Name: antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae
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This study was funded by the Tunisian Ministry of Health and the Tunisian Ministry of Higher Education and Scientific Research.

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
<ol>
	<li>Bacterial strains and growth media 
	NOTE:&#160;Mollicutes species and the walled bacteria used in the present study and their growth media are listed in Table 1.
	<ol>
		<li>Growth of Mollicutes&#160;species
		<ol>
			<li>Inoculate 200 &#181;L from the g...

<ol>
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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 Mycoplasmas17,24. It w...

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.&#160;The cell wall deficiency confers intrinsic resistance to these microorganisms against some antimicrobial agents and is largely responsible for their polymorphism. Mycoplasmas have&#160;small genome and reduced size, which limits their metabolic and biosynthetic capabilities and explains their parasitic and saprophytic nature1.
Mycoplasma pneumoniae is one of the Mycoplasmas that infect&#160;man and is thought to be the most virulent2. M. pneumoniae&#160;colonizes the upper respiratory tract, leading to atypical pneumonia in children and young adults. The clinical signs engendered by M. pneumoniae infection are flu-like, with headache, fever, and cough3. The cytadherence of M. pneumoniae to host cells is mediated by an attachment organelle including P1 major adhesion and several accessory proteins4,5. More clinical manifestations may occur because of local inflammation and stimulation of the host immune system resulting from the intimate adherence of M. pneumoniae to the airway mucosa6. Although pneumonia is a hallmark of M. pneumoniae infection, it has been revealed that infection with this bacterium can also be responsible for a wide spectrum of non-pulmonary manifestations in different anatomical sites such as the central nervous system, heart, skin, and joints7.
As for all Mycoplasma species, the diagnosis of M. pneumoniae&#160;is challenging. The clinical signs evoking mycoplasmosis are mostly inapparent and non-characteristic8. Since it is very hard to diagnose M. pneumoniae infection by only relying on clinical manifestations and symptoms, laboratory screening is of particular interest9. Detecting M. pneumoniae colonies by culture is the gold standard method for a proper diagnosis. However, the fastidious growth requirements and the long time needed for the delivery of definitive results (1-2 weeks) complicate the culture, and thus means it is rarely used for routine diagnosis10. Nucleic acid ampli&#64257;cation technologies were validated in terms of speed and efficiency, although because of their relatively high cost and unavailability in some health care facilities, these molecular techniques are not considered first-line diagnostic tests. It is true that commercial PCR tests are widely used to diagnose M. pneumoniae infections, but they still cannot replace serology. Also, the frequent occurrence of both false negative and false positive results has limited the use of PCR9. Routinely, serology remains the most practiced in laboratories for the diagnosis of M. pneumoniae infection. Several serology approaches have been reported for decades, such as cold hemagglutinins, complement fixation test11, indirect hemagglutination test12, immunofluorescence13, and the technology of ELISA, which was first applied to mycoplasma serology in the early 1980&#39;s14,15,16. One of the major issues encountered when performing ELISA serodiagnosis of M. pneumoniae infection is cross-reactions, which considerably lowers the specificity of the technique. Nonspecific adsorption of human sera with M. pneumoniae antigens was previously reported; in fact, many of the antibodies detected by ELISA in human sera may not always be bound to mycoplasmal antigens17, due to the commonality of M. pneumoniae with some bacteria18,19 and some animal and human tissues20.
Because of the high background readings observed in the conventional ELISA test that was practiced in the laboratory, the interpretation of results was often complicated, and thus the delivery of a proper M. pneumoniae diagnosis was a tough assignment. While facing this issue, we opted to improve the M. pneumoniae ELISA by removing nonspecific reactions of M. pneumoniae antigens with antibodies to be tested. For this purpose, we worked on selective depletion of the nonspecific M. pneumoniae antigens using the adsorption technique. In fact, the main goal of the antigen-capture ELISA is to specifically detect M. pneumoniae&#160;immunoglobulin (Ig) G in human serum samples. The concept of this ELISA consists mainly of the selective capture of M. pneumoniae-specific antigens, before adding the human serum samples. This selectivity is insured by incubating M. pneumoniae crude antigen with a M. pneumoniae polyclonal antiserum, produced in rabbits in the laboratory and rendering it species-specific by adsorption against a panel of heterologous bacteria, belonging or not belonging to the Mollicutes class, sharing antigens with M. pneumoniae species and/or known to colonize the respiratory tract. The adsorption procedure was repeated thrice, and its efficiency to eliminate cross-reactivity was tested by immunoblotting. The developed ELISA assay is a combination of sandwich and indirect ELISA. Briefly, the wells of the ELISA plate are first coated with a polyclonal antiserum specific to M. pneumoniae. Then, M. pneumoniae antigen is added and trapped between the antiserum and the antibodies present in the serum sample to be tested. The formed immunological complex is detected by a secondary enzyme-conjugated antibody (peroxidase-conjugated IgG). The reactions are visualized by the addition of chromogenic substrate, and the absorbance is measured spectrophotometrically. This in-house ELISA is schematically presented in Figure 1. The homemade ELISA proved to be efficient in specifically detecting M. pneumoniae infection and is currently one of the most practiced tests in routine diagnostic activity.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4-chloro-1-naphtol</td>
			<td>Sigma-Aldrich</td>
			<td>C6788-50 TAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>BD</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-50 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate bicarbonate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>C3041-50 Cap</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL1066&#160;</td>
			<td>VWR</td>
			<td>P0058-N1L</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7528-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco PPLO Broth</td>
			<td>BD</td>
			<td>255420</td>
			<td>Frey media</td>
		</tr>
		<tr>
			<td>ELISA plate-Reader</td>
			<td>MULTISKAN GO, Thermo Scientific</td>
			<td>Ref: 51119200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Capricorn Scientific</td>
			<td>FBS-12A</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-human IgG</td>
			<td>Abcam</td>
			<td>ab6759</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat peroxidase-conjugated anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>656120</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid 37%</td>
			<td>Prolab</td>
			<td>2025.290</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2), solution 30%</td>
			<td>Scharlau</td>
			<td>HI01361000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-arginin</td>
			<td>Sigma-Aldrich</td>
			<td>A5006-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Prepared in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Provided by the laboratory of bacteriology of the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Mycoplasma pneumoniae polyclonal antiserum produced in rabbit</td>
			<td>Produced in Pasteur Institute of Tunis</td>
			<td></td>
			<td>Serum was produced by rabbit immunization at the Pasteur Institute of Tunis</td>
		</tr>
		<tr>
			<td>Nicotinamide adenine dinucleotide</td>
			<td>Sigma-Aldrich</td>
			<td>N7004-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Maxisorp flat-bottom 96-well microtiter plate&#160;</td>
			<td>Invitrogen</td>
			<td>44-2404-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin G sodium (1 MIU)</td>
			<td>PANPHARMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>fluka chemika</td>
			<td>77660</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk</td>
			<td>MP Biomedicals</td>
			<td>902887</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Novachim</td>
			<td>PS02805</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4) 95-97%</td>
			<td>Merck</td>
			<td>1007311011</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimerosal</td>
			<td>USB</td>
			<td>22215</td>
			<td></td>
		</tr>
		<tr>
			<td>TMB substrate (3,3&#8217;, 5,5&#8217; TetraMethyl-Benzidine solution)</td>
			<td>Abcam</td>
			<td>ab142042</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T6791-1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>1379-500 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract, powder, Ultrapure</td>
			<td>Thermo Scientific&#160;</td>
			<td>J23547&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

antigen-capture enzyme-linked immunosorbent assay for specific detection of mycoplasma pneumoniae

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.

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&#160;variable. For instance, M. gallisepticum</e...

Watch this Scientific Journal Video about Antigen-Capture Enzyme-Linked Immunosorbent Assay for Specific Detection of Mycoplasma pneumoniae at JoVE.com

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

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.

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

Mycoplasma pneumoniae is a cell wall-deficient prokaryote, mainly known to colonize the human respiratory tract and to be endemic, with epidemic peaks ...

As for all mycoplasm's patients, their diagnosis of Mycoplasm pneumoniae is challenging because of cross-reactions. Our proposed ELISA is based on the selective depletion of non-specific Mycoplasm pneumoniae antigens using an adsorption technique. The in-house antigen capture ELISA described in this video guarantees high spacious specificity.And actually, it has proven to be a reliable screened test for an accurate diagnosis of Mycoplasm pneumoniae infection. Demonstrating the pre-ELISA steps will be Imen Chniba, a PhD student. And the ELISA steps will be Nadine Khadraoui, a technician from our laboratory.To begin the antigen adsorption procedure, pull the 12 heterologous bacterial antigens into a microcentrifuge tube and adjust the protein concentration corresponding to half of the antiserum to be adsorbed. Next, centrifuge the antigen mixture at 14, 000 G for 10 minutes at four degrees Celsius to collect the pellet. Then, re-suspend the pellet with the purified polyclonal anti-Mycoplasma pneumoniae IgG and incubate for two hours at 37 degrees Celsius with slow agitation.At the end of the incubation, centrifuge the suspension again at 14, 000 G for 10 minutes at four degrees Celsius and recover the supernatant corresponding to the specific polyclonal Mycoplasma pneumoniae antiserum. To coat a 96-well ELISA plate with a capture antibody, dilute the antibody to 10 micrograms per milliliter and 0.1 molar carbonate bicarbonate buffer at pH 9.6. Then, add 100 microliters of the diluted antibody to each well of the microplate and incubate overnight at four degrees Celsius.The following day, remove the coating solution and wash the plate five times with the washing buffer. Next, block the unbound surfaces of the coated wells by adding 100 microliters of the blocking solution to each well. After incubating the plate from one hour at room temperature, remove the blocking solution with a pipette and wash the plate as demonstrated previously.Add an equal amount of Mycoplasma pneumoniae whole proteins at a concentration of 10 nanograms per well and allow the antigen-antibody reaction to take place for two hours at room temperature. Following incubation, remove the excess antigen and wash the plate as demonstrated. Next, dilute the human serum test samples and reference positive and negative serum controls.Then, add 100 microliters of the diluted test samples and controls into the appropriate wells and duplicates. Mark the wells containing neither antigen nor sera as blank. After incubating the plate for 90 minutes at room temperature, remove the serum solution and wash the plate five times with the washing buffer.Pipette 100 microliters of the appropriate diluted enzyme conjugated detection antibodies to each well and incubate for one hour at 37 degrees Celsius in the dark. After removing the unbound detection bodies and washing the plate, add 100 microliters of TMB Chromogen solution to visualize the antigen-antibody binding. Let the plate develop for 30 minutes at room temperature.Then, stop the reaction by adding 100 microliters of 7.5%sulfuric acid. Finally, read the absorbance at 450 nanometers using a microplate reader and sort the results based on the calculation of the index of positivity. Immunoblot analysis between the Mycoplasma pneumoniae non adsorbed polyclonal antiserum and heterologous bacterial antigens revealed cross-reactivity but with varying intensity.For instance, Mycoplasma gallisepticum and Mycoplasma imitans antigens displayed the strongest reactivity despite being avian mycoplasmas. In contrast, neither of the two human clinical isolates of Ureaplasma urealyticum showed any reaction. However, antigens of the remaining human genital mycoplasma species and other bacterial antigens yielded considerable cross-reactions with the antiserum.Immunoblot analysis confirmed the efficiency of the Mycoplasma pneumoniae polyclonal antiserum adsorption against the heterologous bacterial antigens. The adsorption procedure eliminated all cross-reactions, thus rendering the Mycoplasma pneumoniae antiserum specific for all subsequent serological and immunoblotting tests. Further, the set of human sera tested using the antigen capture ELISA developed in this study proved positive for Mycoplasma pneumoniae IgG with good specificity.The specificity of this ELISA is mainly insured by the adsorption technique, as it is primordial to perform this critical step carefully and to repeat it at least twice or thrice. The principle of this technique can be applied to diagnose other mycoplasma species or other bacteria in general. However, the selection of bacterial antigens for adsorption should be irrelevant and adequate.

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