International ISSN: 2471-0016 ICPJL

Clinical Pathology Journal
Mini Review
Volume 3 Issue 3 - 2016
Paper Based Vs Conventional Enzyme Linked Immuno-Sorbent Assay: A Review of Literature
Malik Adeel Anwar1*, Muhammad Arslan Tayyab2, Muhammad Kashif1 and Nadeem Afzal1
1Department of Immunology, University of Health Sciences, Pakistan
2Akhtar Saeed Medical and Dental College, Pakistan
Received: December 01, 2016 | Published: December 28, 2016

*Corresponding author: Malik Adeel Anwar, Research Fellow, Department of Immunology, University of Health Sciences, Lahore, Pakistan, Tel: 0092 321 45 123 66; Email:

Citation: Anwar MA, Tayyab MA, Kashif M, Afzal N (2016) Paper Based Vs Conventional Enzyme Linked Immuno-Sorbent Assay: A Review of Literature. Int Clin Pathol J 3(3): 00079. DOI: 10.15406/icpjl.2016.03.00079

Abstract

This brief note addresses the comparison between conventional ELISA and a slightly newer, equally sensitive, specific, less time consuming and cost effective version of its “Paper based ELISA,” which have a few major benefits on the conventional ELISA.

Keywords: Enzyme-linked immunosorbent assays; Radioimmunoassay; Enzyme Immunoassay; Paper microzone plates

Abbreviations

DIA: Dot-Immunobinding Assay; EIA: Enzyme Immunoassay; ELISA: Enzyme-Linked Immunosorbent Assay; RIA: Radio Immuno Assay; P-ELISA: Paper based ELISA.

Introduction of ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) are widely used as diagnostic tools in medicine as well as quality control measures in various industries. They are also used as analytical tools in biomedical research for the detection and quantification of specific antigens or antibodies in a given sample. ELISA is actually derived from a previously known technique called Radio Immuno Assay(RIA). RIA was first described by Yalow and Berson in 1959, a great discovery for which they won a Nobel Prize in Medicine in 1977 [1]. In search for replacing radioactive isotopes, ELISA was introduced in the 1970s [2]. Currently, many ELISA techniques are used in various fields of science and technology. In the typical double antibody sandwich ELISA, antibody attached to the bottom of a well provides both antigen capture and immune specificity, while the second antibody linked to an enzyme provides detection and an amplification factor. Additional advantages of ELISA include the fact that results are highly quantitative and generally reproducible [3]. At the same time, several weaknesses have been recognized for this method because ELISA performance is largely dependent on antibody quality, kit manufacturer’s error, as well as operator skills and experience dealing are of keen importance [4]. ELISA is routinely performed on 96-well plates to allow samples to be tested singly or in duplicate. This essay is fairly simple, requiring only a small amount of serum and is completed within a day, allowing the analysis of a large number of samples in a relatively short time [5].

Principal of Conventional ELISA

Enzyme Immunoassay (EIA) and ELISA uses the basic immunology concept of an antigen binding to its specific antibody, which allows detection of very small quantities of antigens such as proteins, peptides, hormones, or antibody in a fluid sample. EIA and ELISA utilize enzyme-labeled antigens and antibodies to detect the biological molecules. The most commonly used enzymes are alkaline phosphatase and glucose oxidase. The antigen in fluid phase is immobilized, usually in 96-well microtiter plates allowing the antigen to bind to its specific antibody, which is subsequently detected by a secondary, enzyme-coupled antibody. A chromogenic substrate for the enzyme yields a visible color change or fluorescence, indicating the presence of antigen. Quantitative or qualitative measures can be assessed based on such colorimetric reading. Fluorogenic substrates have higher sensitivity and can accurately measure levels of antigen concentrations in the sample [6]. 

ELISA Types and Their Sensitivity and Specificity

The comparison between different types is given below in the Table 1.

Types of ELISA

Screening

Sensitivity

Specificity

Direct [7,8]

Antibody

Low

Very high

Indirect [9]

Antigen/antibody

High

Low

Sandwich [10]

Antigen

Very high

Low/High

Competitive [11]

Antibody

High

High

Table 1: Comparison among different conventional ELISA techniques on basis of screening agent, sensitivity and specificity.

Paper based ELISA

Porous membranes, including nitrocellulose and filter paper, have been used for decades in Dot Immunobinding Assays (DIAs) [12-14]. Though DIAs are the simplest form of immunoassays on paper, they typically require one piece of nitrocellulose strip for each assay; the pieces of nitrocellulose have to be processed individually in Petri dishes, and the assays take several hours to complete [13]. DIAs is typically qualitative, and provides only “yes/no” results [15] but quantitative DIAs has also been reported [16]. Paper based ELISA was first introduced by White strides Research Group from Harvard who found the filter paper can be used for immunoassay for recognition of antigen and antibody [17].

Requirements for Conventional and Paper based ELISA

Conventional ELISA is usually performed in 96-well plates (fabricated by injection molding in plastic) which are quantitative in nature and well-suited for high-throughput assays. But the drawback includes requiring large volumes (20-200μL) of analytes and reagents for each assay, the time required for incubation and blocking (≥1 hour per step, because the reagents must diffuse to the surface of the wells), and the results are usually quantified using a plate reader instrument which typically costs over a $20 000 [13].

Paper microzone plates for ELISA is almost same as conventional ELISA except it uses paper based 96-well plates prepared via wax printing method. In this microzone paper based wells only about 3μL of sample is required, and the results can be measured using a desktop scanner, costing a $100 instrument only. In addition, the time required to complete an entire P-ELISA is less than one hour. This ease of fabrication of paper microzone plates also opens opportunities for a wide range of non-standard formats, and customized connections to carry reagents between zones [18] (Table 2).

Steps Involved

Conventional

Paper Based

Plate type used

96 wells polystyrene plate

96-microzone paper plate

Antigen immobilization

Pre-immobilized

Self-immobilization

Test Sample

Mostly serum

Serum/Lesional fluid

Secondary antibody

May or may not be used

2oAb conjugated HRP

Washing fluid

PBST

PBST

Substrate

TMB+H2O2

TMB+H2O2

Color development & scanning for results

Special optical ELISA reader

Commercial desktop scanner

Table 2: Steps for Conventional and Paper based ELISAs.

HRP: Horseradish peroxidase enzyme;
PBST: Phosphate-Buffered Saline/Tween;
TMB: 3,3',5,5'-Tetramethylbenzidine;
H2O2: Hydrogen per oxide.

Advantages of P-ELISA

We believe that the combination of ELISA and patterned paper will provide a useful new protocol for performing immunoassays. P-ELISA offers three principal advantages over conventional ELISA:

  1. It is more rapid.
  2. It requires only small volumes (1-10μL) of samples and reagents.
  3. It utilizes simple equipment: a pipette, a refrigerator for storing the reagents, and a scanner.

P-ELISA thus extends the range of application of ELISA, especially to small laboratories, and to developing countries.

The most understandable current disadvantage of P-ELISA is that it is less sensitive than conventional ELISA. It may be possible to decrease the limit of detection for the P-ELISA by optimizing the assay to suppress the background signal.

Conclusion

In conclusion, P-ELISA combines the sensitivity and specificity of the ELISA with convenient, low cost and ease-of-use of paper-based platforms; P-ELISA (at its current state of development) is faster and less expensive than the conventional ELISA, but somewhat less sensitive.

References

  1. Yalow RS, Berson SA (1959) Assay of plasma insulin in human subjects by immunological methods. Nature 184(Suppl 21): 1648-1649.
  2. Crowther J (2008) Enzyme linked immunosorbent assay (ELISA). Molecular Biomethods Handbook, pp. 657-617.
  3. Leng SX, McElhaney JE, Walston JD, Xie D, Fedarko NS, et al. (2008) ELISA and multiplex technologies for cytokine measurement in inflammation and aging research. J Gerontol A Biol Sci Med Sci 63(8): 8798-884.
  4. Aziz N, Nishanian P, Mitsuyasu R, Detels R, Fahey JL (1999) Variables that affect assays for plasma cytokines and soluble activation markers. Clin Diagn Lab Immunol 6(1): 89-95.
  5. Lee EH, Kim YH, Kim S, Kim SE, Kim SC (2012) Usefulness of enzyme-linked immunosorbent assay using recombinant BP180 and BP230 for serodiagnosis and monitoring disease activity of bullous pemphigoid. Ann Dermatol 24(1): 45-55.
  6. Gan SD, Patel KR (2013) Enzyme immunoassay and enzyme-linked immunosorbent assay. J Invest Dermatol 133(9): 1-3.
  7. Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8(9): 871-874.
  8. Van Weemen BK, Schuurs AH (1971) Immunoassay using antigen-enzyme conjugates. FEBS Lett 15(3): 232-236.
  9. Lindström P, Wager O (1978) IgG Autoantibody to Human Serum Albumin Studied by the ELISA‐Technique. Scand J Immunol 7(5): 419-425.
  10. Kato T, Ogami K, Shimada Y, Iwamatsu A, Sohma Y, et al. (1995) Purification and characterization of thrombopoietin. J Biochem 118(1): 229-236.
  11. Yorde DE, Sasse EA, Wang TY, Hussa RO, Garancis JC (1976) Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin. Clin Chem 22(8): 1372137-7.
  12. Hawkes R (1986) The dot immunobinding assay. Methods Mol Biol 121: 484-491.
  13. Sumi S, Mathai A, Radhakrishnan VV (2009) Dot-immunobinding assay. Methods Mol Biol 536: 89-93.
  14. Fletcher J (1987) Filter paper dot-immunobinding assay for detection of Spiroplasma citri. Appl Environ Microbiol 53(1): 183-184.
  15. Kalter SS, Heberling RL, Barry JD, Kuramoto IK, Holland PV, et al. (1992) Detection of antibody to immunodeficiency viruses by dot immunobinding assay. J Clin Microbiol 30(4): 993-995.
  16. Jahn R, Schiebler W, Greengard P (1984) A quantitative dot-immunobinding assay for proteins using nitrocellulose membrane filters. Proc Natl Acad Sci U S A 81(6): 1684-1687.
  17. Cheng CM, Martinez AW, Gong J, Mace CR, Phillips ST, et al. (2010) Paper‐Based ELISA. Angew Chem Int Ed Engl 49(28): 4771-4774.
  18. Carrilho E, Phillips ST, Vella SJ, Martinez AW, Whitesides GM (2009) Paper microzone plates. Anal Chem 81(15): 5990-5998.
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