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 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 ...

antigen-capture-enzyme-linked-immunosorbent-assay-for-specific

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

2.3K Views.  Pasteur Institute of Tunis. 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. 

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

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases, which is what we do during our quality control procedures. In this assay, casein acts as a substrate. When the protease we are testing digests casein, the amino acid tyrosine is liberated along with other amino acids and peptide fragments. Folin and Ciocalteus Phenol, or Folin's reagent primarily reacts with free tyrosine to produce a blue colored chromophore, which is quantifiable and measured as an absorbance value on the spectrophotometer. The more tyrosine that is released from casein, the more the chromophores are generated and the stronger the activity of the protease. Absorbance values generated by the activity of the protease are compared to a standard curve, which is generated by reacting known quantities of tyrosine with the F-C reagent to correlate changes in absorbance with the amount of tyrosine in micromoles. From the standard curve the activity of protease samples can be determined in terms of Units, which is the amount in micromoles of tyrosine equivalents released from casein per minute.
To view this article in Chinese, <a href="http://jove.com/index/details.stp?ID=910">click here</a>

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases. Sigma's non-specific protease activity assay may be used as a standardized procedure to determine the activity of proteases.

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

ELIME (Enzyme Linked Immuno Magnetic Electrochemical) Method for Mycotoxin Detection

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous mycotoxins in food commodities. In this protocol we show how to fabricate and interrogate an electrochemical competitive Enzyme linked immunomagnetic assay based on the use of magnetic beads as solid support for the immunochemical chain3 and screen printed electrodes as sensing platform.
Our method aims to determine the total amount of HT-2 and T-2 toxins, mycotoxins belonging to the trichothecenes family and of great concern for human health4. The use of an antibody clone with a cross reactivity of 100% towards HT-2 and T-2 allows to simultaneously detect both toxins with similar sensitivity5.
The first step of our assay is the coating step where we immobilize HT2-KLH conjugate toxin on the surface of magnetic beads. After a blocking step, necessary to avoid non-specific absorptions, the addition of a monoclonal antibody allows the competition between immobilized HT-2 and free HT-2 or T-2 present in the sample or dissolved in a standard solution.
At the end of the competition step, the amount of monoclonal antibody linked to the immobilized HT-2 will be inversely proportional to the amount of toxin in the sample solution.
A secondary antibody labeled with alkaline phosphatase (AP) is used to reveal the binding between the specific antibody and the immobilized HT-2. The final measurement step is performed by dropping an aliquot of magnetic bead suspension, corresponding to a specific sample/standard solution, on the surface of a screen-printed working electrode; magnetic beads are immobilized and concentrated by means of a magnet placed precisely under the screen-printed electrode. After two minutes of incubation between magnetic beads and a substrate for AP, the enzymatic product is detected by Differential Pulse Voltammetry (DPV) using a portable instrument (PalmSens) also able to initiate automatically eight measurements within an interval of few seconds.

ELIME Enzyme Linked Immuno Magnetic Electrochemical Method for Mycotoxin Detection

A protocol to detect trichothecenes (mycotoxins of concern for human health) using a newly developed screening method based on a competitive immunochemical method and a final electrochemical detection is demonstrated.

Immunoassays are a valid alternative to the more expensive and time consuming quantitative HPLC or GC1, 2 methods for the screening detection of hazardous ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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 and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within the brain, ascorbate acts in a neuroprotective and neuromodulatory manner that involves ascorbate cycling between neurons and vicinal astrocytes - a relationship that appears to be crucial for brain ascorbate homeostasis. Additionally, emerging evidence strongly suggests that ascorbate has a greatly expanded role in regulating cellular and systemic iron metabolism than is classically recognized. The increasing recognition of the integral role of ascorbate in normal and deregulated cellular and organismal physiology demands a range of medium-throughput and high-sensitivity analytic techniques that can be executed without the need for highly expensive specialist equipment. Here we provide explicit instructions for a medium-throughput, specific and relatively inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Ascorbate plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. Here we describe a medium-throughput, specific and inexpensive microplate assay for the determination of both intra- and extracellular ascorbate in cell culture.

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

A Multi-detection Assay for Malaria Transmitting Mosquitoes

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

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

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Detection of Protein Ubiquitination

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A Multi-detection Assay for Malaria Transmitting Mosquitoes

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles