Fab Antibody Production Protocol and FAQs

Fab antibody (fragment antibody) refers to the part of the antibody molecule that can bind to the antigen. It consists of a constant region and a variable region on a complete light chain and a heavy chain. The Fab antibody retains the specific binding ability of the antibody. Compared with the full-length antibody, the Fab antibody has better tissue penetration ability and lower immunogenicity. In addition, Fab antibodies do not activate complements or trigger antibody-dependent cell-mediated cytotoxicity (ADCC) through Fc receptors due to the absence of Fc segments, which has advantages in some treatment scenarios.

The Principle of Fab Antibody Production

Enzymatic Extraction of Fab Fragment

The complete IgG antibody is cleaved at a specific site by a specific protease to selectively release the Fab fragment:

Papain

In the presence of reducing conditions (e.g., in the presence of cysteine), the hinge region of IgG is cleaved to generate two Fab fragments (each containing a complete light chain and a partial heavy chain) and one Fc fragment.

Pepsin

IgG is usually cleaved into F (ab ') 2 fragments (two Fabs are linked by disulfide bonds) and degraded Fc fragments under non-reducing conditions, and further reduction is required to obtain a single Fab.

Production of Fab Fragment by Recombinant Expression

The expression vector carrying Fab gene was constructed by genetic engineering, and the Fab fragment was expressed and assembled by host cells (such as E.coli and mammalian cells):

Gene design

The antibody heavy chain (VH + CH1) and light chain (VL + CL) genes were cloned respectively, and the signal peptide (such as pelB guided secretion to the periplasmic cavity), disulfide bond matching, tag (such as His-tag) were introduced to assist purification.

Co-expression system

The light chain and the heavy chain are expressed at the same time, and the Fab is correctly folded by disulfide bond and hydrophobic interaction.

Advantages of Fab Antibody

Small size

Fab antibodies are smaller than full-length antibodies, which allows them to more easily penetrate tissues, including tumor tissues. This permeability is particularly advantageous for the diagnosis and treatment of deep tissue lesions.

Rapid clearance

Due to its small size, Fab antibodies typically have a shorter half-life in the body, which can reduce potential side effects and provide higher contrast during the diagnostic process. In addition, rapid clearance can also help reduce the risk of immunogenicity.

Specificity and affinity

Despite its small size, Fab antibodies still retain specificity and high affinity for binding to antigens. This makes them highly promising for applications that require high specificity recognition, such as targeted drug delivery.

Production flexibility

Fab antibodies can be produced through microbial systems such as bacteria or yeast, and their production cost is lower and speed is faster compared to mammalian cell culture that relies entirely on antibodies. In addition, Fab antibodies can be modified through genetic engineering techniques to enhance their stability and functionality.

The Steps of Fab Antibody Production

Preparation of Fab antibody by Enzyme Digestion

Antibody purification

IgG antibodies were purified from cell culture supernatants or serum using a protein A / G column. IgG was collected after binding and elution.

Enzyme digestion

The IgG solution was mixed with papain and incubated under optimized buffer conditions (such as PBS at pH 7.0). The digestion time and temperature need to be optimized according to the specific antibody and experimental conditions, generally at 37 ° C for 1-4 hours.

Separation and purification

SDS-PAGE was used to detect the digestion effect to ensure that the Fab segment was intact without breaking. The Fab fragment was isolated from the digestion mix by ion exchange or size exclusion chromatography.

Quality detection

SDS-PAGE, Western blot or mass spectrometry were used to confirm the quality of the purified Fab fragment. The binding activity of Fab was detected by ELISA or surface plasmon resonance (SPR).

Figure 1. Schematic representation of mab papain digestion products Fab and Fc antibody fragments. (Reference source: Purification of Fab fragments from a monoclonal antibody papain digest by Gradiflow electrophoresis.）

Preparation of Fab Antibody By Genetic Engineering

Gene cloning

The genes of the light chain and heavy chain variable regions (VL and VH) of the target antibody were extracted. The two gene sequences were amplified by PCR and the appropriate linker peptide gene was added.

Vector construction

VL and VH genes were inserted into the expression vector. Ensure that the vector has a promoter and selection marker suitable for the target host.

Expression and screening

The recombinant vector was transferred into a suitable host cell (such as E.coli or yeast). Fusion Fab antibody was induced to express, and the display of Fab was screened by nylon membrane.

Separation and purification

Fabs can be purified from culture supernatants or cell extracts using Ni-NTA affinity chromatography (if the Fab is His-tagged). Temperature elution and recovery of Fab antibody.

Performance verification

Functional evaluation of Fab, including antigen binding test (such as ELISA, SPR).

application of Fab antibodies

Medical diagnosis

Fab antibodies are widely used in imaging diagnosis, such as PET scanning and SPECT scanning. Their fast clearing feature allows for high contrast imaging to be achieved in a short period of time, thereby improving diagnostic accuracy.

Therapeutic use

In the fields of cancer, infectious diseases and autoimmune diseases, Fab antibody shows a good application prospect as a single therapy or combined with other treatments.

Research tools

In the fields of molecular biology and biotechnology, Fab antibodies serve as research tools for identifying and capturing target proteins, providing strong support for basic research and drug development.

Biosensors

Due to the high specificity and flexibility of Fab antibodies, they are used to develop biosensors for detecting pollutants in the food industry and chemical pollutants in environmental science.

Alpha Lifetech offers Fab antibody production testing specifically designed to meet the complex needs of the biotechnology industry. Our detection method has been carefully designed to ensure the high yield and purity of the Fab fragment, providing researchers with a reliable antibody development tool.

FAQ

1. When using the enzyme digestion method, the common problem is that the incomplete enzyme digestion leads to low yield of Fab segments, or excessive digestion damages the Fab segments.

Solution : Optimize the type, concentration, incubation time and temperature of the enzyme. The optimal digestion conditions can be determined by gradient experiments to ensure the acquisition of high-purity Fab segments.
2. Both enzyme digestion and genetic engineering methods may have the problem of low Fab antibody production.

Solution : For the enzyme digestion method, check the purity and concentration of IgG, and optimize the digestion conditions. For genetic engineering, it may be necessary to optimize the expression system, including increasing the number of vector replication, increasing the promoter strength, or changing the host strain (such as switching from E.coli to yeast expression).
3. The prepared Fab antibody could not effectively bind to the target antigen.

Solution : Evaluate and improve the accuracy of the Fab design to ensure the correctness of the cloned variable region sequence. At the same time, the Fab recombinant expression process was optimized, such as adjusting the folding conditions and purification methods to avoid the removal of key epitopes.
4. In the purification process, Fab antibody may be contaminated by other proteins.

Solution : Increase the accuracy and selectivity of purification steps, such as combining size exclusion chromatography and ion exchange, while using high-performance affinity chromatography. If labelled, metal chelate affinity chromatography (IMAC) can be used to improve purity.
5. Host cells can not effectively express Fab antibodies.

Solution : Try different types of host cells (such as E.coli, Pichia pastoris, CHO cells), and optimize the culture conditions, such as temperature, nutrients, inducer concentration, etc.

reference

[1] Mizote Y, Masumi-Koizumi K, Katsuda T, Yamaji H. Production of an antibody Fab fragment using 2A peptide in insect cells. J Biosci Bioeng. 2020;130(2):205-211. doi:10.1016/j.jbiosc.2020.03.009

[2] Hussain H, Patel T, Ozanne AMS, et al. A comparative analysis of recombinant Fab and full-length antibody production in Chinese hamster ovary cells. Biotechnol Bioeng. 2021;118(12):4815-4828. doi:10.1002/bit.27944

[3] Ohmuro-Matsuyama Y, Mori K, Hamada H, Ueda H, Yamaji H. Electrostatic engineering of the interface between heavy and light chains promotes antibody Fab fragment production. Cytotechnology. 2017;69(3):469-475. doi:10.1007/s10616-016-9955-4

[4] Ohmuro-Matsuyama Y, Mori K, Hamada H, Ueda H, Yamaji H. Electrostatic engineering of the interface between heavy and light chains promotes antibody Fab fragment production. Cytotechnology. 2017;69(3):469-475. doi:10.1007/s10616-016-9955-4

[5] Valedkarimi Z, Nasiri H, Aghebati-Maleki L, Abdolalizadeh J, Esparvarinha M, Majidi J. Production and characterization of anti-human IgG F(ab')2 antibody fragment. Hum Antibodies. 2018;26(4):171-176. doi:10.3233/HAB-180336

[6] Nasiri H, Valedkarimi Z, Aghebati-Maleki L, et al. Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G. Vet Res Forum. 2017;8(4):307-312.

[7] Coleman L, Mahler SM. Purification of Fab fragments from a monoclonal antibody papain digest by Gradiflow electrophoresis. Protein Expr Purif. 2003;32(2):246-251. doi:10.1016/j.pep.2003.07.005