Jackson Immuno Research Inc.
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Whole IgG Affinity-Purified Secondary Antibodies

"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."

Janet Duerr, Ohio University

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Selecting Antibodies for Serological Immunoassays

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:

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.

Figure 1. Immunoassay formats. Sandwich or Indirect.

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.

Table 1: Anti-Human antibodies suitable for the detection of human immunoglobulins in the absence of antibodies from other species.
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

Sandwich Elisa: Elisa plates were coated with either AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (109‑005‑008) or AffiniPure Rabbit Anti-Human IgG, Fcγ fragment specific (390‑005‑008). After blocking with Bovine Serum Albumin (IgG-Free, Protease-Free) (001‑000‑162), ChromPure Human IgG, whole molecule (009‑000‑003) was then titrated across successive wells and the non-binding fraction was washed off. Captured IgG was detected with either Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (109‑035‑008) or Peroxidase AffiniPure Rabbit Anti-Human IgG, Fcγ fragment specific (309‑035‑008) diluted 1:40,000 in assay buffer. Both Goat and Rabbit antibodies displayed almost identical limits of detection and signal strength in this assay.

Figure 3: Human IgM capture and detection using Goat vs Rabbit host Polyclonal Secondary Antibodies

Sandwich Elisa: Elisa plates were coated with either AffiniPure Goat Anti-Human IgM, Fc5μ fragment specific (109‑005‑129) or AffiniPure Rabbit Anti-Human IgM, Fc5μ fragment specific (309‑005‑095). After blocking with Bovine Serum Albumin (IgG-Free, Protease-Free) (001‑000‑162), ChromPure Human IgM (myeloma), whole molecule (009‑000‑012) was then titrated across successive wells and the non-binding fraction was washed off. Captured IgM was detected with either Peroxidase AffiniPure Goat Anti-Human IgM, Fc5μ fragment specific (109‑035‑129) or Peroxidase AffiniPure Rabbit Anti-Human IgM, Fc5μ fragment specific (309‑035‑095) diluted 1:40,000 in assay buffer. Both Goat and Rabbit antibodies displayed similar limits of detection and have strong signals to noise.

Figure 4: Human IgA capture and detection using Goat vs Rabbit host Polyclonal Secondary Antibodies

Elisa plates were coated with AffiniPure Goat Anti-Human Serum IgA, α chain specific (109‑005‑011). After blocking with Bovine Serum Albumin (IgG-Free, Protease-Free) (001‑000‑162), ChromPure Human Serum IgA, whole molecule (009‑000‑011) was then titrated across successive wells and the non-binding fraction was washed off. Captured IgA was detected with either Peroxidase AffiniPure Goat Anti-Human Serum IgA, α chain specific (109‑035‑011) or Peroxidase AffiniPure Rabbit Anti-Human Serum IgA, α chain specific (309‑035‑011) diluted 1:20,000 in assay buffer. Both Goat and Rabbit antibodies displayed similar limits of detection and high signal to noise.

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

SARS-CoV-2 Serology Elisa: The SARS-CoV-2 serology ELISA was carried out following the procedure described in Amanat et al. (2020). Briefly, Elisa plates were coated with SARS-CoV-2 S1 protein (Genscript Z03485) and plates were blocked with 1% Bovine Serum Albumin (IgG-Free, Protease-Free) (001‑000‑162) in PBST. Serial dilutions of Human IgG SARS-CoV-2 Spike S1 Antibody (Genscript A02038) and Human IgM SARS-CoV-2 Spike S1 Antibody (Genscript A02046) were added to successive wells and the non-binding fraction was washed off. Captured IgG and IgM were detected with Peroxidase AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (109‑035‑008) and Peroxidase AffiniPure Goat Anti-Human IgM, Fc5μ fragment specific (109‑035‑129), respectively, diluted 1:20,000 in PBST.

*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

Table 2: Anti-Human antibodies suitable for the detection of more than one human immunoglobulin isotype.
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.

Table 3: Anti-Human antibodies suitable for the detection of human immunoglobulins in the presence of antibodies from other species.
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.

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