Detection of immune response antibodies - the immunoglobulin family - is a powerful research tool to identify responses to infectious agents. product blog
By Ingrid R. Niesman, PhD and Lisa Heiden, PhDInfectious disease is a broad topic with emerging worldwide interest. Here at MyBioSource, our products assist in understanding infectious agents. Our catalog is filled with thousands of relevant ELISA kits to detect infectious agents or antibodies recognizing infectious agents, from ZIKA (ZIKV) and Ebola to infectious parasites like malaria. Our kits utilize direct infectious antigen detection or infectious agent exposure by recognition of sera immune response antibodies. �The presence of antibodies to ZIKV is an indication or presumption an organism was exposed to ZIKV and mounted an immune response,� explains Andrew Torres, Technical Service Scientist at MyBioSource. �We also carry a number of other research tools to study ZIKV virus such as ZIKV Virus ENV-NS1 Recombinant (MBS568704) and pairing antibodies such as MBS568046.� Why would a researcher choose one type of ELISA kit over another? The answer lies in the immune response to the myriad of potential infectious organisms.
Generation of immunoglobulins requires exposure to infectious agents
No conversation on infectious disease can begin without an understanding of the basic immune response to each infectious agent. Immunological responses to bacteria, viruses, fungi, or parasites are mediated by both adaptive and innate mechanisms. The latter, the innate immune system, is critical to initiate primary isolation of the agent, phagocytosis and release of cytokines, but not generation of immunoglobulins. The adaptive immune system is the fallback secondary line of initial defense but is the primary fortification to prevent the invaders from attacking again (immune memory). B-cells, constituents of the adaptive response system, are geared to produce highly specific antibodies � the immunoglobulin family - which bind to infectious agents and allow for recognition by the complement system, leading to containment of disease.
We all have a small percentage of antibodies continually circulating but generation of selective anti-infectious agent immunoglobulins requires exposure to individual microbe-specific antigens. In the early phases of infection, B-cells secret large quantities of IgM, each aimed at specific pathogen antigens. In later phases of infection, some B-cells undergo a classical isotype switching mechanism from IgM production to IgG production � the antibody isotype most capable of activating the complement system in response to infectious agents.
Can we use these specific immunoglobulins for detection of exposure?
Originally developed in the early 1980�s, enzyme-linked immunoassays (ELISA) are the staple of research labs studying infectious disease. Traditional ELISA assays utilize polystyrene microtitre plate designs and competitive or sandwich formats for antigen-antibody binding. For research labs, use of 96 well plates or flexible strips works well. Most research labs have access to automated plate readers to detect the assay results, with plate design formats built into the software. These assays are easy to perform, require only small volumes of sample and can be custom built from available antibody pairs, to meet individual lab requirements.
Quantitative ELISA kits are specially designed to detect presence of an antigen and to compare relative sample antigen abundance to a reference recombinant or purified antigen. Antibodies used in quantitative assays must be carefully matched and validated for individual binding properties to eliminate sample bias.
But we don�t always need precise numbers when we think about infectious diseases; sometimes the answer is much simpler. Qualitative ELISA kits are powerful tools in the identification of specifically generated immunoglobulins in sera and bodily fluids. By binding the antigen, such as inactivated whole virus or viral protein, directly to the plate first, followed by dilutions of sera or other bodily fluids next, immunoglobulin, if present � e.g. IgM or IgG - will bind to the target antigen. In other words, the antigen saturates the plate and the immunoglobulin is the limiting agent. Once the antigen and immunoglobulin are bound, the complex is detected chemically, fluorescently or by luminescence. Comparisons to known positive controls and use of stringent negative controls can determine the signal cut-offs for each assay by individual researchers. By offering qualitative ELISA over quantitative ELISA for infectious disease immunoglobulin detection, our research customers can get a yes or no answer, which may be the important result, rather than how much IgM and/or IgG, for example, is in their sample.�The key question is; did the organism mount an immune response as measured by immunoglobulin production against the target or not.
Targeting detection of specific immunoglobulins against an infectious organism is a different methodology compared to using whole virus or viral proteins as the assay target. In an antigen-targeting scenario, antigen specific antibodies are initially bound to the plate, followed by sera or bodily fluids next. In other words, the antibody saturates the plate and the infectious agent antigen in the sample is the limiting agent. The specific plate bound antibodies are generated and purified from immunized animals and need to be characterized for antigen binding properties.
So why would a researcher chose immunoglobulin over antigen detection?
Researchers studying infectious disease use many different tools to detect exposures. Genetic-based assays, like PCR or RT-PCR, are looking for traces of an infectious organism�s nucleic acids circulating in sera or tissues. And an antigen-based ELISA will only result in positive results if the infectious organism or components of the infectious organism are still present in the sample. Assaying once the organism has been contained and eliminated can result in negative responses for either genetic or protein-based assays. However, infectious disease specific immunoglobulins can circulate for months or years after exposure. Therefore, a recent publication selected our ELISA kit � MBS109003 � as a tool in identifying ZIKV IgM in a sample in conjunction with antigen-based assays and target-PCR. We are working hard to supply additional immune response ELISA kits to many new and emerging infectious diseases to aid researchers. �Our goal at MyBioSource is to provide economical, high quality kits which accelerate our understanding of immune responses to many classes of infectious agents,� Technical Service Scientist Torres adds. In addition to our qualitative ELISA kits, MyBioSource also offers purified recombinant proteins, PCR Kits, antibodies, and other reagents for studying many classes of infectious agents at economical prices, including reagents for labs that would like to customize and build their own assays. Our technical service is a mouse click or phone call away (www.mybiosource.com).
MyBioSource products � some examples of immune response and antigen-based ELISA
• Dengue Virus IgM ELISA Kit (MBS580101)
• Dengue Virus IgG ELISA Kit (MBS580100)
• Ebola Virus IgM (EV-IgM) ELISA Kit (MBS108924)
• Ebola Virus ELISA Kit (MBS047374)
• Recombinant Zaire Ebola Virus Nucleoprotein (MBS1206629)
• Measles IgM ELISA Kit (MBS494302)
• Measles IgG ELISA Kit (MBS494527)
• H1N1 IgM antibody ELISA Kit (MBS9310524)
• H1N1 IgG Antibody (H1N1-IgG) ELISA Kit (MBS108943)
SDS PAGE legend
Manufactured-to-order recombinant proteins have various hosts and applications. The recombinant Zaire Ebola virus nucleoprotein, Cat# MBS1206629, was manufactured in E. coli and has an N-terminal�6xHis-SUMO-tag with a nucleoprotein expression sequence of amino acids 488-739 (LDEDDEDTKP VPNRSTKGGQ QKNSQKGQHI EGRQTQSRPI QNVPGPHRTI HHASAPLTDN DRRNEPSGST SPRMLTPINE EADPLDDADD ETSSLPPLES DDEEQDRDGT SNRTPTVAPP APVYRDHSEK KELPQDEQQD QDHTQEARNQ DSDNTQSEHS FEEMYRHILR SQGPFDAVLY YHMMKDEPVV FSTSDGKEYT YPDSLEEEYP PWLTEKEAMN EENRFVTLDG QQFYWPVMNH KNKFMAILQH HQ). This protein was used as antigen in an immunoassay in Ayouba et al (2017: J Clin Microbiol 55:165�176. https://doi.org/10.1128/JCM.01979-16). The Zaire Ebola virus nucleoprotein can also be manufactured in yeast, baculovirus, or mammalian hosts.
ELISA Legend
Comparison of anti-antigen and anti-antigen antibody ELISA assays. MyBioSource offers a large portfolio of both anti-antigen and anti-antigen antibody ELISA Kits. As an example, from their portfolio, the Papillomavirus (HPV) ELISA Kit, Cat# MBS266745 targets the HPV 16L1 capsid protein antigen in samples. In contrast, the Papillomavirus HPV16L1 capsid protein antibody (IgG) ELISA Kit, Cat# MBS903918 targets HPV16L1 antibodies in samples. That is, MBS266745 detects a viral protein (HPV16L1) whereas MBS903918 detects IgG antibodies against the HPV16L1 viral protein.
Additional reading (MyBioSource product references)
- W-G Li, W-W Chen, L Li, et al, Oncotarget, (2016) doi: 10.18632/oncotarget.8558 / Human Yellow fever virus (YFV) IgM ELISA kit, MBS9310907
- D Focosi, F Maggi, M Pistello. Clin Infect Dis. (2016) doi: 10.1093/cid/ciw210 / Human ZIKV IgM (ZV-IgM) ELISA kit MBS109003 and Human ZIKV IgG (ZV-IgG) ELISA kit, MBS109002
- M Culjat, SE Darling, VR Nerurkar, et al, Clin Infect Dis. (2016) 63 (6): 805-811. doi: 1093/cid/ciw324 / Human ZIKV IgM (ZV-IgM) ELISA kit, MBS109003
- A Ayouba, A Toure, C Butel, et al, J Clin Microbiol. (2017) 55 (1): 165-176. doi: 1128/JCM.01979-16 / Recombinant Zaire Ebola Virus Nucleoprotein, MBS1206629