Functional (FMO2) Protein

20-abx652195 Abbexa
  • 620.00 EUR
  • 272.00 EUR
  • 1859.00 EUR
  • 732.00 EUR
  • 453.00 EUR
  • 100 ug
  • 10 ug
  • 1 mg
  • 200 ug
  • 50 ug

Urease Activity Assay Kit

55R-2261 Fitzgerald 100 assays 689 EUR

Butyrylcholinesterase Activity Kit (Colorimetric)

K516-100 Biovision 664 EUR

Mitochondria Activity Assay Kit

KC310100 Biochain 1 kit 398 EUR

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity

Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region.
Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.

Leave a Reply

Your email address will not be published.