What are differences btw monoclonal anti-human ig G and polyclonal anti-human igG?

Question

What are differences btw monoclonal anti-human ig G and polyclonal anti-human igG?

Answer 1

is a tetrameric immunoglobulin, built of two heavy chains γ and two light chains. Each IgG has two antigen binding sites. It is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids, constituting 75% of serum immunoglobulins in humans.[1] IgG molecules are synthesised and secreted by plasma B cells.

In birds, IgG is replaced with IgY, and is found in serum and yolk.

Functions

This is the only isotype that can pass through the human placenta, thereby providing protection to the fetus in utero. Along with IgA secreted in the breast milk, residual IgG absorbed through the placenta provides the neonate with humoral immunity in its first weeks of life before its own immune system has developed.

It can bind to many kinds of pathogens, for example viruses, bacteria, and fungi, and protects the body against them by complement activation (classic pathway), opsonization for phagocytosis and neutralization of their toxins.

IgG can cause food allergy, and in such causes delayed-onset food allergy, in contrast to food allergy by IgE, whose effects appear rapidly.

Structure

IgG antibodies are large molecules of about 150 kDa composed of 4 peptide chains. It contains 2 identical heavy chains of about 50 kDa and 2 identical light chains of about 25 kDa. The two heavy chains are linked to each other and to a light chain each by disulphide bonds. The resulting tetramer has two identical halves which together form the Y-like shape. Each end of the fork contains an identical antigen binding site.

Name Percent Crosses placenta easily Complement activator Binds to Fc receptors on phagocytic cells

IgG1 66% yes second highest high affinity

IgG2 23% no third highest extremely low affinity

IgG3 7% yes highest high affinity

IgG4 4% yes no intermediate affinity

Meanwhile

Polyclonal antibodies (or antisera) are antibodies that are derived from different B cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope

Production

These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater. An antigen is injected into the mammal. This induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is polyclonal purified from the mammal’s serum.

By contrast, monoclonal antibodies are derived from a single cell line.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines governing animal use and procedures relating to these methodologies are generally oriented around humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other agents while having few if any direct effects when given by themselves) use. This includes adjuvant selection, routes and sites of administration, injection volumes per site and number of sites per animal. Institutional policies generally include allowable volumes of blood per collection and safety precautions including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or personnel.

The primary goal of antibody production in laboratory animals is to obtain high titer, high affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:

surfactants which promote concentration of protein antigens molecules over a large surface area, and

immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein antigens to increase antibody titers and induce a prolonged response with accompanying memory.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.

Animal selection

Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs, hamsters, horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory animal for this purpose. Animal selection should be based upon:

the amount of antibody needed,

the relationship between the donor of the antigen and the recipient antibody producer (generally the more distant the phylogenetic relationship, the greater the potential for high titer antibody response) and

the necessary characteristics [e.g., class, subclass (isotype), complement fixing nature] of the antibodies to be made. Immunization and phlebotomies are stress associated and, at least when using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of such animals can dramatically reduce morbidity and mortality due to pathogenic organisms, especially Pasteurella multocida in rabbits.

Goats or horses are generally used when large quantities of antisera are required. Many investigators favor chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of IgY (IgG) into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive bleeding procedure. One week’s eggs can contain 10 times more antibodies than the volume of rabbit blood obtained from one weekly bleeding. However, there are some disadvantages when using certain chicken derived antibodies in immunoassays. Chicken IgY does not fix mammalian complement component C1 and it does not perform as a precipitating antibody using standard solutions.

Although mice are used most frequently for monoclonal antibody production, their small size usually prevents their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies in mice can be collected from ascites fluid using any one of a number of ascites producing methodologies.

When using rabbits, young adult animals (2.5–3.0 kg or 5.5-6.5lbs) should be used for primary immunization because of the vigorous antibody response. Immune function peaks at puberty and primary responses to new antigens decline with age. Female rabbits are generally preferred because they are more docile and are reported to mount a more vigorous immune response than males. At least two animals per antigen should be used when using outbred animals. This principle reduces potential total failure resulting from non-responsiveness to antigens of individual animals.

Antigen preparation

The size, extent of aggregation and relative nativity of protein antigens can all dramatically affect the quality and quantity of antibody produced. Small polypeptides (<10 ku) and non-protein antigens generally need to be conjugated or cross-linked to larger, immunogenic, carrier proteins to increase immunogenicity and provide T cell epitopes. Generally, the larger the immunogenic protein the better. Larger proteins, even in smaller amounts, usually result in better engagement of antigen presenting antigen processing cells for a satisfactory immune response. Injection of soluble, non-aggregated proteins has a higher probability of inducing tolerance rather than a satisfactory antibody response.

Keyhole limpet hemocyanin (KLH) and bovine serum albumen are two widely used carrier proteins. Poly-L-lysine has also been used successfully as a backbone for peptides. Although the use of Poly-L-lysine reduces or eliminates production of antibodies to foreign proteins, it may result in failure of peptide-induced antibody production. Recently, liposomes have been successfully used for delivery of small peptides and this technique is more efficient than delivery with oily emulsion adjuvants.

Antigen quantity

Selection of antigen quantity for immunization varies with the properties of the antigen and the adjuvant selected. In general, microgram to milligram quantities of protein in adjuvant are necessary to elicit high titer antibodies. Antigen dosage is generally species, rather than body weight, associated. The so called “window” of immunogenicity in each species is broad but too much or too little antigen can induce tolerance, suppression or immune deviation towards cellular immunity rather than a satisfactory humoral response. Optimal and usual protein antigen levels for immunizing specific species have been reported in the following ranges:

rabbit, 50–1000 µg;

mouse, 10–200 µg;

guinea pig, 50–500 µg; and

goat, 250–5000 µg. Optimal “priming” doses are reported to be at the low end of each range.

The affinity of serum antibodies increases with time (months) after injection of antigen-adjuvant mixtures and as antigen in the system decreases. Widely used antigen dosages for “booster” or secondary immunizations are usually one half to equal the priming dosages. Antigens should be free of preparative byproducts and chemicals such as polyacrylamide gel, SDS, urea, endotoxin, particulate matter and extremes of pH.

Peptide Antibodies

When a peptide is being used to generate the antibody, it is extremely important to design the antigens properly. There are several resources that can aid in the design as well as companies that offer this service. Expasy has aggregated a set of public tools under its ProtScale page that require some degree of user knowledge to navigate. For a more simple peptide scoring tool there is a Antigen Profiler tool available that will enable you to score individual peptide sequences based upon a relation epitope mapping database of previous immunogens used to generate antibodies. Finally, as a general rule peptid

Answer 2

Monoclonal antibodies are produced from one single cell line- they are perfect clones of a single, original IgG.  A monoclonal antibody is developed to bind a specific substance.  Polyclonal antibodies are produced from different mother cell lines.  They typically respond to the same antigen, but in different ways.  So there may be a form of IgG that responds to one particular physical/chemical aspect of the antigen while there is another that also responds to it, but from a different physical/chemical approach or aspect.