1. drawback for these traditional LFAs using

1.     Phage Nanoparticle Reporters Lateral Flow Assay

The assay employs both biotinylated anti-norovirus antibodies and gold-labeled anti-norovirus antibodies; when target noroviruses are present in the sample, virions associate with the antibodies while flowing through the strip. A streptavidin test line captures the gold-labeled migrating complexes via the biotinylated anti-norovirus antibodies. Migrating gold-labeled antibodies not bound in the complex are bound later at the control line. The main drawback for these traditional LFAs using colored particles such as blue latex or gold nanoparticles, is the high Limit of Detection (LoD).This assay is specific for Noroviruses.5

 

 

Fig 1. Phage lateral-flow assay detecting Norwalk VLPs. Assay membrane is nitrocellulose (FF80HP, 5×40 mm), sample pad is Fusion 5 (5×20 mm), Absorbent pad is CF5 (10×30 mm). Control line consists of anti-M13 antibodies (0.25 ?g/cm) and test line is anti-Norwalk monoclonal antibodies (1.0 ?g/cm).

 

 

2.     Reverse Transcription Loop-Mediated Isothermal Amplification Assay

In this method, a DNA copy of the viral RNA is generated by reverse transcriptase, and then isothermal amplification is carried out to increase the amount of total DNA. Primer binding sites are chosen so that a series of strand displacement steps allow continuous synthesis of DNA without requiring thermocycling. Reaction products can be detected by adding an intercalating dye to reaction mixtures that fluoresces only when bound to DNA, allowing quantification of product formation by measurement of fluorescence intensity.This assay is capable of detecting HIV-1 subtypes (A,B,C,D & G).12

 

 

3.     ICP 11- Dependent Immunomagnetic Reduction (IMR) Assay

In an IMR assay, a specific antibody that recognizes the target protein is immobilized on magnetic nanoparticles, and thus enables them to specifically bind to the target protein. Magnetic nanoparticles in reagents oscillate with alternating current (AC) magnetic fields via magnetic interaction. Therefore, under the influence of external AC magnetic fields, the original reagent containing homogeneously dispersed magnetic nanoparticles generates a magnetic signal, called multiplefrequency ac magnetic susceptibility ?ac,0 . After target proteins are bound to antibody- labeled magnetic nanoparticles, the resulting magnetic susceptibility is designated ?ac,?  Due to the formation of magnetic clusters, ?ac,? is smaller than ?ac,0.. Thus, the reduction in ?ac of the magnetic reagent is used to determine the concentration of the target protein. Due to cost efficiency, quantification and ease of automatization, the IMR platform is becoming increasingly important as a diagnostic approach. IMR assay is  used to detect White Spot (WSD) and also Nervous Necrosis Virus (NNV), a major viral pathogen for grouper and other fish.7

 

 

 

Fig 2. Overview of immunomagnetic reduction (IMR). (A) Each magnetic nanoparticle, bio-functionalized with antibodies against target proteins, oscillates with the applied alternating current (AC) magnetic field before binding with ICP11. ?ac,0: the original multiple-frequency AC magnetic susceptibility of the magnetic nanoparticles (B) When these magnetic nanoparticles bind to target proteins, they become larger, and some even form clusters. This reduces the AC magnetic susceptibility of the reagent. ?ac,?: the resulting magnetic susceptibility of magnetic nanoparticles after binding with the target proteins

 

4.     Blocking ELISA

For serological diagnosis in pigs, anti-HEV antibodies are universally detected using two methodologies: commercial kits optimized for detection of human anti-HEV antibodies that have been adapted to use for swine and inhouse indirect Enzyme-Linked ImmunoSorbent Assays (iELISAs) using genotype 3 or 4 HEV ORF2 proteins as coating antigens .Unfortunately, these iELISAs have not yet been validated, due to the absence of appropriate “gold standard” for comparison  and studies have shown that iELISAs often provide discordant results and non-specific background signals. In contrast, monoclonal antibody-based blocking ELISA (bELISA) could decrease non-specific binding artifacts and improve the specificity of detection of antibodies in serum samples. Detection of serum antibodies specific for ORF2 was performed by observing their inhibition of binding of HRP-conjugated monoclonal antibody to the coating antigen. As compared with the iELISA, which also used a truncated ORF2 coating antigen, the bELISA had same sensitivity and higher specificity.3

 

 

5.     Lateral Flow Antigen Detection System

A lateral flow assay using mAbs (monoclonal antibodies) which allows for rapid detection of Foot and Mouth Disease (FMD) antigen for all seven serotypes have been demonstrated. The advantage of mAbs is that it is possible to select highly efficient mAbs for high specificity and sensitivity. In this detection system, FMD virus serotyping strip was used which confirmed more sensitive than IS-ELISA. The difference of sensitivity was probably due to the ability of mAb that were used in this system.11

 

 

6.     Antibody Derived Peptides

Current methods to diagnose the presence of the EBOLA virus (EBOV) in biological samples rely mainly on PCR. These methods are able to detect EBOV at low viral loads with high accuracy and reproducibility, but they require special instrumentation and trained personnel, which impose heavy restrictions for use of PCR in Ebola epidemic scenarios. An alternative include use of monoclonal and polyclonal antibodies. However, the production of full length antibodies is a complex process. Thus antibody fragments present several potential advantages over the use of full-length mAbs. Antibody fragments contain the variable regions of a full-length antibody and conceptually they retain the binding specificity of full-length antibodies. Three antibody fragments were designed and produced namely Fab-K252 ,SCFV-13C6,SCFV-13F6 respectively named after their full length counter  parts, mAb K252, mAb 13C6 and mAb 13F6. These binds the mucin like domain in glycoprotein(gp). K252 is a full length mAb originally isolated from EBOLA disease survivor . All these fragments were expressed in E.coli and produced using fed batch culture protocol. These antibody fragments capture glycoprotein.16