Obtaining single-chain antibodies (HCAb)
in camels, alpacas, llamas and sharks

Creation of producer strain of nanoantibodies/ nanobodies / sdAb

Single-chain antibodies (single-domain antibody, sdAb) containing only heavy chains were accidentally found in representative members of the Camelid family (camels, llamas, alpacas) and Shark superorder. A variable region of these antibodies consists only of one domain of 13–15 kDa and shows all properties of classical full-length IgG antibodies. Nanoantibodies are very effectively reproduced in bacterial cells of E. coli thanks to their small size, high hydrophilic nature and thermal stability. Having selected a required sdAb variant from an immunized camel Camelus dromedarium (Double-humped camel) or shark Scyliorhinus canicula (Morgy), we produce VHH and VNAR antibody fragments using a method of phage display with subsequent biopanning.

We produce VHH fragments of camelid, llama, alpaca antibodies, VNAR fragments of shark antibodies

Use of nanoantibodies

Use in pharmacology

Nanoantibodies have an enhanced penetration degree to tissues as compared to classical antibodies.
Nanoantibodies are very stable under conditions of changing temperature and chemical medium,
have a great stability potential in the gastrointestinal tract.
Nanobodies are extraordinarily flexible for subsequent engineering.
Nanoantibodies are characterized by low cost and are easily produced in the industry.
Nanoantibodies became widely used in personalized radionuclide theranostics (therapy and diagnostics)
of cancer diseases. They are used as radiopharmaceuticals for the target delivery of therapeutic
or diagnostic isotopes to the tumor tissue.
In February 2019 FDA approved the first product Caplacizumab, a bivalent nanoantibody
for the treatment of acquired thrombotic thrombocytopenic purpura.

Use in the development of IEA and ICA reagent kits

The use of single-chain antibodies (single-domain antibodies, sdAb) is a rapid and effective alternative
to the production of hybridomas and classical monoclonal antibodies.

Use in science

Fast and inexpensive production of nanoantibodies and their conjugates with fluorescent agents used in cell biology.
The cost and terms of nanoantibody production depend on a specific project and are discussed individually.
The cost and terms of nanoantibody production depend on a specific project and are discussed individually. Contact us now to discuss your project.

Procedure for obtaining VHH fragments of antibodies frpm camel, llama, alpaca

1.

Obtaining the antigen

In order to obtain VHH to a specific target, we immunize a camel or shark with a required antigen. We accumulate a respective antigen in mammalian cells HEK-293 or bacteria E.coli after obtaining an amino acid sequence of the antigen (with its possible modification to lower toxicity, increase the output and facilitate the purification).

2.

Immunization of an animal and obtaining lymphocytes

We immunize a double-humped camel successively with recombinant proteins, peptides or cells starting from small doses, while increasing the dose gradually two times at each subsequent immunization. We use a specialized adjuvant, which is able to cause the high antigen immunogenicity (but without excessive reactogenicity) as an adjuvant. We perform immunization with a one-week or two-week interval while checking the increase of AB titer to a target AG by testing the camel blood serum using the method of direct enzyme-linked immunosorbent assay. We take 150–200 ml of the blood from the camel and isolate lymphocytes from the serum after achieving a required antibody titer and obtaining the hyperimmune serum.

3.

Obtaining the bacteriophage library and biopanning

We obtain cDNA based on RNA isolated from blood lymphocytes of immunized camels. Then we use nested PCR to amplify different sequences of VHH antibody fragments and at the next stage we clone them in a phagemid vector. The final phagemids are accumulated in E. coli cells, than the cells are infected with a bacteriophage. As a result of translation the obtained VHH fragments are merged with p3 protein of M13 phage envelope and then are exposed on the surface of the bacteriophage capsid. The obtained phage library can be subjected to several biopanning rounds to select VHH fragments, which are the most affine to certain antigen.

4.

Obtaining the producer strain and isolation of antibodies

We perform sequencing of the cloned sequence in the selected phagemid after biopenning. We optimize codons in the obtained nucleotide sequence of the target VHH antibody fragment and clone it into an expression vector to accumulate the protein in E. coli strains. We transform the strains with a final plasmid and perform screening in order to determine a suitable producer strain of each specific protein. Then we optimize the culturing and induction conditions of the bacterial culture to obtain the maximum output of the target VHH antibody fragment in the soluble form. Then we scale up the production and accumulate large volumes of the producer strain in the fermenter for subsequent purification of VHH fragments from the wet cell biomass. We isolate a highly purified camelid nanoantibody product from the biomass of the producer strain cells using a whole complex of chromatographic methods.

5.

Analysis of antibodies

Analysis (quality control) of isolated and purified recombinant VHH antibodies is carried out using the following methods:

  • polyacrylamide gel disc electrophoresis under denaturing reducing and non-reducing conditions to determine VHH antibody purity;
  • ELISA to assess the residual protein content of the producer strain;
  • LAL-test for endotoxin content;
  • mass-spectrometric peptide mapping for identification of recombinant VHH antibodies;
  • different variants of immune enzyme assay for specificity of interaction with the antigen (target);
  • biolayer interferometry to determine the dissociation constant (Kd) of obtained antibodies;
  • differential scanning fluorimetry to determine thermal resistance and stability.

6.

Developing the laboratory procedure for nanoantibody production

We develop the experimental and industrial procedure based on the developed laboratory procedure for obtaining nanoantibodies with carrying out the quality control at key stages of the nanoantibody production.

Results of the stages of nanoantibody production

Immunization of an animal
Primer design scheme to obtain a phage library by the example of sdAb of Camelidae family.
Immunization of an animal
 - example 1
Primer design scheme to obtain a phage library by the example of sdAb of Camelidae family.
 - пример 1
Forming a producer strain of VHH antibodies
Forming a producer strain of VHH antibodies
Forming a producer strain of VHH antibodies
 - example 1
Forming a producer strain of VHH antibodies
 - пример 1
Purified VHH antibodies in non-reducing conditions
Purified VHH antibodies in reducing conditions
Purified VHH antibodies in non-reducing conditions
 - example 1
Purified VHH antibodies in reducing conditions
 - пример 1
Detection of the signal by the method of immune fluorescent analysis when a nanoantibody binds with a target protein on BT-474 cells.
No signal in control
Detection of the signal by the method of immune fluorescent analysis when a nanoantibody binds with a target protein on BT-474 cells.
 - example 1
No signal in control
 - пример 1

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