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Title: Nanobodies as potential tools for microbiological testing of live biotherapeutic products

Journal Article · · AMB Express
ORCiD logo [1];  [2];  [1];  [1];  [1];  [1];  [1];  [1];  [1];  [1]
  1. US Department of Agriculture (USDA), Silver Spring, MD (United States)
  2. Univ. of Georgia, Athens, GA (United States)
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Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.

Research Organization:
National Energy Technology Laboratory (NETL), Pittsburgh, PA, Morgantown, WV, and Albany, OR (United States)
Sponsoring Organization:
USDOE Oak Ridge Institute for Science and Education (ORISE)
OSTI ID:
2471196
Journal Information:
AMB Express, Journal Name: AMB Express Journal Issue: 1 Vol. 14; ISSN 2191-0855
Publisher:
SpringerCopyright Statement
Country of Publication:
United States
Language:
English

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Nanobodies as potential tools for microbiological testing of live biotherapeutic products collection January 2024

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60 APPLIED LIFE SCIENCES
Biologics
Lactobacillus
Live biotherapeutic products
Nanobody
Potency assays