Protocols

Antibody Dilution Ratio and Antibody Titer

Abstract:    Antibody dilution ratio and antibody titer are two relevant but completely different concepts. They are easily confused in the experiment. Here, they are subjected to the brief analysis to help you understand the concept.

Keywords:  Antibody Dilution Ratio, Antibody Titer, FineTest Antibodies

1. Introduction

An antibody refers to the protective protein generated from the organism stimulated by the antigen. Globulins which contain antibody activity or has the chemical structure similar to the antibody are called as immunoglobulins(Ig). There are four heterologous polypeptide chains in the natural Ig molecule. Two of them with high molecular mass are known as heavy chain(H). The rest with low molecular mass are known as light chain(L). The amino acid composition of two H chains and L chains in the same Ig molecule is completely identical. The amino acid composition and sequence of heavy chain constant region is different. The antigenicity is also different. Thus, immunoglobulins can be divided into five classes, including IgM, IgD, IgG, IgA and IgE. The corresponding heavy chain is μ, δ, γ, α and ε. Various types of Ig make their characteristics different. Differences include the number and position of intrachain and interchain disulfide bond, the number of structural domain, and the length of hinge region etc. Even in the same Ig, differences also exist, including amino acid composition of hinge region, number and position of heavy chain disulfide bond. Hence, Ig in the same class can be categorized into different subclasses. Antibodies can be applied as test kits, immune drugs or suppressants. In the scientific experiment, antibodies act as the probe or affinity ligand.

2. Conceptual Analysis

2.1. Dilution Ratio

Commercial antibodies are purified protein A/G, or affinity purified antigen. They are lyophilized or stored in storage buffer. Before use, lyophilized antibodies shall be dissolved by storage buffer to achieve a suitable concentration (100ug/ml, 500ug/ml or 1mg/ml etc). Because unpurified antibodies (e.g. antiserum, ascites or cell culture supernatant) include large amount of irrelevant proteins, concentration is not the real antibody concentration at present. Purified or unpurified antibodies in the experiment should be diluted to the certain working concentration according to different applications. The working concentration of antibodies is very important. It is not only related to the affinity ability of antibody and target site, but also depends on antibody's nonspecific binding with other antigens. The nonspecific binding will result in background. The stronger the antibody's affinity ability is, the lower the working concentration is. The low nonspecific binding also decreases working concentration. It’s a waste to adopt over high concentration and light background. If the concentration is too low, target protein or antigen can’t be tested. The working concentration of antibodies should be optimized to obtain the best signal-to-background ratio according to different samples and applications. The table below is the common antibody dilution ratio of various applied experiments. The ratio should be optimized according to detailed experimental results.

Table 1:  Common Antibody Dilution Ratio of Various Applied Experiments

Tissue Culture

Supernatant

Ascites Antiserum Purified Antibodies
WB 1/100 1/1000 1/500 1ug/mL
IHC/ICC/IF undiluted - 1/100 1/100 1/50-1/100 5ug/mL
EIA/ELISA 1/1000 1/10000 1/500 0.1ug/mL
FACS 1/100 1/1000 1/500 1ug/mL
IP - 1/100 1/50-1/100 1-10ug/mL
Estimated IgG Concentration 1-3mg/mL 5-10mg/mL 1-10mg/mL -

2.2. Antibody Titer

Antibody titer is applied in the diagnosis of infectious and other diseases. It is the metrics of the antibody level in the blood sample and used to measure the lowest concentration (max dilution ratio) for recognizing special antigen epitope, which is usually expressed as the max dilution ratio of positive result for blood sample tested by ELISA. The accurate titer value depends on the tested antibody, method and laboratory.

3. FineTest Antibodies

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