Anti-Human Secondary Antibodies and their Importance in Serological Testing

Serological tests are key assays for several health issues, such as crossmatching, screening solid organ transplantation, disease diagnosis, monitoring, and management.

These assays have a vital role in clinical decision-making. Immunoassays are one of the most sensitive and powerful serological tests available, revolving around highly specific binding between antigens and antibodies, allowing quantitative and qualitative detection of analytes, even at low concentrations.

Anti-Human Secondary Antibodies and their Importance in Serological Testing

Image Credit: Jackson ImmunoResearch Laboratories, Inc.

A variety of immunoassays are available, and the detection process varies between techniques. Shown below are the most common techniques, a sandwich (Fig. 1A) and a direct bind (Fig. 1B), respectively.

Considering the species in which the primary antibody was generated and using a secondary antibody that has minimal cross-reactivity to that species is important when choosing an Anti-Human Secondary Antibody for use in sandwich format assays (Fig. 1A).

Anti-Human Secondary Antibodies and their Importance in Serological Testing

Figure 1. Assay formats, A. Sandwich, B. Direct. Image Credit: Jackson ImmunoResearch Laboratories, Inc.

Immunoassays are Important for a Variety of Diseases

There are both advantages and limitations to each immunoassay technique. For example, there are rapid test assays, like lateral flow, which provides qualitative results within minutes, enabling point of care analysis. Alternatively, an ELISA provides quantitative information but can require hours to perform.

Anti-Human Secondary Antibodies and their Importance in Serological Testing

Image Credit: Jackson ImmunoResearch Laboratories, Inc.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the benefits of lateral flow assays. The FDA has even made decisions enabling states to launch new SARS-CoV-2 tests before they have approved them, due to the mortality rates and fast spread of the disease.

This amendment enables the use of rapid test kits, which qualitatively detect IgM and IgG patient-generated antibodies to SARS-CoV-2. This led to the expansion of the SARS-CoV-2 diagnostic capacity in an attempt to reduce the spread of the virus. While there are obvious advantages to lateral flow tests, quantitative data is not obtained via these immunoassays, a key feature of some clinical tests.

Anti-Human Secondary Antibodies are utilized in other critical applications, such as the transplant crossmatch assay. Flow cytometry crossmatch is a crucial immunoassay which determines whether a donor organ is suitable for a transplant patient.

Anti-Human Secondary Antibodies and their Importance in Serological Testing

Image Credit: Jackson ImmunoResearch Laboratories, Inc.

These assays determine the presence of donor-specific crossmatched antibodies in the organ recipient to human leukocyte antigens on the donor’s white blood cells or bead conjugates. Using this, the histocompatibility of both the recipient and the donor can be determined, and suitability for organ transplantation can be assessed by clinicians.

Table 1. The detection process, immunoglobulin isotypes tested, and the advantages and disadvantages of several types of immunoassays.

Immunoassay

Detection process

Immunoglobulin isotypes

Rapid test/ lateral flow

The sample is loaded onto a test membrane and travels by capillary action. Analytes, including hyperimmune antibodies, present in the sample bind to color-coded Anti-Human detection antibodies as they travel. The complexes bind to either an immobilized sandwiching antibody (Fig. 1A) or an antigen in the form of a test line (Fig. 1B). A colored line provides a qualitative positive result. A common method of color-coding reporter antibodies is through conjugation to gold nanoparticles.

A control line is generally also included that indicates the assay is functioning properly.

IgG, IgM

Flow cytometry

A serum sample is added to antigen-bearing cells or coated beads. A secondary antibody conjugated with a fluorescent probe is added and binds to any human antibody captured on the cell or beads (Fig. 1B). The fluorescence can then be analyzed by the flow cytometer to give quantitative data.

IgG, IgM, IgA

Flow Injection Analysis (FIA)

Similar to flow cytometry, the sample is injected into a column containing the immobilized antigen, to which the antibodies of interest in the sample binds (Fig. 1B). A secondary antibody is added and binds to any human antibody captured on the column. This secondary antibody can either be conjugated to a fluorescent probe or an enzyme for colorimetric or fluorescent detection.

IgG, IgM, IgA

ELISA

A primary antibody is immobilized on a solid surface, typically a microtiter plate, which subsequently captures the antigen (Fig. 1A) or the antigen is coated directly onto the plate (Fig. 1B). Human antibodies from hyperimmune serum bind to the antigen. An enzyme-conjugated Anti-Human Secondary Antibody is added which binds to the human antibody from the sample. Finally, an enzyme substrate is added, which results in a colorimetric readout proportional to the analyte concentration.

IgG, IgM, IgA

IPCR

This process is similar to ELISA, but the secondary antibody is conjugated to an oligonucleotide. DNA amplification and detection are then performed by real-time PCR.

IgG, IgM, IgA

Anti-Human Secondary Antibodies are Key Reagents in Immunoassays

While the purpose and function of immunoassays differ, high-quality reagents are essential when manufacturing any serological kit to produce accurate and reliable data that clinicians and patients can trust.

The characteristics and quality of Anti-Human Secondary Antibodies are vital considerations to make when an immunoassay is being developed. Secondary antibodies are favored, as they bind multiple epitopes onto one primary antibody, enhancing the signal for higher selectivity and sensitivity.

In Table 2, some aspects which need to be considered when selecting the best secondary antibodies for an immunoassay are described.

Anti-Human Secondary Antibodies and their Importance in Serological Testing

Image Credit: Jackson ImmunoResearch Laboratories, Inc.

High-Quality Anti-Human Secondary Antibodies from Jackson Immunoresearch

Jackson ImmunoResearch Laboratories, Inc. is a specialist for secondary antibodies and providing them for immunological applications. It hosts a wide range of products, enabling customers to choose the most suitable secondary antibody for developing their serological tests.

Anti-Human Secondary Antibodies are all manufactured to ISO 9001:2015 certification, ensuring the reliability, specificity, and quality of the products. The secondary antibodies can be purchased in both standard and bulk quantities and can be shipped internationally.

Table 2. Considerations for choosing appropriate JIR secondary antibodies and immunoreagents.

Consideration

Jackson ImmunoResearch Secondary Antibodies

Host species

Serological tests for human samples should use Anti-Human Secondary Antibodies to minimize non-specific binding, which can lead to high background signals and false positives.

Anti-Human Secondary Antibodies are developed in a range of host species including alpaca, donkey, goat, mouse, and rabbit.   

Product type

Secondary antibodies are available as whole immunoglobulin (Ig)G, F(ab’)2, and Fab fragments. Whole IgG antibodies are often sufficient, but F(ab’)2 and Fab fragments are useful to avoid binding to live cells with Fc receptors.

Secondary antibodies are available as whole IgG and F(ab’)2 and Fab fragment formats.

Specificity

Depending on the specificity of the immunoassay required, secondary antibodies can be made to be specific to the whole Ig, Fc, or F(ab’)2 domain.

Anti-Human Secondary Antibodies offer specificity for whole Ig, Fc, and F(ab’)2 domains.

Isotype

Most immunoassays test for IgG isotypes, which constitute ~75% of the total serum Ig and are produced as part of the secondary immune response. However, IgM and IgA can be used, as these function in the primary immune response and protect mucus membranes, respectively. It is important that the secondary antibody has specificity for the isotype of the primary antibodies.

Products are available with specificities to IgG, IgM, IgA, IgG+IgM, and IgG+IgM+IgA.

Affinity purification and cross-adsorption

Due to the high structure conservation in Ig domains, it is recommended to use class or species-specific secondary antibodies that have been affinity purified and cross-adsorbed to reduce the possibility of cross-reaction.

Immunoaffinity chromatography is used to isolate affinity-purified antibodies. Antibodies can be purchased which have been cross-absorbed against species and these details are provided in the parentheses of the product description with the term “min X” followed by abbreviations for the relevant species.

Conjugate

Secondary antibodies are often conjugated to a reporter molecule e.g. an enzyme, fluorescent probe, or colored particle. The detection system of the immunoassay will determine the type of conjugate.

Anti-Human Secondary Antibodies are available unconjugated or conjugated to a range of reporter types, including biotin, alkaline phosphatase, horseradish peroxidase, fluorophores, and colloidal gold nanoparticles.

Sources and Further Reading

  • O’Connell, K. (2018). What is Serology?
  • Vashist, S.K, et al. (2018). Handbook of Immunoassay Technologies: Approaches, Performances and Applications.
  • Assaygenie.com. (2020). COVID-19 Rapid POC CE-IVD Test (25 Tests).
  • Globalbiodefence.com. (2020). FDA Pandemic Response: States Can Now Approve COVID-19 Diagnostics, Serology Testing.
  • Tait, B.D. (2016). Detection of HLA Antibodies in Organ Transplant Recipients – Triumphs and Challenges of the Solid Phase Bead Assay. Frontiers in Immunology.

About Jackson ImmunoResearch Laboratories, Inc.

Jackson ImmunoResearch Laboratories, Inc. specializes in the production and conjugation of affinity-purified secondary antibodies and purified immunoglobulins. Our products are sold primarily to scientists in universities and research institutes throughout the world who are conducting research in the plant, animal, and biomedical sciences.

The company is located in rural Pennsylvania, near the Amish country, about 40 miles west of Philadelphia. It was started in 1982 by scientists whose education and previous experience included research in cell biology, protein chemistry, microbiology, and product development in immunology. Our goal is to provide other scientists with the newest, largest selection of, and highest quality of secondary reagents, with the best technical and customer services possible. The products are for research use only, and not for diagnostics or therapeutics. They are not primary antibodies, nor are they medical devices.

We hope this site will provide not only information about our company and our products, but also answer some technical questions you may have regarding the use of our reagents.


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Last updated: Nov 26, 2020 at 4:53 AM

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