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

As for all mycoplasm's patients, their diagnosis of Mycoplasm pneumoniae is challenging because of cross-reactions. Our proposed ELISA is based on the selective depletion of non-specific Mycoplasm pneumoniae antigens using an adsorption technique. The in-house antigen capture ELISA described in this video guarantees high spacious specificity.

And actually, it has proven to be a reliable screened test for an accurate diagnosis of Mycoplasm pneumoniae infection. Demonstrating the pre-ELISA steps will be Imen Chniba, a PhD student. And the ELISA steps will be Nadine Khadraoui, a technician from our laboratory.

To begin the antigen adsorption procedure, pull the 12 heterologous bacterial antigens into a microcentrifuge tube and adjust the protein concentration corresponding to half of the antiserum to be adsorbed. Next, centrifuge the antigen mixture at 14, 000 G for 10 minutes at four degrees Celsius to collect the pellet. Then, re-suspend the pellet with the purified polyclonal anti-Mycoplasma pneumoniae IgG and incubate for two hours at 37 degrees Celsius with slow agitation.

At the end of the incubation, centrifuge the suspension again at 14, 000 G for 10 minutes at four degrees Celsius and recover the supernatant corresponding to the specific polyclonal Mycoplasma pneumoniae antiserum. To coat a 96-well ELISA plate with a capture antibody, dilute the antibody to 10 micrograms per milliliter and 0.1 molar carbonate bicarbonate buffer at pH 9.6. Then, add 100 microliters of the diluted antibody to each well of the microplate and incubate overnight at four degrees Celsius.

The following day, remove the coating solution and wash the plate five times with the washing buffer. Next, block the unbound surfaces of the coated wells by adding 100 microliters of the blocking solution to each well. After incubating the plate from one hour at room temperature, remove the blocking solution with a pipette and wash the plate as demonstrated previously.

Add an equal amount of Mycoplasma pneumoniae whole proteins at a concentration of 10 nanograms per well and allow the antigen-antibody reaction to take place for two hours at room temperature. Following incubation, remove the excess antigen and wash the plate as demonstrated. Next, dilute the human serum test samples and reference positive and negative serum controls.

Then, add 100 microliters of the diluted test samples and controls into the appropriate wells and duplicates. Mark the wells containing neither antigen nor sera as blank. After incubating the plate for 90 minutes at room temperature, remove the serum solution and wash the plate five times with the washing buffer.

Pipette 100 microliters of the appropriate diluted enzyme conjugated detection antibodies to each well and incubate for one hour at 37 degrees Celsius in the dark. After removing the unbound detection bodies and washing the plate, add 100 microliters of TMB Chromogen solution to visualize the antigen-antibody binding. Let the plate develop for 30 minutes at room temperature.

Then, stop the reaction by adding 100 microliters of 7.5%sulfuric acid. Finally, read the absorbance at 450 nanometers using a microplate reader and sort the results based on the calculation of the index of positivity. Immunoblot analysis between the Mycoplasma pneumoniae non adsorbed polyclonal antiserum and heterologous bacterial antigens revealed cross-reactivity but with varying intensity.

For instance, Mycoplasma gallisepticum and Mycoplasma imitans antigens displayed the strongest reactivity despite being avian mycoplasmas. In contrast, neither of the two human clinical isolates of Ureaplasma urealyticum showed any reaction. However, antigens of the remaining human genital mycoplasma species and other bacterial antigens yielded considerable cross-reactions with the antiserum.

Immunoblot analysis confirmed the efficiency of the Mycoplasma pneumoniae polyclonal antiserum adsorption against the heterologous bacterial antigens. The adsorption procedure eliminated all cross-reactions, thus rendering the Mycoplasma pneumoniae antiserum specific for all subsequent serological and immunoblotting tests. Further, the set of human sera tested using the antigen capture ELISA developed in this study proved positive for Mycoplasma pneumoniae IgG with good specificity.

The specificity of this ELISA is mainly insured by the adsorption technique, as it is primordial to perform this critical step carefully and to repeat it at least twice or thrice. The principle of this technique can be applied to diagnose other mycoplasma species or other bacteria in general. However, the selection of bacterial antigens for adsorption should be irrelevant and adequate.