Number of distinct features of IgY compared to IgG have turned the pages towards the application of these antibodies in diagnostic assays and kits. The major point that has to be kept in mind when it comes to diagnosis is the avidity of protein towards the target antigen i.e., antigen-binding specificity. Mammalian proteins are highly conserved and when these are used as antigen in mammals, it might pose less or no antigenicity. Due to this phylogenetic evolutionary difference, IgG usage in medical diagnostics might lead to false positive results. Adding more, proteins in human serum might activate the complement system thereby interfering with the result. Thus, to avoid these hurdles, usage of IgY antibodies have been a better option 17. These IgY antibodies when conjugated with nanoparticles have a great potential in conferring increased sensitivity and accuracy for different immunoassays 18.
Among the different nanoparticles that are used for diagnostic applications, gold nanoparticles are widely used owing to its good optical and electronic properties. Gold nanoparticles serve as an excellent platform in biosensing applications as they have high surface area to volume ratio and biocompatibility. Gold nanoparticle-based immunoassays have been largely benefitting biologists as they are suitable cell targeting vectors for qualitative and quantitative analysis. In order to sense the analyte, the surface of nanoparticles is fabricated using specific moieties to increase sensitivity and selectivity. Back in 2009, Lisa et al., developed an immune dipstick to detect organochlorine pesticide at nanogram level. For detecting, IgY antibodies were made specifically against this pesticide and were then immobilized onto the strip. The results were then evaluated using visual pictures, demonstrating the suitability of this immunoassay approach for the quick and accurate detection of Dichlorodiphenyltrichloroethane (DDT). Further, studies can be extended to other pesticides and interference of other antigens in the sample need to be investigated as well. Hence, this study enabled rapid, specific and on-site detection of the analyte with a detection limit of about 27 ng/mL 19. Another study by Zhang et al., detected Swimming crab reovirus (SCRV), a causative agent that causes severe haemorrhaging in swimming crab leading to 100% mortality within 10 days thereby affecting the entire crab industry. With the aim of detecting SCRV, authors developed IgY-gold complexes to monitor and control diseases in crab farm. This study was more like a one-step immunochromatographic (ICA) strip enabling on-site rapid detection of the antigen. Polycloncal antibodies usually results in cross-reactivity and low specificity hampering the intended outcome. But conjugation of gold nanoparticles with IgY antibodies increased specificity and selectivity towards the different aquatic-associated pathogens. In fact, IgY being more hydrophobic in nature, interacts and adsorbs better on the surface of gold nanoparticle through hydrophobic bonds. Additionally, its sensitivity, feasibility, and speed were satisfactory. Additionally, with the detection time of less than 10 min, the ICA strip had a high consistency of about 98.28% 20. Egea et al., developed a diagnostic kit for the detection of indoor dust mite allergens using polyvalent IgY antibodies conjugated to colloidal gold nanoparticles. The ability of conjugates to detect the allergen was evaluated using immune-dot blot assay. Results showed that these conjugates were able to recognize the allergens with a detection limit of 0.78 microgram 21. Other studies reported by Yeristyan, Selvakumar, Nie and Yaghubi used IgY conjugated gold nanoparticles for different purposes 21-23, 26. Chemiluminescence immunoassay is widely used for the detection of human IgG levels in humans. Owing to its high cost, lack of sensitivity and selectivity due to physiochemical parameters, it’s highly imperative to search for an alternative. And one such study by Yeritsyan21 et al., have opened up avenues in this aspect. The authors used IgY antibodies conjugated onto gold nanoparticles for the detection of human IgG. IgY antibodies being more acidic than rabbit IgG is expected to destabilize gold nanoparticles at higher concentrations thereby providing sufficient of antibodies for immunoassay. By immobilizing IgY antibodies over the gold nanoparticles, the authors were able to achieve a detection limit of 30 ng/mL. Though this approach gave good results by increasing detection range and sensitivity, this seem to be time-consuming 22. Selvakumar et al., developed an immunodipstick based gold nano sensor to detect vitamin B12 in fruits and energy drinks and were able to achieve 1 ng/mL detection limit. The presence of vitamin B12 was identified on the basis of the colour intensity obtained. The intensity of colour developed was inversely proportional to the amount of analyte present in the food sample. The results obtained from this dipstick based immunosensor were comparable to the results obtained from standard ELISA method thereby proving its efficacy, reproducibility, stability and sensitivity. In fact, this methodology doesn’t require hands of skilled persons. This study has a full potential in emerging as a biosensor tool for detection of various analytes like toxins, adulterants, microbes and for monitoring purpose 23. Another similar study using gold nanoparticles to detect food borne pathogens, E. coli O157:H7 was reported by Yaghubi et al., The fabricated localized surface plasmon resonance based nano biosensor helps in optically detecting the presence of bacteria by measuring the change in LSPR band. It showed high specificity, speed, limit of detection of about 10 CFU/mL and precision with no requirement of specialized hands thereby serving as a great tool for detection of other clinical specimens. A different strategy i.e., rapid immunochromatographic assay using Fe3O4/Au composite nanoparticles were synthesised for the detection of same pathogen (E. coli O157:H7) 24. This study by Xue et al., made anti-E. coli O157:H7 IgY and conjugated that to a core/shell structured super-paramagnetic composite nanoparticle. The pathogens were detected along quantitative measurement of concentration based on the magnetic signal intensities obtained and were able to achieve a detection limit of 103 CFU/mL 25.
For a change, researchers started to employ gold nanoparticles along with other nanoparticles to improve the detection limit, sensitivity and selectivity. In that aspect, a study by Liu et al., demonstrated the detection of a food-borne pathogen - Vibrio parahaemolyticus, using a fluorescence quenching system by using gold nanoparticles. CdSe/ZnS quantum dots were conjugated to IgY-gold conjugates. With no pre-requirement of sample preparation, the developed fluorescence turn-on assay seemed to be highly sensitive and selective under optimum conditions with a limit of detection of 10 CFU/mL 26. Yet another study reported by Nie et al., (is similar to the aforementioned study) for the detection of a different pathogen. IgY against EV71 virus were produced and conjugated to gold nanoparticles. a fluorescence quenching system was formed thereafter using quantum dots and IgY-gold nanoparticle conjugates. In the presence of target virus, the IgY-gold nanoconjugates aggregated together emitting fluorescence signal without quenching. Based on the intensity of signal, the concentration of virus was found and was able to achieve a detection limit of 104 CFU/mL. This strategy is a potential tool for routine screening of any infection causing virus 27.
On the other hand, three different studies were postulated to use gold nanoparticles for detecting a bacterial pathogen, Staphylococcus aureus (SA) that causes gastrointestinal infection. A report by pang et al., was distinguishing wherein they had used gold nanorods as they are shown to absorb more biological moieties than gold nanospheres, to which IgY and urease enzymes were bound. Using these dual labelled gold nanorods, a rapid colorimetric detection probe was developed. Not only do they bind to bacteria, but also hydrolyse urea. Subsequent mixing of this probe in non-specific magnetic beads lead to the formation of sandwich-type immunocomplex, that is, dual labelled gold nanorod probe-SA-non-specific magnetic bead (iron oxide nanoparticle). These immunocomplexes are separated in the presence of external magnetic field subsequently leading to change of colour of test paper based on the presence of target bacteria. This is a novel, rapid and specific strategy as they have used two different nanoparticles with two different biological entities. In addition to the detection value obtained, the result can also be visually observed. This technique with limit of detection of 460 cfu/mL has a huge potential in food safety application for screening bacteria 28.
The next study by Yao et al., used IgY- nanoconjugates made of gold and iron oxide nanoparticles instead of rods, also, making changes in the mode of detection. Herein, the colorimetric assay developed for detection was based on etching-enhanced peroxidase-like catalytic activity of gold nanoparticles. No change of colour was observed after wet etching in the presence of hydrogen peroxidase in the sample containing the target bacteria. The lowest level of bacteria that it was able to detect was about 10 cfu/mL in addition to visual confirmation. Both these studies by (yao & pang) have attained a different limit of detection just by changing the mode of detection and shape of nanoparticles 29. Yet another study by Roushani et al., developed gold nanoparticle with modifications. The authors had attached IgY on to the gold nanoparticle which was modified using glassy carbon electrode. IgY was able to better capture the bacteria due to high sensitivity and selectivity, while with the increased surface area of modified gold nanoparticle they were able to significantly amplify the obtained electrochemical signal. This immunosensor was able to accurately detect bacteria with detection limit of 3.3 cfu/mL. This enhanced limit of detection can be attributed to the deposition of gold nanoparticles on the glassy-carbon electrode. This could have enhanced the immobilization of IgY-conjugated gold nanoparticles thereby improving its outcome 30.
The next widely used nanoparticles in the field of diagnostics are quantum dots because of their inherent optical and electronic properties. Moreover, conjugation of biomolecules with quantum dots helps achieve high specificity, sensitivity and biocompatibility. Quantum dots are easily excitable, produce strong luminescence thereby possessing high photostability. Chouhan et al., developed a diagnostic kit by conjugating IgY antibodies onto Cadmium telluride quantum dot for sensitive and specific detection of methylparathion (pesticide). This was shown to be effective over HPLC, ELISA and other techniques because of the combinatorial effect of antigen-antibody interaction and highly fluorescent CdTe quantum dot. This rapid and reliable sensor was able to achieve a limit of detection of about 0.1 ng/mL 31. Kannagasubulakshmi et al., developed mercaptopropionic acid capped cadmium telluride quantum dots for visual detection of insecticidal proteins. IgY antibodies were conjugated onto the nanoparticles for enabling the detection of transgenic proteins whereas nitrocellulose membrane was used as assay matrix to enable fluorescence signals from quantum dots. By this technique, the authors were able to achieve a limit of detection of 2.91 ng/mL. This was a unique strategy of developing a reliable and a highly photostable platform for detection of chemical and biological moieties 32. A lateral flow immunochromatographic strip (LCS) was developed by Zhang et al., using IgY conjugated quantum dots (CdTe/ZnS core shell quantum dots) wherein the extracted IgY was developed to detect rhein, a component found in traditional Chinese medicine. The mode of detection was based on competitive-immunoassay principle which showed qualitative detection limit of 98.2 ng/mL. The results were good and corresponding to the results obtained from the conventional techniques like HPLC. In fact, in a similar study by the same authors 33 this detection range was almost same to that obtained from LCS conjugated with IgG. This correlation makes IgY antibodies a promising component for providing best results with no interference with mammalian proteins or the rheumatoid factors. Not only that, use of IgY antibodies help in reduction of production cost in addition to the promotion of animal-welfare unlike IgG antibodies 34.
Very interestingly, a study by Li et al., reported the development of a detection method that uses fluorescence signal to detect a member of intracellular bacteria, Brucella melitensis 16M in food samples based on peptide-mediated magnetic separation (PMS) technology and Au nanoparticles (AuNPs)-mediated bio-barcode assay technology assembled by quantum dots (QDs). This study stands distinguishing owing to its remarkable methodology of combining both gold nanoparticles and quantum dots along with fluorescence signal. IgY was conjugated to DNA-attached gold nanoprobe. On the other hand, complementary DNA probe was attached to quantum dots. Upon exposure, IgY on gold nanoparticles bind to the bacteria while the oligonucleotide chains hybridise with the complementary chains found in quantum dots. The unbound quantum dots-DNA probe in the supernatant are used to indirectly measure the concentration of bacteria present in the sample by detecting the fluorescence. With this strategy, the authors were able to achieve a limit of detection of 1.07x102 CFU/mL in milk and 1.72 x 102 CFU/mL in lamb leach 35.
One other study by Liu et al., developed a colorimetric assay using gold nanoparticles along with silver nanoclusters to rapidly detect a food-borne pathogen, Listeria monocytogens. Most of the colorimetric assays are based on aggregation of nanoparticles which might give out false-positive results. Hence, this study focused on silver nanoclusters which could potentially act as artificial enzyme on to which IgY antibodies and aptamer attached magnetic beads. Based on the oxidation mediated by silver nanoclusters, gold nanoparticles redisperse in the solution leading to colour change whose intensity is proportional to the concentration of the pathogen. The achieved limit of detection was as low as 10 CFU/mL 33.
Same authors developed a different nanoparticle for the detection of same pathogen in food. Liu et al., described a multicolorimteric assay using gold nanorods along with aptamer-modified magnetic beads and IgY coated MnO2 nanoparticles. IgY attached nanoparticles recognise the bacteria which is further separated using magnetic beads. These sandwich complexes attached to bacteria catalyse the chromatographic substrate, TMB. Gold nanorods act upon (etch) this catalysed substrate giving out different colours based on the concentration of pathogen present in food. By this, the authors were able to detect the bacteria at a detection limit of 10 CFU/mL 36.
There were other studies which used different nanoparticles unlike the usual ones. A study by Kilinc et al., employed latex nanoparticles. IgY was synthesized against M2e peptide. Latex nanoparticles were conjugated to anti-M2e IgY antibody in the latex agglutination assay for diagnosing influenza A virus. This methodology was able to specifically recognize two different strains of influenza virus, that is, H3N2 and H1N1. Though this study has potential in diagnosis, prevention and treatment of influenza A virus, the limit of detection, its specificity and selectivity are yet to be discussed (Table 2) 37.
(Here Table 2)