Llama Immunization for Anti-CD28 Nanobodies

EXAMPLE 35

Two llamas (No. 45 and No. 46) were immunized with 6 boosts (100 or 50 μg/dose at weekly intervals) of CD28-Fc fusion (R&D Systems, Minneapolis, MN, US), formulated in adjuvant Stimune (Cedi Diagnostics, the Netherlands), according to standard protocols. At week 4, sera were collected to define antibody titers against CD28 by ELISA. In short, 96-well Maxisorp plates (Nunc Wiesbaden, Germany) were coated with hCD28-Fc. After blocking and adding diluted sera samples, the presence of anti-CD28 Nanobodies was demonstrated by using rabbit anti-llama immunoglobulin antiserum and anti-rabbit immunoglobulin alkaline phosphatase conjugate. The titer exceeded 16000 for both animals.

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US11858997B2. Amino acid sequences that modulate the interaction between cells of the immune system (2024-01-02). Ablynx N.V. [BE]. Inventors: Guy Hermans [BE], Peter Verheesen [BE], Edward Dolk [NL], Hendricus Renerus Jacobus Mattheus Hoogenboom [NL], Michael John Scott Saunders [BE], Hans De Haard [NL], Renee de Bruin [NL].

Patent 2024

CD28-Fc fusion Maxisorp plates

Adjuvant Alkaline phosphatase Animals Anti immunoglobulin Antibody Antiserum Diagnostics Elisa Immunizations Llama Nanobodies Rabbit Sera

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Variable analysis

independent variables

Number of boosts (100 or 50 μg/dose at weekly intervals) of CD28-Fc fusion

dependent variables

Antibody titers against CD28 by ELISA

control variables

Standard protocols for immunization
Use of adjuvant Stimune

controls

Positive control: Sera from the two immunized llamas (No. 45 and No. 46)
Negative control: Not explicitly mentioned

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