Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae

Summary

In Mycoplasma pneumoniae infection, serology tests can generate good results, yet with low specificity because of immunological cross-reaction. The in-house antigen-capture ELISA, described in this paper, guarantees high species specificity and has been shown to be a reliable screening test for accurate diagnosis of M. pneumoniae.

Abstract

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks every 6 years, in older children and young adults. Diagnosis of M. pneumoniae is challenging because of the fastidious nature of the pathogen and the possibility of asymptomatic carriage. Laboratory diagnosis of M. pneumoniae infection based on antibody titration in the serum samples of patients remains the most practiced method. Because of the potential problem of immunological cross-reactivity with the use of polyclonal serum for M. pneumoniae, an antigen-capture enzyme-linked immunosorbent assay (ELISA) has been developed to improve the specificity of serological diagnosis. ELISA plates are coated with M. pneumoniae polyclonal antibodies, raised in rabbits and rendered specific after adsorption against a panel of heterologous bacteria that share antigens with M. pneumoniae species and/or are known to colonize the respiratory tract. The reacted M. pneumoniae homologous antigens are then specifically recognized by their corresponding antibodies in the serum samples. Further optimization of the physicochemical parameters to which the antigen-capture ELISA is subjected led to a highly specific, sensitive, and reproducible ELISA.

Introduction

Mycoplasmas are among the smallest and simplest known prokaryotes. They are mainly distinguished from other bacteria by the lack of a cell wall structure. Thus, Mycoplasmas were classified in a separate class named Mollicutes¹. The cell wall deficiency confers intrinsic resistance to these microorganisms against some antimicrobial agents and is largely responsible for their polymorphism. Mycoplasmas have small genome and reduced size, which limits their metabolic and biosynthetic capabilities and explains their parasitic and saprophytic nature¹.

Mycopla....

Protocol

The present study has been conducted in conformity with ethical aspects established by the ethical committee of Pasteur Institute of Tunis.

1. Pre-ELISA steps: Prerequisites and preprocessing

1. Bacterial strains and growth media
   NOTE: Mollicutes species and the walled bacteria used in the present study and their growth media are listed in Table 1.
   1. Growth of Mollicutes species
      1. Inoculate 200 µL from the glycerol stock of each species in 1,800 µL of the media.
      2. Grow Mollicutes species statically at 37 ˚C with 5% CO₂ for 2....

Results

The immunoblotting activity of the non-adsorbed polyclonal Mycoplasma pneumoniae antiserum to heterologous bacteria
Cross-reactivity does indeed exist as showcased in the immunoblotting results (Figure 2) and compared to the positive control (Lane 1), some of the M. pneumoniae antigens are shared with the screened bacteria. The intensity of these cross-reactions was variable. For instance, M. gallisepticum and .......

Discussion

This paper presents a general description of an in-house ELISA mainly developed to ensure specific screening of M. pneumoniae infection. The details about the protocol of the ELISA assay itself as well as some pre- and post-processing steps are provided. The specificity of this assay is ensured by the adsorption technique. This procedure was previously described in ELISA tests developed for the diagnosis of human and avian Mycoplasmas^17,24. It w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-chloro-1-naphtol	Sigma-Aldrich	C6788-50 TAB
Bacto peptone	BD	211677
Bacto tryptone	BD	211705
Bovine serum albumin	Sigma-Aldrich	A9647-50 G
Carbonate bicarbonate buffer	Sigma-Aldrich	C3041-50 Cap
Casein	Sigma-Aldrich	C7078-1 KG
CMRL1066	VWR	P0058-N1L
D-Glucose	Sigma-Aldrich	G7528-1KG
Difco PPLO Broth	BD	255420	Frey media
ELISA plate-Reader	MULTISKAN GO, Thermo Scientific	Ref: 51119200
Fetal bovine serum	Capricorn Scientific	FBS-12A
Goat peroxidase-conjugated anti-human IgG	Abcam	ab6759
Goat peroxidase-conjugated anti-rabbit IgG	Life Technologies	656120
Hydrochloric Acid 37%	Prolab	2025.290
Hydrogen peroxide (H2O2), solution 30%	Scharlau	HI01361000
L-arginin	Sigma-Aldrich	A5006-1KG
LB broth	Prepared in Pasteur Institute of Tunis		Provided by the laboratory of bacteriology of the Pasteur Institute of Tunis
Mycoplasma pneumoniae polyclonal antiserum produced in rabbit	Produced in Pasteur Institute of Tunis		Serum was produced by rabbit immunization at the Pasteur Institute of Tunis
Nicotinamide adenine dinucleotide	Sigma-Aldrich	N7004-10G
Nunc Maxisorp flat-bottom 96-well microtiter plate	Invitrogen	44-2404-21
Penicillin G sodium (1 MIU)	PANPHARMA
Phenol red	fluka chemika	77660
Skim milk	MP Biomedicals	902887
Sodium bicarbonate	Sigma-Aldrich	S6297-1KG
Sodium carbonate	Sigma-Aldrich	S7795-1KG
Sodium chloride (NaCl)	Novachim	PS02805
Sulfuric acid (H2SO4) 95-97%	Merck	1007311011
Thimerosal	USB	22215
TMB substrate (3,3’, 5,5’ TetraMethyl-Benzidine solution)	Abcam	ab142042
Trizma base	Sigma-Aldrich	T6791-1 KG
Tween 20	Sigma-Aldrich	1379-500 ML
Yeast extract, powder, Ultrapure	Thermo Scientific	J23547