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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 17  |  Issue : 2  |  Page : 53-55

A simple and sensitive immunoassay method for the detection of low-molecular-weight proteins and neuropeptides


1 Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, Kogi State University, Kogi State; Department of Anatomy, Faculty of Medical Sciences, Federal University, Nasarawa State, Nigeria
2 Department of Anatomy, Faculty of Science; Centre of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok; Aquatic Animal Biotechnology Research Center, Surat Thani Campus, Surat Thani, Thailand

Date of Web Publication22-Jul-2019

Correspondence Address:
Dr. Abayomi Ajayi
Department of Anatomy, Faculty of Basic Medical Sciences, College of Health Sciences, Kogi State University, Anyigba, PMB 1008, Anyigba, Kogi State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jeca.jeca_36_17

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  Abstract 


BACKGROUND: Low-molecular-weight proteins and peptides perform numerous regulatory roles in biological systems and could be useful as biomarkers. Hence, qualitative and quantitative measurement of peptide levels has become increasingly important.
AIM: This present study was aimed at developing a simple and sensitive dot immunoblot assay (DIA) by modifying existing protocols.
MATERIALS AND METHODS: Using polyclonal antibody generated against FMRFamide peptide, at a dilution ratio of 1:10,000 in buffer, as a primary antibody, alkaline phosphatase-conjugated goat anti-rabbit antibody as secondary antibody and nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate development system, peptides were dotted on 0.2μm nitrocellulose membrane in volumes of 1μl, 0.5μl and 0.2μl.
RESULTS: Results show immunoreactivity of FMRFamide antibody to small quantities of dotted neuropeptide as low as 0.2ng and 33.4pM.
CONCLUSION: This indicates that this protocol is sensitive with the additional advantage of simplicity, speed, small sample volume, low cost, and production of nonhazardous waste. This DIA protocol could be useful in resource-poor settings and laboratories with low budgets.

Keywords: Dot immunoblot assay, low-molecular-weight proteins, neuropeptides, peptides


How to cite this article:
Ajayi A, Withyachumnarnkul B. A simple and sensitive immunoassay method for the detection of low-molecular-weight proteins and neuropeptides. J Exp Clin Anat 2018;17:53-5

How to cite this URL:
Ajayi A, Withyachumnarnkul B. A simple and sensitive immunoassay method for the detection of low-molecular-weight proteins and neuropeptides. J Exp Clin Anat [serial online] 2018 [cited 2019 Nov 20];17:53-5. Available from: http://www.jecajournal.org/text.asp?2018/17/2/53/263166




  Introduction Top


Low-molecular-weight proteins and peptides are small polypeptides, normally ranging from 3 to 100 amino acid residues (DeWied, 1969; Kastin et al., 1979; Strand, 1999). They perform numerous regulatory roles in biological systems and could be useful as biomarkers (Schulte et al., 2005). Hence, qualitative and quantitative measurements of low-molecular-weight proteins and peptide levels have become increasingly important (Rubakhin and Sweedler, 2008; Gillette and Carr, 2013).

One of the major problems associated with the assessment of low-molecular-weight proteins and peptides is their small molecular mass and often low quantity which makes them difficult to detect in samples. For example, FMRFamide neuropeptide, which was first discovered in the mollusc (Price and Greenberg, 1977), is a peptide with very small molecular weight. Attempt at isolating FMRFamide-related peptides (FaRPs) in tissue samples of the sea cucumber, Holothuria scabra, has led to the development of a sensitive detection technique for this peptide. In this paper, we present a simple and sensitive immunoassay method for the detection of low-molecular-weight proteins and peptides, as applied in the study of FaRPs.


  Materials and Methods Top


Synthetic peptide

Synthetic FMRFamide peptide (>98% pure) was purchased from GenScript (USA).

Antisera

Polyclonal antibody, raised against the tetrapeptide FMRFamide, was used in the experiment. Commercially available rabbit polyclonal antibody against FMRFamide was procured from ImmunoStar Inc. (USA). The antibody was raised against FMRFamide–thyroglobulin conjugate and preabsorbed with 100 μg thyroglobulin, according to the manufacturer.

Immunoassay

An 8 cm × 6 cm size membrane cut from Amersham 0.2 μm nitrocellulose membrane (GE Healthcare Life Sciences) was divided into “wells” using a pencil and ruler. Serial dilutions of 1 μg/μl of FMRFamide neuropeptide were spotted on the membrane in volumes of 1 μl, 0.5 μl, and 0.2 μl. The membrane was air-dried at room temperature for 30 min and baked at 100°C for 30 min to fix the peptides to the membrane. After cooling to room temperature, the membrane was washed for 10 min in a small volume of 0.1M Tris buffer (pH 7.4) with 0.05% Tween 20 (v/v; buffer 1), and then incubated for 60 min in a blocking solution containing 3% bovine serum albumin in 0.1M Tris buffer (pH 7.4). The membrane was then incubated overnight in anti-FMRFamide polyclonal antibody (1:10,000 in buffer 1) and washed three times for 5 min each in buffer 1 before incubation in alkaline phosphatase-conjugated goat anti-rabbit secondary antibody (1:500 in buffer 1) for 1 h. The membrane was washed again three times for 5 min each in buffer 1 and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate (1:100 in development buffer). After about 15 min, the membrane was washed in distilled water, air-dried, and photographed. The number of mole of neuropeptide in each well was calculated using the following formula:




  Results Top


The concentrations of dotted peptide on the nitrocellulose membrane are provided in [Table 1] and [Table 2]. The results, as presented in [Figure 1], show immunoreactivity of FMRFamide antibody to different concentrations of FMRFamide neuropeptide at 1 μl, 0.5 μl, and 0.2 μl with the least detectable neuropeptide concentration at about 0.0002 μg or 0.2 ng which corresponds to 3.34 × 10−13 M or 33.4 pM of FMRFamide.
Table 1: Volume and concentration of dotted peptide

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Table 2: Molar mass of dotted peptide

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Figure 1: Dot immunoblot assay of FMRFamide peptide

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  Discussion Top


In the present study, dot immunoblot assay (DIA) was capable of detecting FMRFamide neuropeptide at concentrations as low as 0.2 ng and 33.4 pM but not beyond 0.02 ng or 0.334x fM. The lowest sample volume of 0.2 μl was also observed to have the highest sensitivity. Three reasons could be responsible for the high degree of sensitivity shown by this technique. First is the low sample volume dotted on the membrane and second is the dilution factor with which the primary antibody 1:10,000 in the buffer. Another possible contributing factor is the development system used. Our preliminary study with under the same conditions has shown that alkaline phosphatase-conjugated antibody is more sensitive with lower background than 3,3', 5, 5'-tetramethylbenzidine. This DIA can be used in the isolation of neuropeptides from tissues (Ajayi and Withyachumnarnkul, 2013). In addition, when compared to similar protocols such as that of Salzet et al., with sensitivity of 1.32 pmole (Salzet et al., 1993; Loi et al., 1997), we find this protocol to be equally sensitive. It has the additional advantage of simplicity, speed, small sample volume, low cost, and production of nonhazardous waste. This DIA protocol is recommended for laboratories with low budgets and might be useful in resource-poor settings.


  Conclusion Top


This sensitive protocol is simple, is fast, requires small sample volume, is cost-effective, and does not produce any hazardous waste. Hence, this DIA protocol is recommended for laboratories with low budgets, and this might be useful in resource-poor settings.[10]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Ajayi A., Withyachumnarnkul B. (2013). Presence and distribution of FMRFamide-like immunoreactivity in the sea cucumber Holothuria scabra (Jaeger, 1833). Zoomorphology 132:285-300.  Back to cited text no. 1
    
2.
DeWied D. (1969). Effects of peptide hormones on behavior. In: Ganong W.F., Martini L, editors. Frontiers in Neuroendocrinology. Oxford University Press, New York, p. 97-140.  Back to cited text no. 2
    
3.
Gillette M.A., Carr S.A. (2013). Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry. Nat Methods 10:28-34.  Back to cited text no. 3
    
4.
Kastin A.J., Olson R.D., Scally A.V., Coy D.H. (1979). CNS effects of peripherally administered brain peptides. Life Sci 25:401-14.  Back to cited text no. 4
    
5.
Loi P.K., McGraw H.F., Tublitz N.J. (1997). Peptide detection in single cells using a dot immunoblot assay. Peptides 18:749-53.  Back to cited text no. 5
    
6.
Price D.A., Greenberg M.J. (1977). Structure of a molluscan cardioexcitatory neuropeptide. Science 197:670-1.  Back to cited text no. 6
    
7.
Rubakhin S.S., Sweedler V.J. (2008). Quantitative measurements of cell-cell signaling peptides with single-cell MALDI MS. Anal Chem 80:7128-36.  Back to cited text no. 7
    
8.
Salzet M., Bulet P., Van Dorselaer A., Malecha J. (1993). Isolation, structural characterization and biological function of lysine-conopressin in the central nervous system of the pharyngobdellid leech Erpobdella octoculata. FEBS Lett 217:897-903.  Back to cited text no. 8
    
9.
Schulte I., Tammen H., Selle H., Schulz-Knappe P. (2005). Peptides in body fluids and tissues as markers of disease. Expert Rev Mol Diagn 5:145-57.  Back to cited text no. 9
    
10.
Strand F.L. (1999). Neuropeptides: Regulators of physiological processes. MIT Press, Cambridge.  Back to cited text no. 10
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2]



 

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