Challenges in Detection of Serum Oncoprotein: Relevance to Breast Cancer Diagnostics

Figures & data

Figure 1 Diagrammatic representation of different types of ELISA.

Table 1 Advantages and Disadvantages of Different Types of ELISA

Type of ELISA	Advantages	Disadvantages
Direct	● Rapid and easy set-up ● Simple procedure	● Less specific since it employs single antibody. ● Detection antibody should be conjugated. ● Potential for high background signal.
Indirect	● Amplification of signal using a secondary antibody. ● High sensitivity as compared to direct ELISA. ● Cost effective	● Longer protocol due to greater number of steps. ● Potential for cross-reactivity caused by secondary antibody leading to non-specific signal.
Competitive	● Ability to quantitate small molecules with low analyte concentration. ● Sample purification not required prior to use.	● Low sensitivity and specificity
Sandwich	● Highest specificity and sensitivity Antigen purification not required prior to use.	● Costly and time-consuming. ● Requires matched antibody pairs. ● Long procedure.

Figure 2 Three-dimensional structure of Her2-antibody complexes. (Left) Structure of the extracellular domain of Her2 is shown in ribbon representation. Subdomains I–IV are shown in pale-green, orange, green and purple colors, respectively. (Middle) Structure of Her2-Trastuzumab complex is shown. Trastuzumab (blue) binds to Sbd-IV. (Right) Structure of Her2-Pertuzumab complex is shown. Pertuzumab (pink) binds to Sbd-II (PDB: 1N8Z ¹⁵⁹ and 1S78 ¹⁶⁰).

Figure 3 Schematic representation of plasmonic ELISA.

Figure 4 Schematic representation of FACTT. Reproduced from Zhang H, Cheng X, Richter M, Greene MI. A sensitive and high-throughput assay to detect low-abundance proteins in serum. Nat Med. 2006;12(4):473–477.¹⁵

Table 2 Different Methods for Detection of Analytes and Signal Amplification

S.No.	Target	Detection Method Employed	Amplification Method Used	Detection Limit	Reference
1.	Prostate Specific Antigen (PSA) and HIV-1 Capsid antigen	Plasmonic ELISA	Gold nanoparticles (Au NP)	1 × 10^−18 g/mL	[^Citation11]
2	Bovine Serum Albumin (BSA)	Immuno-PCR	Streptavidin-Protein A chimera attached to biotinylated DNA was amplified by PCR and detected by ethidium bromide staining	260bp fragment observed with 580 antigen molecules (9.6 × [10.^-22] mol)	[^Citation12]
3.	Nucleocapsid protein of SFTSV	Gold nanoparticle-based ELISA	Au NP labeled with HRP-labeled monoclonal antibody	0.9pg/mL	[^Citation13]
4.	hCG	Gold nanoparticle-based ELISA	Assembly of Au NP induced by functional polyamidoamine dendrimers	0.03IU/L	[^Citation14]
5.	HER2	FACTT (Fluorescent Amplification catalyzed by T7 polymerase Technique)	T7 RNA polymerase amplifies the DNA template in the streptavidin-biotin-dsDNA template module bound to Ag-Ab complex	0.08fM	[^Citation15]
6.	Gliadin (in Celiac disease)	Indirect ELISA using Au NP as probes	Au NP labeled with HRP-labeled antibody	180pg/mL	[^Citation110]
7.	E. coli 0157:H7	Magnetic ELISA	Poly-L-Lysine mediated brushes on Magnetic NP (MNP) binding to Ab-HRP	8cfu/mL	[^Citation121]
8.	Prostate Surface Antigen (PSA)	Enzyme free ELISA	Enzyme free signal amplification based on gold vesicles encapsulated with Pd-Ir NP	31fg/mL	[^Citation129]
9.	Prostate Surface Antigen (PSA)	Loop mediated isothermal amplification (LAMP) in combination with Solid Phase-Proximity Ligation Assay (SP-PLA)	LAMP	0.0001pM	[^Citation135]
9.	Prostate Surface Antigen (PSA)	Immuno PCR in combination with Solid Phase-Proximity Ligation Assay (SP-PLA)	Real time PCR	0.1pM	[^Citation135]
10.	Shiga Toxin producing E. coli	Immuno-PCR	Immuno capture of Shiga toxin 2 and amplification of a DNA marker using real time PCR	0.1pg/mL	[^Citation137]
11.	MUC1 (tumor protein Mucin 1)	Sandwich immunoassay coupled to EXPAR (isothermal exponential amplification reaction)	EXPAR signal amplification and real-time quantitative PCR (rt-qPCR)	1.63pM	[^Citation140]
12.	Respiratory Syncytial virus (RSV)	Nanoparticle amplified-Immuno PCR (NPA-IPCR)	Signal amplification using multivalent Au NP followed by real time PCR	4.1 PFU/mL	[^Citation143]

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