Relatively few multiplex lateral flow immunoassays (LFIAs) are available today for detecting viral pathogens, primarily due to technical and manufacturing hurdles. However, several of these assays are being developed. The detection of antibodies against more than one agent is probably the easiest type of multiplex assay to manufacture. The key components of such assays are to have specific antigen targets immobilized on a membrane strip which can be detected by the antibodies in a patient’s sera. However, the demand for such tests need to by evaluated as to their market need and whether it is commercially feasible to offer multiplexed assays, rather than monospecific assays for each target.

Anfossi et al1Anfossi et al., 2018. Multiplex Lateral Flow Immunoassay: An Overview of Strategies towards High-throughput Point-of-Need Testing. Biosensors 9 (2), 1-14., give an excellent synopsis of developing multiplex assays using either spatially separated areas (test lines) on the same strip, or a platform which uses multiple strips for each analyte to be detected. A wide variety of multiplex LFIA have been developed to detect antibiotics, drugs, chemicals, poisons and toxins; Song et al.2Song et al., 2015. Multiplex Lateral Flow Immunoassay for Mycotoxin Determination. Anal. Chem. 86, 4995-5001., Peng et al.3Peng et al., 2016. Multiplex lateral flow immunoassay for five antibiotics detection based on gold nanoparticle aggregations. RSC Adv. 6, 7798-7805., and Xing et al44) Xing et al., Ultrasensitive immunochromatographic assay for the simultaneous detection of five chemicals in drinking water. Biosens. Bioelectron. 66, 445-453.. Far fewer have been developed to detect viral pathogens. An example for one of the multiplex LFIA for a viruses is the two-color assay developed by Lee et al.5Lee et al., 2016. Two-color Lateral Flow Assay for Muliplex Detection of Causative Agents Behind Acute Febrile Illness. Anal. Chem. 88, 8359-8363.. Their goal was to use a patient’s serum antibodies to detect 2 different pathogens (dengue and Chikungunya viruses) on the same test strip which also detected both IgM (an indicator of a recent infection) and IgG (an indicator for a past infection) against each virus. This novel assay is actually a 4-plex system, detecting both IgG and IgM for both dengue and Chikungunya viruses. To accomplish this, they used colored latex beads to detect each antibody (blue latex for anti-IgG and red latex for anti-IgM). Lee describes this assay as: the sample pad for accepting the sample, the conjugate pad for storing the detection probes, the nitrocellulose membrane for immobilizing the test and control region reagents, and the absorbent pad for collecting the waste mixtures. The samples (blood/plasma/serum) and chase buffer are added to the sample pad. Once wetted, the blue and red detection probes which have been dry-stored on the conjugate pad are released and become free to interact with the sample IgGs and IgMs which have bound to their respective recombinant viral protein targets in the test line. Further downstream on the strip, secondary antibodies (i.e. anti-goat IgG) capture both blue anti-IgG and red anti-IgM detection probes based on their host species (i.e. goat) and develop mixed colors to confirm that the test has completed successfully.

The paper by Lee et al.6Lee et al., 2016. Two-color Lateral Flow Assay for Muliplex Detection of Causative Agents Behind Acute Febrile Illness. Anal. Chem. 88, 8359-8363., was the first demonstration of the ability to detect both IgG and IgM for more than one pathogen on a single strip. They showed that by observing the color and location of the CHIKV and DENV test regions, their assay could indicate the presence of anti-CHIKV IgG/IgM and anti-DENV IgG/IgM. This approach should be applicable for detecting other multiple pathogen recombinant proteins on a single strip. However, it is mentioned that, as the number of test regions increases there may be a need for a simple analyzer detection system to accommodate users who may have color-perception difficulties.