Antibodies are used in a myriad of applications (summarized in the Labome article Antibody applications). Most commercially available antibodies have been purified and are shipped either lyophilized or in a buffered stock solution. The lyophilized antibodies are generally reconstituted with a buffer (typically PBS, TBS) to create a stock solution which can be further diluted prior to use. Concentrations of antibody stock solutions (prior to the final dilution), either from commercial suppliers or reconstituted, are often 100 μg/ml, 200 μg/ml (for example, SCBT GATA-4 antibody), 500 μg/ml, or 1 mg/ml (for example, Thermo Fisher Scientific CD3 antibody). For comparison, pharmaceutical monoclonal antibodies are usually formulated at ≥ 100 mg/ml concentration [1]. However, such antibody stock concentration measures are only meaningful if the antibody has been purified, either by protein A or through affinity chromatography. If the antibody has not been purified, the measured concentration of the stock solution will be dramatically higher than the actual concentration of the antibody of interest due to the presence of many unrelated proteins. This point is especially important for any cost calculation of the antibodies.

Both unpurified antibodies (in serum, ascitic fluid or cell culture supernatant) and purified antibodies (in buffers) need to be diluted to working concentrations for different applications. The proper final working concentration is critical to the success of each experiment. The optimal final concentration is a balance between the affinity of the antibody for its targeted epitope and the non-specific binding between the antibody and other antigens (that is, the cross-reactivity) that generates background noise, obscuring the signal of interest. Higher antibody affinity and/or lower cross-reactivity allows for a lower final working concentration. If the working concentration is too low, the target protein or antigen will not be detectable. If the working concentration is too high, the background noise will be high and its wastes your potentially costly antibodies. The proper dilution for a working concentration needs to be optimized for each antibody, application, and sample to obtain the best signal-to-noise ratio.

Labome curates antibody information from the literature. In addition, Labome partner suppliers provide antibody application information obtained from the literature for their antibody products. In the literature, antibody working concentrations are typically given in ratio notations, such as 1:100, or 1:1000, to describe the dilution of the provided or recommended stock solution, for example, alpha-tubulin (Sigma, T9026) at 1:400,000 [2] or flow cytometry at 1:200 [3]. This practice should be discouraged in favor of the final concentrations such as 1 ug/ml [4], or in the case of a ChIP experiment in ug / million cells etc. [5]. Labome dilution data (as of January 2020) show that out of 99,560 incidences of antibody applications with dilution information from 23,949 articles (211,561 incidences are without dilution information), 94,498 used ratio notations, with a dilution of 1:1000 being most common with 23,575 occurrences followed by 1:100 at 14,636, 1:200 at 12,409 and 1:500 at 11,103 occurrences. It is critical to understand that such dilution notations apply only to dilution of the stock solution of an antibody and not to the actual antibody concentration. For example, a 1:100 dilution of an antibody stock solution at 100 μg/ml would have the same measured final concentration (1 μg/ml) as a 1:1000 dilution of an antibody stock solution of 1 mg/ml. In the Labome-surveyed antibody dilution data, final working concentrations, rather than than dilutions, are reported on 6,216 occasions. Table 1 lists the distributions of final antibody working concentrations for different applications, based on surveyed data. Table 2 lists the same concentration use distribution information but is limited to monoclonal antibodies only. The most commonly used working concentrations across the applications were 1 μg/ml and 2 μg/ml, which thus makes this range a good starting point for optimization/titration experiments.

While some antibodies have a wide range of acceptable working concentrations, many antibodies produce an acceptable signal-to-noise ratio only at a fairly precise concentration. Relevant literature should always be consulted first: some articles list dilutions in detail (see [6] for example). An optimal working concentration for an antibody for each assay should generally be established through a titration experiment, with serial dilution of the antibody stock solution. Rigorous statistical criteria have been proposed to establish the optimal dilution of antibodies for flow cytometry application [7]. During a titration study for immunohistochemistry, repeated staining of the same section with progressively less diluted antibody has been evaluated [8].

Disclaimer: this article and the information that follows is not medical advice. Any person suspecting/having contracted a disease should consult with licensed medical professionals. This article is intended for individuals interested in understanding the basics of clinical diagnosis of various diseases.

Antibody titer is a different concept from antibody dilution. It is used in the diagnosis of infectious and other diseases. The following discusses some of the common questions.

Antibody titer indicates the level of the antibodies in a blood sample, defined as the greatest dilution (lowest concentration) of the blood sample at which an antibody assay, such as ELISA, still produces a detectable positive result. The higher the antibody concentration in the blood, the greater the dilution that will produce a detectable signal. The actual titer value for an antibody will also vary based on the antibody being test, the method used, and the actual laboratory performing the test.

Antinuclear antibodies (ANA) describes a group of antibodies that react to various components of cell nuclei. ANA testing is used for the diagnosis of several auto-immune diseases, such as systemic lupus erythematosus. ANA tests are performed through indirect immunofluorescence microscopy or ELISA. The indirect immunofluorescence assay is usually performed on the HEp-2 human tumor cell line. Titers of 1:80 or higher are considered positive in the HEp-2 assays [9]. If the immunofluorescence assay is instead performed on rodent tissue substrate, titers of 1:20 or 1:40 or higher have been considered positive [9].

Several antibodies against the Hepatitis B virus (HBV) are measured for the diagnosis and prognosis of acute and chronic hepatitis. They include antibodies against HBV surface antigen (anti-HBs), antibodies against HBV core antigen (anti-HBc), and an antibody against HBV e antigen (anti-HBe). Comprehensive resources are available about these applications from the CDC. Commonly the detection of these antibodies is done through standardized, and often automatic, assay systems, such as Abbott Architect Anti-HBc II for anti-HBc antibodies [10] or Bio-Rad MonoLISA Anti-HBs kit [11].