Designing human serological immunoassays: Key considerations for antibody selection
Introduction
High-quality reagents are essential to ensure accuracy, sensitivity, and reproducibility - the key requirements of a reliable diagnostic test. Here we describe some of the critical considerations necessary for selecting the right detection reagent for your immunoassay.
What type of assay are you developing?
Assay development will be governed by the sensitivity, speed, and ease of use you require, alongside access to equipment and how the test is to be administered. The most common serological tests are:
- Bead-based
- ELISA
- Lateral Flow
- Western Blot
- Nephelometric/Turbidimetric
- Flow Cytometry
Want to know more about serological testing? View our serology page here.
What Format will you use?
Immunoassays can detect a diverse array of molecules such as a structural motif on a virus particle or allergen or immunoglobulins from biological fluids of hyperimmune patients, such as serum or saliva. Assays can set up in a variety of formats such as, direct, indirect, sandwich, and competitive. Indirect and sandwich assays are the more commonly used formats. They offer a higher level of sensitivity by way of the conjugated secondary antibody amplifying the signal from the target molecule.
Why choose polyclonal for your detection reagent?
Polyclonal antibodies are ideally suited for the detection component of the immunoassay. Their ability to bind multiple epitopes on one primary antibody results in inherent amplification which leads to higher test sensitivity and selectivity.
Human patients? Choose JIR Anti-Human antibodies
If you’re looking to create diagnostic tests for human patients it is likely that you’ll need anti-human Ig antibodies at some stage of the test.
Veterinary testing?
It’s not only human patients that need accurate and sensitive diagnostic and surveillance testing. With zoonotic disease disrupting people’s lives across the world, animal testing will become more prevalent. We manufacture antibodies with specificity against a wide range of other species, from farm animals such as pigs, cattle, sheep, and poultry, through to companion animals such as cats and dogs.
Are other host species present in your assay?
Serological tests typically do not have species to species cross-reactivity complications due to the nature of their design. Table 1 details suggested anti-human antibodies suitable for immunoassays where cross-reactivity from other species proteins will not be present.
| Product Code | Description | Detects |
|---|---|---|
| 109‑005‑008 | Goat Anti-Human IgG, Fcγ fragment specific | IgG only |
| 109‑005‑129 | Goat Anti-Human IgM, Fc5μ fragment specific (min X Bov Sr Prot) | IgM only |
| 109‑005‑011 | Goat Anti-Human Serum IgA, α chain specific | IgA only |
Does host species have an impact on your assay?
JIR manufactures Anti-Human Antibodies with strong affinity and avidity in mice, rabbits, alpacas, and donkeys, but those most commonly used for serological tests are made in goats. Larger animals facilitate the production of greater volumes of raw material. With the increased pressures of the COVID 19 response, JIR production has increased to accommodate highly requested items related to the outbreak. Our goal is to ensure the best products are selected for large scale assay and kit development with cost-per-assay in mind.
We compared two popular host species, Rabbit and Goat to identify any difference in detection of human IgG, IgM, and IgA by ELISA.
The following ELISAs demonstrate that the specificity and sensitivity of JIR Goat Anti-Human and Rabbit Anti-Human antibodies are comparable for detecting IgG, IgM or IgA.
Figure 2: Human IgG capture and detection using Goat vs Rabbit host Polyclonal Secondary Antibodies
Figure 3: Human IgM capture and detection using Goat vs Rabbit host Polyclonal Secondary Antibodies
Figure 4: Human IgA capture and detection using Goat vs Rabbit host Polyclonal Secondary Antibodies
Detecting specific classes of immunoglobulin
When immunoglobulin specificity needs are paramount you can trust JIR secondary antibodies to differentiate with exquisite accuracy. Figure 5 shows the specificity of JIR Anti-Human IgG (109‑035‑008) and JIR Anti-Human IgM (109‑035‑129) for their intended target Ig in the presence of other immunoglobulins.
Figure 5: JIR Anti-Human IgG and IgM Demonstrate Exquisite Specificity and Sensitivity in a Serological ELISA
*Serological assay conditions based on Mt. Sinai EUA April 15, 2020 (Amanat et al., 2020)
Pan-Specific antibody detection
Pan specific antibodies allow the measurement of total Ig of two or more immunoglobulin classes. These products will not differentiate IgG, IgM, and IgA
| Product Code | Description |
|---|---|
| 109‑005‑127 | Goat Anti-Human IgG + IgM (H+L) (min X Bov Sr Prot) |
| 109‑005‑064 | Goat Anti-Human IgA + IgG + IgM (H+L) |
Do you have antibodies from species other than human in your assay?
In many serological assays, antibodies from additional species may be present. For example, a mouse or rabbit antibody may be used for capturing antigen to which a human antibody may bind. In these cases, background signal is possible due to cross-reactivity of the anti-human antibody with the antibody from the additional species. For human serological assay designs utilizing antibodies from other species, JIR offers antibodies cross-adsorbed (min X) against common primary antibody hosts to reduce the risk of cross-reactivity. Table 3 details popular Goat Anti-Human antibodies cross-adsorbed against a range of common species including, bovine, horse, mouse and rabbit.
| Product Code | Description | Cross-Reactivity Considerations |
|---|---|---|
| 109‑005‑190 | Goat Anti-Human IgG, Fcγ fragment specific (min X Bov, Ms Sr Prot) | Only in the absence pf rabbit and horse serum proteins |
| 109‑005‑098 | Goat Anti-Human IgG, Fcγ fragment specific (min X Bov, Hrs, Ms Sr Prot) | Will cross-react with rabbit serum proteins if present |
| 109‑005‑170 | Goat Anti-Human IgG, Fcγ fragment specific (min X Bov, Ms, Rb Sr Prot) | Only in the presence of rabbit serum proteins |
Custom and bulk capabilities
We can manufacture and supply most standard inventory secondary antibodies in bulk volumes upon request.
Customers can be completely confident of product quality, consistency and minimal lot to lot variation. Our goal is providing the highest quality of product with outstanding service.
Jackson ImmunoResearch Laboratories, Inc. is certified by BSI to ISO 9001:2015 under certificate number FM 545248.
Contact our Sales team for more information.
