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

Summary

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

Abstract

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

Introduction

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

Mycoplasma pneumoniae is one of the Mycoplasmas that infect man and is thought to be the most virulent². M. pneumoniae 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 proteins^4,5. 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 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 amplification 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's^14,15,16. 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 bacteria^18,19 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 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.

Protocol

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

1. Pre-ELISA steps: Prerequisites and preprocessing

1. Bacterial strains and growth media
   NOTE: Mollicutes species and the walled bacteria used in the present study and their growth media are listed in Table 1.
   1. Growth of Mollicutes species
      1. Inoculate 200 µL from the glycerol stock of each species in 1,800 µL of the media.
      2. Grow Mollicutes species statically at 37 ˚C with 5% CO₂ for 2-4 days until a pH change is observed (the pH indicator is phenol red).
      3. Scale up the cultures to 10 mL by adding a 1/10^th 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's medium demonstrates the presence of M. gallisepticum, while its alkalinization proves the growth of M. imitans.
      4. Spread 50 µL of the cultures on agar plates to confirm the growth of the bacteria. Maintain the agar plates at 37 ˚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).
   2. Growth of non-Mollicutes species
      1. Culture the non-Mollicutes bacteria in 3 mL of the media at 37 ˚C overnight (shaking at 200 rpm).
      2. Compare the turbidity of the cultures with the control media to confirm growth.
      3. Scale up the cultures to 10 mL by adding a 1/100^th dilution of the culture to the media and allow them to grow again in the same conditions.
2. Antigen preparation
   1. Antigen preparation of Mollicutes species
      1. 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 ˚C for 30 min.
      2. 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.
      3. Resuspend each antigen in 500 µL of PBS.
   2. Antigen preparation of non-Mollicutes species
      1. Centrifuge the overnight grown cultures of the non-Mollicutes bacteria at 1,500 x g for 15 min.
      2. Resuspend each bacterial pellet in 500 µL 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 ˚C until their subsequent usage.
3. Cross-reactivity screening by immunoblotting
   1. Denature the bacterial proteins by mixing 8 µL 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 ˚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.
   2. Subject the denatured proteins (100 µg protein/well) to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by Laemmli's method²² with 12% separating gel and 5% stacking gel. After that, transfer the proteins electrophoretically to the nitrocellulose membrane by Towbin et al.'s method²³.
   3. 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.
   4. 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.
4. 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.
   1. 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).
   2. Centrifuge the antigen mixture at 14,000 x g for 10 min at 4 °C, collect the pooled pellet, and incubate it with the purified polyclonal anti-M. pneumoniae IgG for 2 h at 37 °C with slow agitation.
   3. After incubation, centrifuge the suspension at 14,000 x g for 10 min at 4 °C and recover the supernatant (which corresponds to the specific polyclonal M. pneumoniae antiserum).
      ​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.

2. ELISA steps: The assay itself

1. Microplate coating with the capture antibody
   1. Dilute the capture antibody (M. pneumoniae pre-adsorbed polyclonal antiserum) to a concentration of 10 µg/mL in 0.1 M carbonate-bicarbonate buffer (pH 9.6).
   2. Coat the 96-well ELISA plate by adding 100 µL of the diluted capture antibody to each well.
   3. After overnight incubation at 4 ˚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).
2. Blocking
   1. Block the remaining binding surfaces of the coated wells by adding 100 µL of the blocking solution (0.5% casein in PBS) to each well.
   2. Incubate for 1 h at room temperature.
   3. Remove the blocking solution with a pipette and wash the plate five times with the washing buffer.
3. Application of the antigen
   1. Add an equal amount of M. pneumoniae whole proteins to all coated wells (10 ng/well).
   2. Allow the reaction to take place for 2 h at room temperature.
   3. Remove the excess of the antigen solution and wash the plate five times with the washing buffer.
4. Application of the test samples and controls
   1. 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.
   2. Add 100 µL 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.
   3. After incubating the plate for 90 min at room temperature, remove the serum solutions and wash the plate five times with the washing buffer.
5. Application of the enzyme-conjugated detection antibody
   1. Dilute the enzyme-conjugated detection antibodies, HRP-conjugated anti-rabbit, and anti-human IgG to 1/10,000 in PBS-Tween 20.
   2. Pipette 100 µL of the appropriately diluted detection antibody to each well.
   3. 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.
6. Detection and data analysis
   1. Add 100 µL of the 3,3', 5,5' Tetramethyl-benzidine (TMB) chromogen solution to visualize the antigen-antibody binding.
   2. Allow the plate to develop for 30 min at room temperature then add 100 µL of the stop solution (7.5% H₂SO₄) to stop the enzymatic reaction.
   3. 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 < 0.7: Negative result
      IP = 0.7: Test should be repeated within 2 weeks
      IP > 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.

3. Post-ELISA steps: Results assessment and test validation

1. 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).

Results

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

Discussion

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

Materials

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