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 glycerol stock of each species in 1,800 &#181;L of the media.</li>
			<li>Grow Mollicutes species statically at 37 &#730;C with 5% CO2 for 2-4 days until a pH change is observed (the pH indicator is phenol red).</li>
			<li>Scale up the cultures to 10 mL by adding a 1/10th dilution of the culture to the media and allow them to grow again in the same conditions. 
			NOTE: According to the metabolism, some human Mollicutes species (M. pneumoniae, M. genitalium, and M. fermentans) acidify the medium due to glucose fermentation, and some others (M. hominis and Ureaplasma) alkalinize the medium by arginine hydrolysis or urea hydrolysis, respectively. Regarding avian mycoplasmas, the acidification of Frey&#39;s medium demonstrates the presence of M. gallisepticum, while its alkalinization proves the growth of M. imitans.</li>
			<li>Spread 50 &#181;L of the cultures on agar plates to confirm the growth of the bacteria. Maintain the agar plates at 37 &#730;C with 5% CO2 and observe them regularly under the microscope for the appearance of typical fried egg colonies (Mycoplasmas) and dark urchin-like colonies (Ureaplasmas).</li>
		</ol>
		</li>
		<li>Growth of non-Mollicutes&#160;species
		<ol>
			<li>Culture the non-Mollicutes bacteria in 3 mL of the media at 37 &#730;C overnight (shaking at 200 rpm).</li>
			<li>Compare the turbidity of the cultures with the control media to confirm growth.</li>
			<li>Scale up the cultures to 10 mL by adding a 1/100th dilution of the culture to the media and allow them to grow again in the same conditions.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Antigen preparation
	<ol>
		<li>Antigen preparation of Mollicutes species
		<ol>
			<li>Harvest the whole proteins (present in confirmed cultures of M. pneumoniae and the other Mollicutes species) by centrifugation at 40,000 x g at 4 &#730;C for 30 min.</li>
			<li>Discard the supernatant and wash the pellets three times with 1 mL of 1x phosphate-buffered saline (PBS, pH 7.4) by a series of three centrifugations with the same conditions.</li>
			<li>Resuspend each antigen in 500 &#181;L of PBS.</li>
		</ol>
		</li>
		<li>Antigen preparation of non-Mollicutes&#160;species
		<ol>
			<li>Centrifuge the overnight grown cultures of the non-Mollicutes bacteria at 1,500&#160;x g for 15 min.</li>
			<li>Resuspend each bacterial pellet in 500 &#181;L of PBS. 
			NOTE: Protein concentration is determined using the classic Bradford quantitation method with bovine serum albumin as standard21. The protein concentration of Mollicutes species usually ranges between 0.7-4.8 mg/mL. For the other bacterial species, protein concentration can reach 20 mg/mL. All antigens are stored at -20 &#730;C until their subsequent usage.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cross-reactivity screening by immunoblotting
	<ol>
		<li>Denature the bacterial proteins by mixing 8 &#181;L of each antigen with an equal volume of sample buffer (0.5 M Tris-HCl; pH 6.8, 10% glycerol [v/v], 10% SDS [w/v], and 0.2% bromophenol blue), incubate the mixture at 100 &#730;C for 5 min. 
		NOTE: Denaturation is performed to cover the proteins with negative charge and to break their secondary structures, which allows them to run across the polyacrylamide gel and to be separated according to their molecular weights.</li>
		<li>Subject the denatured proteins (100 &#181;g protein/well) to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by Laemmli&#39;s method22 with 12% separating gel and 5% stacking gel. After that, transfer the proteins electrophoretically to the nitrocellulose membrane by Towbin et al.&#39;s method23.</li>
		<li>Immerse the nitrocellulose membrane in 5% skim milk in PBS for 30 min to block the unoccupied surfaces. Incubate the protein blots at room temperature for 2 h with rabbit polyclonal M. pneumoniae antiserum (produced at Pasteur Institute of Tunis) diluted to 1/200 in PBS-Tween 20, then with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG diluted to 1/2,000 in PBS-Tween 20 for 1 h at room temperature.</li>
		<li>Wash off the unbound antibodies with PBS and obtain the results by exposing the nitrocellulose sheet to the substrate solution (4-chloro-1-naphthol, H2O2). Stop the reaction by adding water after 5-15 min.</li>
	</ol>
	</li>
	<li>Adsorption procedure 
	NOTE: To eliminate cross-reactions between polyclonal M. pneumoniae antiserum and heterologous bacteria antigens, the adsorption procedure previously described by Ben Abdelmoumen and Roy24 is employed.
	<ol>
		<li>Prepare an antigen pool of the 12 heterologous bacteria (suspected to share antigens with M. pneumoniae), mix it in a microcentrifuge tube, and adjust the protein concentration corresponding to half of the antiserum to be adsorbed in the experiment. 
		NOTE: The volume and the concentration vary between the different assays. This step mainly depends on the concentration of the antibody to be adsorbed. For example, if the antibody concentration = 3.56 mg/mL, the concentration of the antigen pool (composed of the 12 heterologous bacteria) should be 1.78 mg/mL (therefore, approximately 0.148 mg of each antigen).</li>
		<li>Centrifuge the antigen mixture at 14,000 x g for 10 min at 4 &#176;C, collect the pooled pellet, and incubate it with the purified polyclonal anti-M. pneumoniae IgG for 2 h at 37 &#176;C with slow agitation.</li>
		<li>After incubation, centrifuge the suspension at 14,000 x g for 10 min at 4 &#176;C and recover the supernatant (which corresponds to the specific polyclonal M. pneumoniae antiserum). 
		&#8203;NOTE: To ensure specificity of the polyclonal M. pneumoniae antiserum, repeat the adsorption procedure three times. Before it can be used in the ELISA assay, the adsorbed polyclonal M. pneumoniae antiserum should be tested by immunoblotting for the absence of cross-reactions against the included set of bacteria.</li>
	</ol>
	</li>
</ol>
2. ELISA steps: The assay itself
<ol>
	<li>Microplate coating with the capture antibody
	<ol>
		<li>Dilute the&#160;capture antibody (M. pneumoniae pre-adsorbed polyclonal antiserum) to a concentration of 10 &#181;g/mL in 0.1 M carbonate-bicarbonate buffer (pH 9.6).</li>
		<li>Coat the 96-well ELISA plate by adding 100 &#181;L of the diluted capture antibody to each well.</li>
		<li>After overnight incubation at 4 &#730;C, remove the coating solution and wash the plate five times with the washing buffer (composition [per L]: 146.29 g of NaCl, 39.4 g of Tris-HCl, 0.2 g of Thimerosal, and 0.5 mL of Tween 20; pH 7.3).</li>
	</ol>
	</li>
	<li>Blocking
	<ol>
		<li>Block the remaining binding surfaces of the coated wells by adding 100 &#181;L of the blocking solution (0.5% casein in PBS) to each well.</li>
		<li>Incubate for 1 h at room temperature.</li>
		<li>Remove the blocking solution with a pipette and wash the plate five times with the washing buffer.</li>
	</ol>
	</li>
	<li>Application of the antigen
	<ol>
		<li>Add an equal amount of M. pneumoniae whole proteins to all coated wells (10 ng/well).</li>
		<li>Allow the reaction to take place for 2 h at room temperature.</li>
		<li>Remove the excess of the antigen solution and wash the plate five times with the washing buffer.</li>
	</ol>
	</li>
	<li>Application of the test samples and controls
	<ol>
		<li>Dilute the test samples (human sera) and controls (reference positive and negative sera) to 1/200, 1/400, and 1/800 in PBS-Tween 20.</li>
		<li>Add 100 &#181;L of the diluted samples and controls into the appropriate wells. Carry out each reaction in duplicate. Mark the wells containing neither antigen nor sera as blank.</li>
		<li>After incubating the plate for 90 min at room temperature, remove the serum solutions and wash the plate five times with the washing buffer.</li>
	</ol>
	</li>
	<li>Application of the enzyme-conjugated detection antibody
	<ol>
		<li>Dilute the enzyme-conjugated detection antibodies, HRP-conjugated anti-rabbit, and anti-human IgG to 1/10,000 in PBS-Tween 20.</li>
		<li>Pipette 100 &#181;L of the appropriately diluted detection antibody to each well.</li>
		<li>After incubating the plate for 1 h at room temperature in the dark, remove the unbound detection antibodies and wash the plate five times with the washing buffer.</li>
	</ol>
	</li>
	<li>Detection and data analysis
	<ol>
		<li>Add 100 &#181;L of the 3,3&#39;, 5,5&#39; Tetramethyl-benzidine (TMB) chromogen solution to visualize the antigen-antibody binding.</li>
		<li>Allow the plate to develop for 30 min at room temperature then add 100 &#181;L of the stop solution (7.5% H2SO4) to stop the enzymatic reaction.</li>
		<li>Read the absorbance at 450 nm wavelength using the microplate reader and sort out the results based on the calculation of the index of positivity (IP). 
		IP = Absorbance of the tested serum / Absorbance of the cut-off 
		IP &#60; 0.7: Negative result 
		IP = 0.7: Test should be repeated within 2 weeks 
		IP &#62; 0.7: Positive result 
		NOTE: The IP formula is conceived in the laboratory and the 0.7 value is set up after optimization. For duplicate reactions, the mean value of absorbance is calculated. Optimum serum dilution and antigen concentration are established by the ELISA checkerboard titration method (data not shown). Cut-off value = OD (optical density) of a reference positive serum (diluted at the same dilution as the sample). This OD should be at least three times higher than the OD value of the negative control.</li>
	</ol>
	</li>
</ol>
3. Post-ELISA steps: Results assessment and test validation
<ol>
	<li>The ability of the in-house ELISA to specifically diagnose M. pneumoniae infection in the tested patients is evaluated through supplementary immunoblots using antigens of M. pneumoniae and the heterologous bacteria to confirm the positivity of the tested human sera in M. pneumoniae antibodies and their negativity in the other suspected bacteria (data not shown).</li>
</ol>

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The authors declare that there are no competing interests.

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><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&amp;nbsp;</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 (H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;), 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>&lt;em&gt;Mycoplasma pneumoniae&lt;/em&gt; 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&amp;nbsp;</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&amp;nbsp;</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 (H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;) 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&amp;rsquo;, 5,5&amp;rsquo; 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&amp;nbsp;</td><td>J23547&amp;nbsp;</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 ...

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2.3K Views.  Pasteur Institute of Tunis. Zoals voor alle patiënten met mycoplasma, is hun diagnose van Mycoplasma pneumoniae een uitdaging vanwege kruisreacties. Onze voorgestelde ELISA is gebaseerd op de selectieve uitputting van niet-specifieke Mycoplasma pneumoniae antigenen met behulp van een adsorptietechniek. De in-house antigeen capture ELISA die in deze video wordt beschreven, garandeert een hoge ruimtelijke specificiteit.En eigenlijk heeft het bewezen een betrouwbare gescreende test te zijn voor een nauwkeurige diag...

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

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

Immunology and Infection

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

A Rapid, Multiplex Dual Reporter IgG and IgM SARS-CoV-2 Neutralization Assay for a Multiplexed Bead-Based Flow Analysis System

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel pathogens, such as SARS-CoV-2. Multiplex serological testing can be particularly useful because it can simultaneously analyze antibodies to multiple antigens that optimizes pathogen coverage, and controls for variability in the organism and the individual host response. Here we describe a SARS-CoV-2 IgG 3-plex fluorescent microsphere-based assay that can detect both IgM and IgG antibodies to three major SARS-CoV-2 antigens-the spike (S) protein, spike angiotensin-converting enzyme-2 (ACE2) receptor-binding domain (RBD), and nucleocapsid (Nc). The assay was shown to have comparable performance to a SARS-CoV-2 reference assay for IgG in serum obtained at &#8805;21 days from symptom onset but had higher sensitivity with samples collected at &#8804;5 days from symptom onset. Further, using soluble ACE2 in a neutralization assay format, inhibition of antibody binding was demonstrated for S and RBD.

A flow analysis system for bead-based multiplexed assays which provides a two-reporter readout was used for the development of multiplex serological and antibody neutralization assays that can simultaneously measure neutralizing IgG and IgM antibodies for SARS-CoV-2.

The COVID-19 pandemic has underscored the need for rapid high-throughput methods for sensitive and specific serological detection of infection with novel ...

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Quantifying Myeloperoxidase-DNA and Neutrophil Elastase-DNA Complexes from Neutrophil Extracellular Traps by Using a Modified Sandwich ELISA

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures composed of DNA with granule proteins, such as myeloperoxidase (MPO) and neutrophil elastase (NE), and cytoplasmic and cytoskeletal proteins. Although interest in NETs has increased recently, no sensitive, reliable assay method is available for measuring NETs in clinical settings. This article describes a modified sandwich enzyme-linked immunosorbent assay to quantitatively measure two components of circulating NETs, MPO-DNA and NE-DNA complexes, which are specific components of NETs and are released into the extracellular space as breakdown products of NETs. The assay uses specific monoclonal antibodies for MPO or NE as the capture antibodies and a DNA-specific detection antibody. MPO or NE binds to one site of the capture antibody during the initial incubation of samples containing MPO-DNA or NE-DNA complexes. This assay shows good linearity and high inter-assay and intra-assay precision. We used it in 16 patients with COVID-19 with accompanying acute respiratory distress syndrome and found that the plasma concentrations of MPO-DNA and NE-DNA were significantly higher than in the plasma obtained from healthy controls. This detection assay is a reliable, highly sensitive, and useful method for investigating the characteristics of NETs in human plasma and culture supernatants.

We present a protocol for a modified sandwich enzyme-linked immunosorbent assay technique to quantitatively measure two components of neutrophil extracellular trap remnants, myeloperoxidase conjugated-DNA and neutrophil elastase conjugated-DNA complexes,derived from activated neutrophils.

Certain stimuli, such as microorganisms, cause neutrophils to release neutrophil extracellular traps (NETs), which are basically web-like structures ...

Analysis of SARS-CoV-2 Using Dual-Reporter and Multiplex Technology

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

Author Spotlight: Advancing Antiviral Strategies Through Novel Immunocapture and Mass Spectrometry Techniques

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...

Dynamic Monitoring of Seroconversion using a Multianalyte Immunobead Assay for Covid-19

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes on the same analyte remain limited. This report describes the development and optimization of a multiplexed immunobead assay for serological testing of common immunoglobulin isotypes (e.g., IgA, IgM, and IgG) associated with an immune response to SARS-CoV-2 infection or vaccination. Assays were accomplished using a flow-based, multiplex fluorescent reader with dual-channel capability. Optimizations focused on analyte capture time, detection antibody concentration, and detection antibody incubation time. Analytical assay performance characteristics (e.g., assay range (including lower and upper limits of quantitation); and intra- and inter-assay precision) were established for either IgG/IgM or IgA/IgM serotype combination in tandem using the 'dual channel' mode. Analyte capture times of 30 min for IgG, 60 min for IgM, and 120 min for IgA were suitable for most applications, providing a balance of assay performance and throughput. Optimal detection antibody incubations at 4 &#181;g/mL for 30 min was observed and are recommended for general applications, given the overall excellent precision (percent coefficient of variance (%CV) &#8804; 20%) and sensitivity values observed. The dynamic range for the IgG isotype spanned several orders of magnitude for each assay (Spike S1, Nucleocapsid, and Membrane glycoproteins), which supports robust titer evaluations at a 1:500 dilution factor for clinical applications. Finally, the optimized protocol was applied to monitoring Spike S1 seroconversion for subjects (n = 4) that completed a SARS-CoV-2 vaccine regimen. Within this cohort, Spike S1 IgG levels were observed to reach maximum titers at 14 days following second dose administration, at a much higher (~40-fold) signal intensity than either IgM or IgA isotypes. Interestingly, we observed highly variable Spike S1 IgG titer decay rates that were largely subject-dependent were observed, which will be the topic of future studies.

This article describes a convenient method for monitoring dynamic changes in antibody titers for two immunoglobulin isotypes concurrently (either IgA, IgM, or IgG) resulting from an immune response to SARS-CoV-2 infection or vaccination. This 'Multianalyte Covid-19 Immune Response Panel' employs three indirect immunoassays built on coded microspheres that are read using a flow-based multiplex reader with 'dual-channel' capability.

Multiplex technologies for interrogating multiple biomarkers in concert have existed for several decades; however, methods to evaluate multiple epitopes ...

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

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