All methods used in immunochemistry rely on the antibody molecule or derivatives of it. Antibodies are incredibly useful molecules that can be designed to detect an almost limitless number of antigens. They are adaptable and will operate in many conditions. They can be used in both diagnostic and therapeutic scenarios. Antibodies can be made in various ways and the choice of which method to use is very much dependent on the final assay format. For an antibody to be of use, it has to have a defined specificity, affinity and avidity, as these are the qualities that determine its usefulness in the method to be used.

Polyclonal Antibody Production

Polyclonal antibodies are raised in appropriate donor animals, generally mice, rats or rabbits for smaller amounts and sheep or goats for larger quantities. It is important that animals are free of infection, as this would raise unwanted antibodies.

Usually, antigens are mixed with an appropriate adjuvant prior to immunising the animals. Adjuvants increase the immunogenicity of the antigen, thereby reducing the amount of antigen required, as well as stimulating specific immunity to it. Adjuvants may be detergents, oils or complex proprietary products containing bacterial cell walls or preparations of them. Prior to immunising an animal, pre-immune blood samples are taken such that the baseline IgG level can be appraised (Figure 1). Immunisations are spaced at intervals between 2 and 4 weeks to maximise antibody production. Blood samples are taken throughout the immunisation program and the serum is tested for specific activity to the antigen by enzyme-linked immunosorbent assay (ELISA) or other methods. Typically, a range of doubling serum dilutions are made (1/100–1/12 800) and tested against the antigen. Detection of antigen at 1/6400 dilution indicates high levels of circulating antibody. Western blot analysis of the serum can confirm the specificity of the antibodies produced. Once a high level of the desired antibody is detected in test bleeds, larger samples can be taken. Animal welfare legislation governs permissible amounts and frequency of bleeds. Blood donations are allowed to clot and the serum collected. Serum can be stored at 4 °C or lower for longer periods.

Fig1. Immunisation schedule.

Occasionally, antigens that give a poor response in mammals can give much higher yields in chickens. Chickens secrete avian immunoglobulin Y ( IgY) into their eggs to provide protection for the developing embryo. This can be utilised for effective poly clonal antibody production. The chickens are immunised three or four times with the antigen and the immune status is monitored by test bleeds. Eggs are collected and can yield up to 50 mg of antibody per yolk. The antibody has to be purified from the egg yolks prior to use and a number of proprietary kits can be used to do this.

Very pure polyclonal antibodies can be produced in rats and mice in ascitic fluid , which is the intra-peritoneal fluid extracted from mice with a peritoneal tumour. Non-secretory myeloma cells are injected into the peritoneal cavity and the growing tumour cells cause the animal to produce ascitic fluid which contains very high levels of the antibodies that the animal is currently secreting in its blood. Animals that are immunised with a particular antigen of interest will produce high titers of the respective polyclonal antibodies in the ascitic fluid, which can be removed by aspiration with a syringe and needle.

Monoclonal Antibody Production

Mice, rats or other rodents are the donor animal of choice for monoclonal antibody production, as they are low cost and easy to manage and handle. Balb/C is the mouse strain typically used for monoclonal antibody production and most of the tumour cell lines used for fusions are derived from this mouse.

Mice are immunised, usually three or four times over the course of 3–4 months, by the intraperitoneal route using antigen mixed with an appropriate adjuvant (Figure2). Samples from test bleeds can be taken to monitor the immune status of the animals. Once the mice are sufficiently immune, they are left for 2–3 months to rest. This is important as the cells that will be used for the hybridoma production are memory B cells and require the rest period to become quiescent.

Fig2. Monoclonal antibody production.

Mice are sacrificed and the spleens removed; a single spleen typically provides sufficient cells for two or three cell fusions. Three days prior to cell fusion, the partner cell line NS-0 is cultured to provide a log phase culture. For cell fusions, a num ber of methods are established, but one of the most commonly used is fusion by centrifugation in the presence of polyethylene glycol (PEG). Cells from spleen and fusion partner are mixed in a centrifuge tube, PEG is added to solubilise the cell membranes and the fusion is carried out by gentle centrifugation. In order to sup press functional interference, PEG levels are lowered by dilution with culture medium and clones are grown from single recombinant parent cells on microtitre wells. Cell lines used for fusion partners have a defective enzyme pathway and thus allow for selection after cell fusion. NS-0 cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase ( HGPRT), which prevents them from using a nucleoside salvage pathway when the primary pathway is disabled due to the presence of the antibiotic aminopterin. Accordingly, the tissue culture additive HAT, which contains hypoxanthine, aminopterin and thymidine is used to select for hybridomas after cell fusion. They inherit an intact nucleoside salvage pathway from the spleen cell parent, which allows them to grow in the presence of aminopterin. Unfused NS-0 cells are unable to assimilate nucleosides and die after a few days. Unfused spleen cells are unable to divide more than a few times in tissue culture and will die as well. Two weeks after the cell fusion, the only cells surviving under the above conditions are hybridomas. The immunisation process ensures that many of the spleen cells that have fused will be secreting antibody targeting the antigen used for immunisation; however, this cannot be relied upon and rigorous screening is required to ensure that the hybridomas selected are secreting an antibody of interest. Screening is often carried out by ELISA, but other antibody assessment methods may be used. It is important that hybridomas are assessed repeatedly as they can lose the ability to secrete antibody after a few cell divisions. This occurs as chromosomes are lost during division to return the hybridoma to its normal chromosome quota.

Once hybridomas have been selected they have to be cloned to ensure that they are truly monoclonal. Cloning involves the derivation of cell colonies from individual cells grown isolated from each other. In limiting dilution cloning, a cell count is carried out and cells are diluted into media, ensuring that only one cell is present in each well of the tissue culture plate. The plates are incubated for 7 days and cell growth assessed after this time. Colonies derived from single cells are then tested for antibody production by ELISA. It is desirable that a cell line should exhibit 100% cloning efficiency in terms of antibody secretion, but some cell lines are inherently unstable and will always produce a small number of non-secretory clones. Provided such cell lines are not subcultured excessively, this problem may not be substantial, though. Recloning these lines regularly ensures that cultures are never too far from an authenticated clone.

It is very important to know the antibody isotype of the hybridomas and a number of commercial kits are available to determine this. Most are based on lateral flow technology. Once the isotype of the antibody is established and it is clonally stable, cultures can be grown to provide both cell banks and antibody for use in testing or for reagent development. It is absolutely essential to record the pedigree of every cell line and to be vigilant in handling and labelling flasks to prevent cross-contamination of cell lines.

Cell Banking

Cell banks are established from known positive clones and are produced in a way that maximises reproducibility between frozen cell stocks and minimises the risk of cellular change ( Figure 3). A positive clone derived from a known positive clone is rapidly expanded in tissue culture until enough cells are present to produce 12 vials of frozen cells simultaneously. This master cell bank is stored at −196 °C under liquid nitrogen vapour. The working cell bank is derived from the master cell bank. One of the frozen vials from the master cell bank is thawed and rapidly grown until there are enough cells to produce 50 vials of frozen cells simultaneously. This is the working cell bank and it is also stored at −196 °C under liquid nitrogen vapour. This strategy ensures that all of the vials of the working cell bank are identical. Each of the 12 identical vials of the master cell bank will provide a new working bank, providing 550 further identical working vials before the process of deriving a new master cell bank by selecting a positive clone for expansion from the last master cell bank vial is required.

Fig3. Cell banking.

Growing Hybridomas for Antibody Production

 All monoclonal antibodies are secreted by growing hybridomas into the tissue culture media and a number of methodologies exist for optimising antibody production with respect to yield, ease of purification and cost. The simplest method for antibody production relies on static bulk cultures of cells growing in T flasks. T flasks are designed for tissue culture and have various media capacities and cell culture surface areas. For most applications, a production run requires between 250 ml and 1000 ml of medium. Most cell lines produce between 4 and 40 mg of antibody per litre. The cells from a working cell bank vial are thawed rapidly into 15 ml medium containing  10% fetal bovine serum and placed in an incubator at 37 °C supplemented with 5% CO 2 . Once cell division has started, the culture sizes are increased using medium supplemented with 5% fetal bovine serum until the desired volume is reached. Once the working volume has been reached, the cells are left to divide until all nutrients are utilised and cell death occurs; this takes about 10 days. The cell debris can then be removed by centrifugation and the antibody harvested from the tissue culture medium. For some applications, the antibody can be used in this form without further processing.

Monoclonal antibodies can also be produced in ascitic fluid in mice by injecting hybridoma cells into the peritoneum. The hybridoma cells grow to high densities and secrete high levels of the monoclonal antibody of interest into the ascitic fluid, which is harvested by aspiration with a syringe and needle. Nude mice have no T cells and thus possess a poor immune system. They are often used for ascitic fluid production as they do not mount an immune response to the implanted cells. The mice should be naïve , i.e. not be immunised prior to use, as it is important that the only antibody present in the ascitic fluid is derived from the implanted cells.

A number of in vitro bioreactor systems have been developed to produce high yields of monoclonal antibody in small volumes of fluid, which mimics ascitic fluid production (Figure 4). All of them rely on physically separating the cells from the culture medium by a semi-permeable membrane that allows nutrient transfer, but prevents monoclonal antibodies from crossing. The culture medium can be changed to maximise cell growth and health, and fluid can be removed from around the cells to harvest antibody. Some are based on a rotating cylinder with a cell-growing compartment at one end and separated from the media container by a membrane. Others have capillary systems formed from membrane running through the cell culture compartment and in these, the media is pumped through the cartridge to facilitate nutrient and gas exchange. These systems do produce high yields of antibody, but their set up and maintenance requires a significant effort. Nevertheless, they are ideal where large quantities of monoclonal antibody are needed and space is at a premium. They are, however, prone to contamination by yeasts and great care must be exercised when handling them. Cells are grown in bioreactors for up to 6 weeks, so the clone used must be stable and it is advisable to monitor its long-term growth prior to embarking on this form of culture. The major advantage of bioreactor cultures is that the antibody is produced in high concentration without the presence of media components, hence making it easy to purify. Total quantities per bioreactor run may be several hundred milligrams to gram quantities.

Fig4. Bioreactors for antibody production. (a) Rotating bioreactor utilising two compartments to separate growth medium and cells from antibodies. (b) Hollow fibre bioreactor utilising a capillary network to separate growth medium from cells and antibodies.

Antibodies Recognising Small Molecules

The immune system will recognise foreign proteins if they have a molecular mass above 2000–5000 Da. The magnitude of the response increases proportionally to the molecular mass. If an antibody is to be generated against a smaller molecule, it has to be conjugated to a carrier in order to effectively increase its size above the threshold for immune surveillance. A small molecule that is not antigenic by itself, but is eliciting an immune response when attached to a large carrier molecule is called a hapten . Carriers are macromolecules that bind these small haptens and enable them to induce an immune response. Most carriers are secretory or cell surface proteins that are naturally exposed to the immune system. Additionally, some polymers are known to act as carriers. If a polyclonal antibody is generated, it is advisable to change the carrier protein at least once in the immunisation procedure as this favours more antibody being made to the hapten and less to each of the carrier proteins. If a monoclonal antibody is generated, then the carrier protein can be the same throughout the immunisations. When screening hybridomas for monoclonal antibody production, it is necessary to screen against the hapten and carrier separately and to select antibodies responding to the hapten only.

Anti-Idiotypic Antibodies

An idiotope is the unique set of epitopes of the variable portion of an antibody. An antibody can have multiple idiotopes, which together form the idiotype of the anti body. If a new antibody is generated that specifically binds to an idiotope of a previously described antibody, it is termed an anti-idiotypic antibody. The binding site of an anti-idiotypic antibody is a copy of an original epitope. Some human cancers have unique cell surface tumour antigens that can be targeted for antibody therapy. Anti idiotypic vaccines contain mouse monoclonal antibodies with designated idiotopes, mimicking these antigen sequences and thus triggering an immune response against the tumour antigen. By way of example, the recently developed anti-idiotypic vaccine racotumomab acts as a therapeutic vaccine for the treatment of solid tumours.

Phage Display for Development of Antibody Fragments

Bacteriophage or, in short, phage, are viruses that infect and replicate within bacteria. They can be engineered by molecular methods to express proteins, and if the nucleotide sequence corresponding to the foreign protein is fused to the coat protein gene, the foreign protein will be expressed on the virus surface. It is possible to iso late the variable (V) antibody coding genes from various sources and insert these into the phage, resulting in single-chain antigen-binding (scFv) fragments . Whereas whole antibodies are too large and complex to be expressed by this system, the scFv fragments can be expressed and used for diagnostic purposes. The DNA used in this process may come from immunised mouse B cells. The V genes are cloned into the phage producing a library, which is then screened to isolate clones that specifically bind antigen immobilised onto a solid surface. During the library screen, the desirable clones bind to the antigen and those that do not bind (no recognition) are washed off. Bound clones are eluted and put through further screens. The repeated cycling of these steps results in a phage mixture that is enriched with relevant (i.e. binding) phage. This process is called panning , referring to the gold-panning technique used by nineteenth-century prospectors. Selected phages can be multiplied in their host bacterium, sequenced and the ‘best’ scFv fragments can subsequently be used to develop ELISA and other immunoassays.

Antibody Purification

 The choice of method used for the purification of antibodies depends very much on the fluid that they are in. Antibody can be purified from serum by the addition of chaotropic ions, typically ammonium sulfate. At around 60% saturation with ammonium sulfate, a precipitate is obtained that mainly contains antibody, thus providing a rapid method for IgG purification. This method does not work well in tissue culture supernatant, since media components such as ferritin are coprecipitated. However, ammonium sulfate precipitation may be used as a preparatory method prior to further chromatographic purification.

Prior to purification by affinity chromatography , the tissue culture supernatant is often concentrated, as a reduced volume simplifi es the process. Tangential fl ow devices and centrifugal concentrators may be used to reduce the volume to 10% of the starting amount ( Figure 5). Antibodies from both polyclonal and monoclonal sources can be purified by similar means. In both cases, the antibody type is IgG, which allows purification by protein A/G affinity chromatography. Protein A/G is a recombinant fusion protein that combines the IgG binding domains of both protein A and protein G. These proteins are derivatives of bacterial cells and the ability to reversibly bind IgG molecules forms the pivotal step in this affinity purification. Binding to the column occurs at neutral pH and the pure antibody fraction can be eluted at pH 2.0. Fractions are collected and neutralised to yield pH 7.0. Antibody-containing fractions are identified by spectrophotometry using absorbance at 280 nm (specific wavelength for protein absorbance) and are pooled. A solution of IgG at 1 mg cm −3 has an absorbance reading of approximately 1.4 at 280 nm. This can be used to calculate the amount of antibody in specific aliquots after purification.

Fig5. Affinity chromatography of antibodies using immobilised protein A/G.

Purified antibody should be adjusted to 1 mg cm −3 and kept at 4 °C, or −20 °C for long-term storage. Typically, 0.02% sodium azide is added to the antibody solution in order to increase shelf-life by suppressing the growth of adventitious microorganisms. Antibodies can be stored for several years at 4 °C, and for decades if kept below −20 °C, without losing activity.

Antibody Modification

For most of the common immunoassays, Western blotting, ELISA, immunofluorescence, immunohistochemistry and immunocytochemistry, as well as flow cytometry, there are numerous antibody labelling protocols for the detection and quantification of antigens. The label can be attached by one of two approaches:

• direct: the label is attached via a covalent bond to the primary antibody

• indirect: unlabelled primary antibody is bound to the antigen and a secondary labelled reagent is used for quantification.

Antibodies can be labelled by the addition of a marker enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Linkage is achieved by cross-linking chemistry (e.g. glutaraldehyde) to provide stable antibody–enzyme conjugates. Conjugation of an antibody to HRP is carried out in two stages. First, glutaraldehyde is coupled to reactive amino groups on the enzyme. The glutaraldehyde-tagged HRP is then purified by size-exclusion chromatography to remove unreacted cross-linker. In a second step, glutaraldehyde-tagged HRP is added to antibody solution, and the cross-linker reacts with amino groups on the antibody, thus forming a covalent link between the antibody and HRP. Alkaline phosphatase can be linked to the antibody by glutaraldehyde using a one-step conjugation.

A number of proprietary labelling reagents are also available for preparing anti body–enzyme conjugates. Fluorescent labels are required in immunofluorescent assays; here, fluorescein is usually the molecule of choice. Often, the derivative used for labelling is fluorescein isothiocyanate (FITC). In FITC, an isothiocyanate group (–N=C=S) is incorporated as a reactive group that readily couples to primary amines and thus yields fluorescent conjugates of antibodies.

The interaction of biotin and avidin or streptavidin is one of the strongest known non-covalent interactions between two proteins and has been exploited for use in many applications, including detection of proteins, nucleic acids and lipids, as well as protein purification. Biotin is a small molecule that when introduced to biologically active macromolecules, such as antibodies, in most cases doesn’t change activity. Streptavidin is a tetrameric protein, with each subunit binding one molecule of biotin. The bond is highly specific and very strong. Due to these properties, the streptavidin–biotin bridge as a very high affinity protein–ligand interaction and often utilised to link proteins in immunoassays. In typical applications, the antibody is conjugated to biotin, and streptavidin is conjugated to a fluorophore or enzyme, thereby providing signal amplification and increased sensitivity so that antigens that are expressed at low levels are more likely to be detected ( Figure 6).

Fig6. Streptavidin–biotin complex.

It is possible to link antibodies to gold particles for use in immunosorbent electron microscopy (ISEM) and lateral flow devices. Gold particles are prepared by citrate reduction of auric acid. The size of particle is predictable and can be controlled by pH manipulation. The gold particles are reactive and will bind antibodies to their surface, thus forming immunogold. The immunogold particles are stable and can be stored at 4 °C until required.

Similarly, rare earth elements (lanthanides) can be used as labels and because each lanthanide fluoresces at a different wavelength, a single assay can detect the presence of two or three different antibodies that can be individually visualised at the same time (multiplexing). The lanthanides are attached to the antibodies as a chelate, with the most common one being diethylenetriamine pentaacetate (DTPA).

Antibodies attached to latex particles can be used either as the solid phase for an immunoassay or as markers in lateral flow devices. Magnetised latex particles are available, allowing the easy separation of the latex particle–antibody–antigen complex from a liquid phase. There are also variants of latex and magnetic particles that have protein A covalently attached to their surface. Since protein A binds the Fc portion of an antibody, the latter is orientated such that the antigen binding site is facing outwards, thus maximising the chance of successful encounter.

اخر الاخبار

اخبار العتبة العباسية المقدسة

وفد العتبة العباسية المقدسة يزور شعبة الإعداد البدني للاطّلاع على برنامج تأهيل المنتسبين الجدد

قسم بين الحرمين يقدم جهودًا مضاعفة لإعادة المفقودات إلى الزائرين

بعد برامج تدريبية متخصصة.. متحف الكفيل يبيّن منجزات ملاكاته في فن الخاتم (الأرابيسك)

شعبة مدارس الكفيل الدينية تعقد الاجتماع التحضيري الأول لمهرجان روح النبوّة الثقافي

المزيد

الآخبار الصحية

ماذا يحدث إذا تناولنا الطعام مرة واحدة فقط في اليوم

اكتشاف عوامل خطر غير متوقعة لمرض الكبد الدهني

فيروس هانتا والسفينة الموبوءة.. تحديث من الصحة العالمية

طبيب يحسم الجدل السجائر الإلكترونية وعلاقتها بالسرطان

المزيد

الآخبار التكنلوجية

لأول مرة في الولايات المتحدة.. تشغيل مفاعل مصغر نووي يغذي الذكاء الاصطناعي بالطاقة

الكشف عن تفاصيل أول عملية ناجحة لاستعادة انتاج الحيوانات المنوية

أطلس يكشف خفايا الجسم البشري بدقة أرفع بـ50 مرة من شعر الإنسان

المتحف المصري الكبير يتحول إلى "أيقونة خضراء" بدعم ياباني

المزيد

مواضيع ذات صلة

Harnessing the Immune System for Antibody Production

Electrophoretic Techniques : Support Media and Buffers

Principles of Liquid Chromatography

Preliminary Purification Steps

Cell Disruption and Production of Initial Crude Extract

Determination of Protein Concentrations

Molecular Biotechnology and Applications

The Biology of Restriction Enzymes

The Isolation of DNA

MODERN BIOTECHNOLOGY

ANCIENT BIOTECHNOLOGY

DEFINITIONS OF BIOTECHNOLOGY