Nano-bodies

Heavy-chain antibodies (HCAbs) without light chains naturally exist in the blood of the Camelidae family of

animals.

The variable domain of HCAb, referred to as a nanobody (Nb or VHH), is currently the smallest, fully

functional antigen-binding fragment.

Their novel assembles in targeted research, which may bring novel perspectives for tumor-targeted diagnosis and therapy

IgG of Camel has 3 subclasses: IgG1 – IgG2 – IgG3

Nano-body characters and advantages

1- Tiny body has only a single domain for

antigen-binding with high affinity and very tightly

2-Has high activity to penetrate into cells and tissues thus,

Very important for intracellular Ag as Hepatitis –c

3-Highly resistance to acidity and alkalinity

e.g. Hilobacter pylori resist acidic PH in the submucosa of the stomach and first part of the duodenum,

that’s why it induce peptic and duodenal ulcers where nanbody can be used there to eliminate this bacteria.

4-VHH portion of Camel is similar to that of Human,

Act as transporter can be used to treat breast cancer, through preparation of specific nanbody against cancer cell,

Loading it with anticancer drug specific to breast cancer without immune interference.

5-Easily to bind with other molecules like antibiotics and/or anticancer drug.

6-As it can bind to and internalized by cerebrovascular endothelial cells are able to cross blood brain barrier and transmigrate into the tissues of CNS

Disadvantages

The tumor therapeutic efficacy of many Nbs is often limited as a result of poor

pharmacokinetics, mainly as a consequence of fast clearance from blood due to their small size.

Recently, they have been used as biomedical vehicles to target drugs to tumors where they can be engineered to form conjugates with

other molecules in order to gain additional function, or

coupled to the surface of drug delivery systems for effective tumor targeting.

Preparation of Nano-body

As Hepatitis-C virus has envelope protein V1- V2

1-Hyperimmunization: Inject V1- V2  3-5 times in mice

2-Collect peripheral blood

3-Separate T-lymphocytes, B-lymphocytes and plasma cells

4-RNA extraction: rupture of plasma cells and get its all RNA

5-cDNA: convert RNA into cDNA through RT-PCR

6-Apply PCR to VHH gene and constant area adhere to it

7-Apply nested PCR for amplification of VHH gene

8-Separate VHH gene and insert it into vector, e.g. Bacteriophage plasmid called phagmid

9-Gene expression of VHH, VHH extracted and purified

Nano bodies Uses

Therapy

A-Nbs as targeting vehicles in tumor targeting therapy

1-Nbs as targeting vehicles in conjugates

The monomeric behavior and small exon size (~400 bp) confer Nbs with the characteristic of easily being fused to partners,

Leading to the design of fusion constructs composed of Nbs and novel molecules represent a new class of antibody-based therapeutics

The Nbs will target the conjugate to its antigen; whereas the partner of the sdAb will launch an additional activity depending on its identity 

(2) Nbs as targeting vehicles in drug delivery system

Targeted delivery of drugs to tumor tissues is an effective strategy that can increase antitumor efficacy and reduce

toxicity by the avoidance of normal tissues

Application of sdAb fragments In diagnostic and immune-analytical methods

Due to its effective penetration into the tissue, make Nb good candidate for imaging probes used for monitoring of tumor, metastasis lesions etc.

Such immune-imaging probes are prepared by labeling Nbs with short life isotopes with,

 rapid antigen targeting and faster clearance from the blood compared to radilolabelled MAB

resulting in contrast enhancing imaging signal and reduced accumulation of radiolabelled fragment minimizing both liver affection or any other radiation burden.

The main limitation is the unspecific uptake by kidney and bladder

-Chromobodies: is the fusing Nb to fluorescent protein that expressed in the living cells as chromobodies offer the possibilities to trace intracellular Ag e.g viruses,

And to modulate protein function in the living cells.

Uses in immunoassay and biosensors

Due to smaller size and lower aggregation propensity enable immobilization of extremely high no of Nbs molecules on the surface of,  the well of microtiter plate or biosensor transducer leading to substantial increase in sensitivity of immune detection system.

Help in:

Detection contamination and toxic in foods

Diagnosis of serious human diseases