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Isotyping Immunoglobulin ELISA Kits and Multiplex Immunoassays |
Characterizing and quantitating immunoglobulins
Determine antibody isotypes with ease and accuracy using the Invitrogen immunoglobulin-specific and isotyping ELISA kits, ProQuantum high-sensitivity immunoassays, and ProcartaPlex multiplex immunoassays. Select single-isotype or multi-isotype (isotyping) kits involving IgG, IgA, IgE, and IgM subclasses and isotypes from human, mouse, and rat species.
Overview of isotyping and immunoglobulin characterization
The various antibodies produced by plasma cells are classified by their isotypes that differ in function and antigen responses. Five major classes or antibody isotypes have been identified in placental mammals: IgA, IgD, IgE, IgG, and IgM (Figure 1 and Table 1). Antibody isotypes are categorized according to differences in their amino acid sequence in the constant region (Fc) of the antibody heavy chains. The antibody isotypes IgG and IgA are further grouped into subclasses (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) based on additional small differences in their amino acid heavy chain sequences. Determination of individual subclasses is relevant in assessing primary immunodeficiencies or immune responses, especially if the total IgG or IgA concentration is not altered.
Learn more about each isotype: Immunoglobulin Structure and Classes
Figure 1. Annotated diagram of immunoglobulin structure.
Table 1. Human immunoglobulin isotypes and their properties.
| Species | Immunoglobulin | Serum level (Adult [mg/mL]) | Molecular mass (kD) | Activation/Binding of complement | Binding ability to Protein A |
|---|---|---|---|---|---|
| Human | IgA1 | 0.6–3 | 160 (monomer) | – | + |
| Human | IgA2 | 0.06–0.6 | 160 (monomer) | – | + |
| Human | IgM | 0.045–2.5 | 970 (pentamer) | +++ | + |
| Human | IgE | 0.00005 | 188 | – | + |
| Human | IgG1 | 3.8–11.4 | 146 | ++ | ++ |
| Human | IgG2 | 1.5–6.9 | 146 | + | ++ |
| Human | IgG3 | 0.2–1.70 | 165 | +++ | – |
| Human | IgG4 | 0.08–1.4 | 146 | – | ++ |
+++: Very Strong Affinity; ++: Strong affinity; +: Moderate/slight affinity; –: No affinity
Characterization of immunoglobulins
There are multiple methods that can be used for antibody isotyping and the chosen method will depend on the scale and throughput of antibody production, the nature of results needed (i.e., quantitative versus qualitative analysis), assay costs, detection instrumentation, and technical expertise required. These methods include singleplex and multiplex immunoassays, lateral flow assays, microagglutination titer assays, western blotting, indirect immunofluorescence assay (IIFA), and flow cytometry.
Enzyme-linked immunosorbent assays (ELISA) are among the methods of choice for large-scale investigations and can provide accurate quantitative results. Invitrogen isotyping ELISA kits are a fast, sensitive, and convenient method for quantitation of immunoglobulin isotypes during and after hybridoma development and to assess immune response. The Invitrogen ProcartaPlex antibody isotyping panels enable screening titers of individual immunoglobulin isotypes from serum, plasma, and cell culture supernatant samples using Luminex magnetic bead technology.
Learn more about available products for Antibody Isotyping and Characterization
Common applications
Hybridoma screening is one of the most important steps in monoclonal antibody development and it is performed at multiple stages of the process to help ensure that the most productive and specific clones are selected for further evaluation. Antibody isotyping provides a powerful tool for hybridoma development allowing for rapid screening at the various development stages of fusion, cloning, and subcloning to characterize each potential clone. With Invitrogen isotyping ELISA and ProcartaPlex assays, you can accurately and specifically identify which heavy and light chain your hybridoma is producing and if it is monoclonal or not.
Immunization with vaccines induces the production of antibodies that provide protection against pathogens. To better understand the effects on the immunogenic properties of a vaccine and its adjuvants, profiling sera and plasma can help provide useful insight into immune response. Since each antibody isotype possesses unique effector functions, the examination of individual antibody isotypes first produced following vaccination can help elucidate or indicate a primary immune response to a vaccine. For example, IgE is characteristic in allergic reactions, and therefore useful during the assessment of an immune response. If there is no allergic reaction, IgE is usually barely above the limit of detection whereas a strong IgE response could be an indicator of acute-onset reactions to the vaccine. IgA and IgM levels also increase early on and wane after a few weeks, but IgG levels are longer lasting for long-term immunity.
Learn more about SARS-CoV-2 Serology Assays
Isotyping and immunoglobulin ELISA kits
Antibody isotyping ELISA kits are a fast, sensitive, and convenient method for quantitation of immunoglobulin isotypes to assess or to gauge the immune response. Compared to membrane-based isotyping tests, ELISAs provide an accurate quantitative result with a higher throughput for testing more samples at a time. Using these assays, antibody levels of individual isotypes can be easily assessed for individual isotype determination or for multiple isotypes using a single sample.
Invitrogen ELISA kits for popular targets such as IgA, IgE, IgG1, IgM etc. are listed in Table 2. Plate layout for typical Mouse and Rat Ig Isotyping Kits is shown in Figure 2.
Search all isotyping and immunoglobulin ELISA kits
Learn more about ELISA kits and components
Popular isotyping and immunoglobulin protein targets and ELISA performance data
Table 2. View our ELISA kits for the following targets:
Figure 2. Plate layout for typical Mouse and Rat Ig Isotyping Kits. Invitrogen Ig Isotyping Kits are the optimal match for screening your hybridoma in a 96-strip well (8x12) ELISA format. Up to 12 individual samples can be tested for anti-IgG1, IgG2a, IgG2b, IgG2c or IgG3, IgA, and IgM, as well as light chain kappa and lambda.
Isotyping and immunoglobulin ProQuantum high sensitivity immunoassays
ProQuantum high-sensitivity immunoassay kits for immunoglobulin detection are designed to easily provide quantitative measurements in small sample volumes of 1–2 μL. Utilizing proximity-based amplification technology, the assay combines the analyte specificity of high-affinity antibody-antigen binding with the signal detection and amplification capabilities of real-time PCR to achieve a simple yet powerful next-generation protein quantitation platform. A user-friendly, no-wash workflow combined with intuitive software for analytics enables sample-to-answer in just two hours.
Invitrogen ProQuantum immunoassay kits for popular targets such as Tau, Tau pT181 and Troponin I are listed in Table 3. Standard curve of IgE using Human IgE ProQuantum Immunoassay Kit is shown in Figure 3.
Find isotyping and immunoglobulin-related ProQuantum assays
Learn more about how ProQuantum immunoassays work
Read BioProbes Journal article: Introducing ProQuantum High-Sensitivity Immunoassays—The new generation of target-specific protein quantitation
Popular immunoglobulin research protein targets and ProQuantum assay performance data
Table 3. Immunoglobulin-related ProQuantum immunoassays. View our ProQuantum immunoassay kits for the following popular targets:
Figure 3.Representative standard curve of Human IgE. The standard curve for human IgE using Human IgE ProQuantum Immunoassay Kit shows a broad dynamic range (0.00768–3,000 ng/mL) of IgE protein.
Isotyping and immunoglobulin ProcartaPlex multiplex immunoassays
Invitrogen ProcartaPlex isotyping assays are based on the principles of a sandwich ELISA but utilize Luminex xMAP (multianalyte profiling) bead-based technology for the simultaneous detection and quantitation of multiple antibody isotypes targets in a single 25–50 μL sample from plasma, serum, and hybridoma culture supernatants.
The ProcartaPlex isotyping assays are available as preconfigured panels in a ready-to-use format that has been extensively tested for analyte combinability, interference, and cross-reactivity to provide the highest level of validation and precision (Table 4). Panels can also be easily designed to specific research needs by using the ProcartaPlex Panel configurator tool below to make your customized selections.
Comparison data of ProcartaPlex Human Antibody Isotyping Panel, 7plex and IgG4 Human ELISA Kit in human serum samples shows a correlation factor of R2 = 0.98 (Figure 4).
ProcartaPlex Panel Configurator
Learn more about ProcartaPlex multiplex immunoassays
Preconfigured isotyping and immunoglobulin multiplex immunoassay panels and performance data
Figure 4. Performance comparison of ProcartaPlex assay with coated ELISA. Human serum samples were tested in parallel using ProcartaPlex Human Antibody Isotyping Panel, 7plex and IgG4 Human ELISA Kit. The measured concentration of IgG4 shows high correlation (R2>0.95) demonstrating that ProcartaPlex panels achieve quantitative results comparable to traditional ELISA kits with the additional benefits of multiplexing, including reduced sample volume, shorter assay/hands-on time and lower costs per data point.
Table 4. Preconfigured ProcartaPlex multiplex immunoassay panels for isotyping and immunoglobulins.
| Product Name | Size | Cat. No. |
| ProcartaPlex Human Antibody Isotyping Panel, 7plex Target list [bead region]: IgG1 [12], IgG2 [20], IgG3 [34], IgG4 [42], IgA [55], IgE [48], IgM [62] | 96 tests | EPX070-10818-901 |
| ProcartaPlex Mouse Antibody Isotyping Panel 1, 7plex Target list [bead region]: IgA [38], IgE [42], IgG1 [12], IgG2a [18], IgG2b [21], IgG3 [25], IgM [52] | 96 tests | EPX070-20815-901 |
| ProcartaPlex Mouse Antibody Isotyping Panel 2, 7plex Target list [bead region]: IgA [38], IgE [42], IgG1 [12], IgG2b [21], IgG2c [43], IgG3 [25], IgM [52] | 96 tests | EPX070-20816-901 |
| ProcartaPlex Rat Antibody Isotyping Panel, 6plex Target list [bead region]: IgG1 [25], IgG2a [28], IgG2b [34], IgG2c [44], IgA [61], IgM [48] | 96 tests | EPX060-30123-901 |
Overview of isotyping and immunoglobulin characterization
The various antibodies produced by plasma cells are classified by their isotypes that differ in function and antigen responses. Five major classes or antibody isotypes have been identified in placental mammals: IgA, IgD, IgE, IgG, and IgM (Figure 1 and Table 1). Antibody isotypes are categorized according to differences in their amino acid sequence in the constant region (Fc) of the antibody heavy chains. The antibody isotypes IgG and IgA are further grouped into subclasses (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) based on additional small differences in their amino acid heavy chain sequences. Determination of individual subclasses is relevant in assessing primary immunodeficiencies or immune responses, especially if the total IgG or IgA concentration is not altered.
Learn more about each isotype: Immunoglobulin Structure and Classes
Figure 1. Annotated diagram of immunoglobulin structure.
Table 1. Human immunoglobulin isotypes and their properties.
| Species | Immunoglobulin | Serum level (Adult [mg/mL]) | Molecular mass (kD) | Activation/Binding of complement | Binding ability to Protein A |
|---|---|---|---|---|---|
| Human | IgA1 | 0.6–3 | 160 (monomer) | – | + |
| Human | IgA2 | 0.06–0.6 | 160 (monomer) | – | + |
| Human | IgM | 0.045–2.5 | 970 (pentamer) | +++ | + |
| Human | IgE | 0.00005 | 188 | – | + |
| Human | IgG1 | 3.8–11.4 | 146 | ++ | ++ |
| Human | IgG2 | 1.5–6.9 | 146 | + | ++ |
| Human | IgG3 | 0.2–1.70 | 165 | +++ | – |
| Human | IgG4 | 0.08–1.4 | 146 | – | ++ |
+++: Very Strong Affinity; ++: Strong affinity; +: Moderate/slight affinity; –: No affinity
Characterization of immunoglobulins
There are multiple methods that can be used for antibody isotyping and the chosen method will depend on the scale and throughput of antibody production, the nature of results needed (i.e., quantitative versus qualitative analysis), assay costs, detection instrumentation, and technical expertise required. These methods include singleplex and multiplex immunoassays, lateral flow assays, microagglutination titer assays, western blotting, indirect immunofluorescence assay (IIFA), and flow cytometry.
Enzyme-linked immunosorbent assays (ELISA) are among the methods of choice for large-scale investigations and can provide accurate quantitative results. Invitrogen isotyping ELISA kits are a fast, sensitive, and convenient method for quantitation of immunoglobulin isotypes during and after hybridoma development and to assess immune response. The Invitrogen ProcartaPlex antibody isotyping panels enable screening titers of individual immunoglobulin isotypes from serum, plasma, and cell culture supernatant samples using Luminex magnetic bead technology.
Learn more about available products for Antibody Isotyping and Characterization
Common applications
Hybridoma screening is one of the most important steps in monoclonal antibody development and it is performed at multiple stages of the process to help ensure that the most productive and specific clones are selected for further evaluation. Antibody isotyping provides a powerful tool for hybridoma development allowing for rapid screening at the various development stages of fusion, cloning, and subcloning to characterize each potential clone. With Invitrogen isotyping ELISA and ProcartaPlex assays, you can accurately and specifically identify which heavy and light chain your hybridoma is producing and if it is monoclonal or not.
Immunization with vaccines induces the production of antibodies that provide protection against pathogens. To better understand the effects on the immunogenic properties of a vaccine and its adjuvants, profiling sera and plasma can help provide useful insight into immune response. Since each antibody isotype possesses unique effector functions, the examination of individual antibody isotypes first produced following vaccination can help elucidate or indicate a primary immune response to a vaccine. For example, IgE is characteristic in allergic reactions, and therefore useful during the assessment of an immune response. If there is no allergic reaction, IgE is usually barely above the limit of detection whereas a strong IgE response could be an indicator of acute-onset reactions to the vaccine. IgA and IgM levels also increase early on and wane after a few weeks, but IgG levels are longer lasting for long-term immunity.
Learn more about SARS-CoV-2 Serology Assays
Isotyping and immunoglobulin ELISA kits
Antibody isotyping ELISA kits are a fast, sensitive, and convenient method for quantitation of immunoglobulin isotypes to assess or to gauge the immune response. Compared to membrane-based isotyping tests, ELISAs provide an accurate quantitative result with a higher throughput for testing more samples at a time. Using these assays, antibody levels of individual isotypes can be easily assessed for individual isotype determination or for multiple isotypes using a single sample.
Invitrogen ELISA kits for popular targets such as IgA, IgE, IgG1, IgM etc. are listed in Table 2. Plate layout for typical Mouse and Rat Ig Isotyping Kits is shown in Figure 2.
Search all isotyping and immunoglobulin ELISA kits
Learn more about ELISA kits and components
Popular isotyping and immunoglobulin protein targets and ELISA performance data
Table 2. View our ELISA kits for the following targets:
Figure 2. Plate layout for typical Mouse and Rat Ig Isotyping Kits. Invitrogen Ig Isotyping Kits are the optimal match for screening your hybridoma in a 96-strip well (8x12) ELISA format. Up to 12 individual samples can be tested for anti-IgG1, IgG2a, IgG2b, IgG2c or IgG3, IgA, and IgM, as well as light chain kappa and lambda.
Isotyping and immunoglobulin ProQuantum high sensitivity immunoassays
ProQuantum high-sensitivity immunoassay kits for immunoglobulin detection are designed to easily provide quantitative measurements in small sample volumes of 1–2 μL. Utilizing proximity-based amplification technology, the assay combines the analyte specificity of high-affinity antibody-antigen binding with the signal detection and amplification capabilities of real-time PCR to achieve a simple yet powerful next-generation protein quantitation platform. A user-friendly, no-wash workflow combined with intuitive software for analytics enables sample-to-answer in just two hours.
Invitrogen ProQuantum immunoassay kits for popular targets such as Tau, Tau pT181 and Troponin I are listed in Table 3. Standard curve of IgE using Human IgE ProQuantum Immunoassay Kit is shown in Figure 3.
Find isotyping and immunoglobulin-related ProQuantum assays
Learn more about how ProQuantum immunoassays work
Read BioProbes Journal article: Introducing ProQuantum High-Sensitivity Immunoassays—The new generation of target-specific protein quantitation
Popular immunoglobulin research protein targets and ProQuantum assay performance data
Table 3. Immunoglobulin-related ProQuantum immunoassays. View our ProQuantum immunoassay kits for the following popular targets:
Figure 3.Representative standard curve of Human IgE. The standard curve for human IgE using Human IgE ProQuantum Immunoassay Kit shows a broad dynamic range (0.00768–3,000 ng/mL) of IgE protein.
Isotyping and immunoglobulin ProcartaPlex multiplex immunoassays
Invitrogen ProcartaPlex isotyping assays are based on the principles of a sandwich ELISA but utilize Luminex xMAP (multianalyte profiling) bead-based technology for the simultaneous detection and quantitation of multiple antibody isotypes targets in a single 25–50 μL sample from plasma, serum, and hybridoma culture supernatants.
The ProcartaPlex isotyping assays are available as preconfigured panels in a ready-to-use format that has been extensively tested for analyte combinability, interference, and cross-reactivity to provide the highest level of validation and precision (Table 4). Panels can also be easily designed to specific research needs by using the ProcartaPlex Panel configurator tool below to make your customized selections.
Comparison data of ProcartaPlex Human Antibody Isotyping Panel, 7plex and IgG4 Human ELISA Kit in human serum samples shows a correlation factor of R2 = 0.98 (Figure 4).
ProcartaPlex Panel Configurator
Learn more about ProcartaPlex multiplex immunoassays
Preconfigured isotyping and immunoglobulin multiplex immunoassay panels and performance data
Figure 4. Performance comparison of ProcartaPlex assay with coated ELISA. Human serum samples were tested in parallel using ProcartaPlex Human Antibody Isotyping Panel, 7plex and IgG4 Human ELISA Kit. The measured concentration of IgG4 shows high correlation (R2>0.95) demonstrating that ProcartaPlex panels achieve quantitative results comparable to traditional ELISA kits with the additional benefits of multiplexing, including reduced sample volume, shorter assay/hands-on time and lower costs per data point.
Table 4. Preconfigured ProcartaPlex multiplex immunoassay panels for isotyping and immunoglobulins.
| Product Name | Size | Cat. No. |
| ProcartaPlex Human Antibody Isotyping Panel, 7plex Target list [bead region]: IgG1 [12], IgG2 [20], IgG3 [34], IgG4 [42], IgA [55], IgE [48], IgM [62] | 96 tests | EPX070-10818-901 |
| ProcartaPlex Mouse Antibody Isotyping Panel 1, 7plex Target list [bead region]: IgA [38], IgE [42], IgG1 [12], IgG2a [18], IgG2b [21], IgG3 [25], IgM [52] | 96 tests | EPX070-20815-901 |
| ProcartaPlex Mouse Antibody Isotyping Panel 2, 7plex Target list [bead region]: IgA [38], IgE [42], IgG1 [12], IgG2b [21], IgG2c [43], IgG3 [25], IgM [52] | 96 tests | EPX070-20816-901 |
| ProcartaPlex Rat Antibody Isotyping Panel, 6plex Target list [bead region]: IgG1 [25], IgG2a [28], IgG2b [34], IgG2c [44], IgA [61], IgM [48] | 96 tests | EPX060-30123-901 |
Additional resources for isotyping and immunoglobulin characterization
Immunoassay instruments
Stylesheet for Classic Wide Template adjustments
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