Advanced antibody profiling tools, such as protein microarrays and luciferase immunoprecipitation systems (LIPS), have revolutionized immunology research because of their ability to evaluate antibody responses to several hundreds to even thousands of antigens at one time.1
This approach particularly plays an important role in vaccine research by providing important insights into the overall immunoreactivity against the entire proteome of certain pathogens.
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The Role of Antibody Profiling in Immunology
Antibody profiling plays a significant role in immunology research.2 Advanced antibody profiling tools have increased the range of antigens tested and enabled a high-quality assessment of humoral responses.
Researchers evaluate antibody response to assess specific clinical symptoms of specific pathogens, enabling disease diagnosis. In vaccine research, antibody titers against specific proteins (infectious agents) indicate the extent of protection induced by the vaccine.3
Antibody profiling enables the identification of B-cell responses against antigens, particularly against conformational epitopes that offer wide-ranging protection for a specific infection. This information is essential to assess the efficacy of a newly developed vaccine, predominantly regarding the duration of protection the vaccine delivers.
Although conventional immunoassay methods, such as the enzyme-linked immunosorbent assay (ELISA) and Western blotting, can efficiently detect antibodies, these methods are tedious as they can only analyze one antigen at a time.
Therefore, it is not easy to identify diagnostically important antigens and those that trigger robust immune responses.
Scientific communities have shown immense interest in assessing antibody responses to whole proteomes for some infectious agents. If conventional immunoassay methods, such as ELISA, were used, this task could be time-consuming and labor-intensive.
In contrast to ELISA, solid-phase antigen arrays can detect immunodominant antigens much more quickly, this method provides limited information regarding conformational epitopes.
Furthermore, it is not regarded as an efficient diagnostic strategy because of its low sensitivity, which results from the presence of impure antigens in the background. Conventional immunoassays also present a narrow dynamic range of detection.
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Advanced Antibody Profiling Tools
Recent high-throughput antibody profiling technologies have enabled large-scale analysis of protein antigens.4 These methods enable the screening of many recombinant protein antigens, ranging between 80 and 2000, to investigate humoral responses against infectious agents. Some of the advanced protein profiling tools are discussed below.
Protein microarrays
Protein microarrays enable antigen target identification from complex proteomes in an unbiased manner.5 This technique was used to analyze sera from syphilis-infected patients, where a total of 900 different Treponema pallidum antigens were arrayed on microtiter plates for antibody profiling.
This enabled the identification of 34 additional proteins with antigenic potential. Notably, this technique identified TP0136 as a protective antigen.6 Besides syphilis, this strategy has also been used to diagnose severe acute respiratory syndrome (SARS) infection.
Antibody array profiling technologies provide useful insights into monitoring and assessing vaccine responses.
For instance, an antibody array using 430 distinct recombinant proteins and chemically synthesized short peptides was used to evaluate antibody responses in a simian model for human HIV infection.7
This strategy helped understand B-cell responses to different HIV multiprotein vaccines. A convergence of immunodominant antibody responses to several linear epitopes was detected in the envelope protein. It must be noted that such information cannot be drawn using conventional ELISA or western blotting approaches.
In vitro antigen arrays
Antigen arrays have been developed based on in vitro transcription/translation (IVTT). This is a relatively simple method that uses polymerase chain reaction (PCR) technique to generate the necessary antigen expression vector constructs compatible with IVTT expression.
A newly developed IVTT antigen array enabled the efficient identification of new antigenic targets.8 For example, the H3L envelope protein was identified as a major antigenic target of neutralizing antibodies induced via smallpox vaccination.
Luminex microsphere immunoassays
The Luminex microsphere immunoassays enable the detection and quantification of multiple antibodies. Scientists have multiplexed Luminex assays to detect antibodies to different pathogens, such as Haemophilus influenzae type b, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheria, and papillomavirus.7
Radiobinding assay (RBA) is a type of solution-phase immunoassay that exhibits high sensitivity and specificity in detecting autoantibodies in autoimmune diseases.
Currently, RBA for a variety of human pancreatic β-cell targets, including IA-2 and GAD65, are used to diagnose and monitor autoantibodies in Type 1 diabetes.9 LIPS is a nonradioactive solution-phase assay that can generate high-quality antibody titer data for most protein antigens.7
Machine learning approach
Implementation of machine learning (ML) has accelerated the antibody profiling process in a cost-efficient manner.
The reduction in experiment cost is mainly associated with the subsequent lowering of the number of experiments that are required in conventional approaches. Scientists experience difficulties in ML-guided antibody design and development (D&D) due to a lack of standardized data sets and evaluation methods.10
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Commercially available antibody profiling assays
Infinity Bio is a technology company that offers a proprietary antibody profiling platform. It provides custom antigen libraries and comprehensive profiling services that significantly helped scientists develop novel vaccines and immunotherapy against many diseases.
Infinity Bio’s antibody reactome analyses enable scientists and clinicians to understand immune patterns essential to developing futuristic personalized treatments across a variety of diseases.
The antibody reactome analyses provide important insights into an individual’s or a population’s immune response against a particular pathogenic protein or vaccine component.
These constitute invaluable information for uncovering complex disease mechanisms, which aid in developing effective diagnosis and treatment for the disease. Their antibody profiling platform enables the discovery of molecular targets and uncover reactivity patterns of autoimmune diseases, such as scleroderma, lupus, and multiple sclerosis.
Other companies involved in manufacturing antibody profiling assays include Infinity Bio, Bio-Rad, Cell Signaling Technology, and Thermo Fischer Scientific. The antibody response profiling assay developed by JPT also plays an important role in vaccine development and immunotherapy.
References
- Burbelo PD, et al. LIPS arrays for simultaneous detection of antibodies against partial and whole proteomes of HCV, HIV and EBV. Mol Biosyst. 2011;7(5):1453-62. doi: 10.1039/c0mb00342e.
- Rezaei M, et al. Immune cell profiling and antibody responses in patients with COVID-19. BMC Infect Dis. 2021;21(1):646. doi: 10.1186/s12879-021-06278-2.
- Earle KA, et al. Evidence for antibody as a protective correlate for COVID-19 vaccines. Vaccine. 2021;39(32):4423-4428. doi: 10.1016/j.vaccine.2021.05.063.
- Lee HE, et al. Development, High-Throughput Profiling, and Biopanning of a Large Phage Display Single-Domain Antibody Library. Int J Mol Sci. 2024;25(9):4791. doi: 10.3390/ijms25094791.
- Berrade L, Garcia AE, Camarero JA. Protein microarrays: novel developments and applications. Pharm Res. 2011;28(7):1480-99. doi: 10.1007/s11095-010-0325-1.
- Brinkman MB, et al. A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin. Infect Immun. 2008;76(5):1848-57. doi: 10.1128/IAI.01424-07.
- Burbelo PD, et al. Antibody-profiling technologies for studying humoral responses to infectious agents. Expert Rev Vaccines. 2010;9(6):567-78. doi: 10.1586/erv.10.50.
- Oulton T, et al. Plasmodium falciparum serology: A comparison of two protein production methods for analysis of antibody responses by protein microarray. PLoS One. 2022 Aug 29;17(8):e0273106. doi: 10.1371/journal.pone.0273106.
- Orban T, et al. Pancreatic islet autoantibodies as predictors of type 1 diabetes in the Diabetes Prevention Trial-Type 1. Diabetes Care. 2009;32(12):2269-74. doi: 10.2337/dc09-0934.
- Khuat T T, et al. Applications of machine learning in antibody discovery, process development, manufacturing and formulation: Current trends, challenges, and opportunities. Computers & Chemical Engineering. 2024; 182, 108585. https://doi.org/10.1016/j.compchemeng.2024.108585
Further Reading