Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

The definitive identification of mycoplasmas is usually based on serological procedures, including growth inhibition (1,2), metabolic inhibition (3,4), immunofluorescence (5–7), and immunobinding assays (8–14). The technique most commonly adopted for the routine identification of mycoplasma species isolated from clinical material is, at present, the immunobinding assay involving either broth culture (12,14), mycoplasma colonies on agar plates, or the imprints of colonies (8–13). Several procedures with polyclonal or monoclonal antibodies (MAb) have been described. All these assay systems are based on the detection of mycoplasma surface antigens, which are believed to be highly specific.

At present, immunobinding assays are the most reliable tests for mycoplasma identification, but specificity and sensitivity can be affected in certain circumstances. Shared antigens between some species can lead to cross-reactions, although the use of specific MAbs can greatly improve specificity. However, recently it has become apparent that many mycoplasma species are able to undergo high-frequency surface antigenic variation (15–18). In practical terms, this peculiarity has two main consequences: first, many mycoplasma species may be antigenically highly heterogeneous, so that the selection of reagents, including MAb, which are simultaneously specific and representative of all antigenic variants within the species, is difficult.; second, the usual laboratory practice involving filter cloning and propagation by subcultivation of randomly selected agar-grown subpopulations may result in rapid antigenic drift of the reference strains, as has already been proven.

Since the number of mycoplasma species is increasing, each isolate has to be tested with several sera for complete identification and immunobinding assays involving mycoplasma colonies, or imprints of colonies, are becoming highly laborious. Immunobinding assays using polyclonal antibodies with broth culture bound onto nitrocellulose paper, on the other hand, can be affected by a high level of background staining. The technique of dot immunobinding on membrane filtration in microplates (MF dot) (14) eliminates background staining problems and offers further advantages over other tests, such as practicality, rapidity, ready standardization, and the possibility of treating many samples against several sera simultaneously. MF dot immunobinding test is described here.

MF dot immunobinding is performed with special 96-well microplates whose well bottoms are made of a durapore 0.22-μm membrane filter (low-protein affinity). These plates allow the removal of well fluids by vacuum filtration. In this way, mycoplasmas are separated from broth media by trapping them on the filtration membranes, and the broth proteins that do not bind to the membrane are easily removed. The membranes are then incubated with hyperimmune rabbit sera. The unbound immunoglobulins (IgG) are removed by filtration as above, and the bound antibodies are detected by means of an enzyme-conjuguated antirabbit IgG. MF dot immunobinding is an easy and reliable test for the identification of mycoplasmas, but in light of the high rate of surface antigenic variability occurring in many mycoplasmas, criteria must urgently be defined for the standardization of mycoplasma strains and diagnostic antisera to ensure that reproducible results can be obtained in different laboratories.

2 Materials

  1. 1.

    96-Well plates (millititer GV 0.22-μm durapore, Millipore).

  2. 2.

    Vacuum holder (Millititer, Millipore).

  3. 3.

    Vacuum pump with manometer.

  4. 4.

    TBS: 6.057 g Tris, 11.688 g NaCl, in 1 L distilled H2O adjusted to pH 7.4 with HCl. Store at +4°C, and use within 15 d.

  5. 5.

    Washing buffer (TBS-T): Freshly prepared TBS containing 0.05% of Tween 20.

  6. 6.

    Blocking solution (TBS-B): Freshly prepared solution of 10% normal horse serum in TBS filtered through 0.45 μm.

  7. 7.

    Rabbit hyperimmune sera for the various mycoplasma species: Store lyophilized for long-term use and in 10 μL–20 μL aliquots at −20°C for short-term experiments. Do not freeze and thaw each aliquot more than four times.

  8. 8.

    Conjugate: affinity-isolated swine antirabbit IgG conjugated to horseradish peroxidase (HRP) diluted in TBS-B to a predetermined optimum concentration.

  9. 9.

    Color reagent: 3,3′ diaminobenzidine tetrachloride (DAB) in powder (C12H14N4, 4 HCl). DAB is unstable and light-sensitive, store dry at −20°C, and replace frequently (harmful by inhalation and contact with skin; a possible carcinogen).

  10. 10.

    Enzyme substrate: H2O2 30%, store at −4°C in a large volume, light-sensitive.

  11. 11.

    Substrate solution: dissolve 25 mg DAB in 50 mL TBS and then add 50 μL H2O2. Freshly prepare.

3 Methods

  1. 1.

    Before use, wash the plates once with TBS-T and then three times with TBS without incubation. After each wash, remove the fluids by applying a vacuum, and after the last rinse, remove any drops on the plate bottom with disposable tissues.

  2. 2.

    Use broth mycoplasma cultures without any preparation. Pipet the mycoplasma cultures to be identified and the reference cultures in 200-μL aliquots/well (see Notes 1 and 2 ). Usually, 12 cultures/plate and 1/column are tested. Filter the well contents by vacuum suction, and remove any drops on the plate bottom with tissue.

  3. 3.

    Add blocking solution in 200-μL aliquots/well and leave for 30 min of incubation with slow agitation. Filter the well contents as in step 2, and remove any drops on the plate bottom.

  4. 4.

    Dispense rabbit hyperimmune sera, and diluted in TBS-B, in 200-μL aliquots/well (see Note 3 ). Cultures to be identified are usually tested againts eight sera, 1/line. After 30 min of incubation with slow agitation, remove the fluids by vacuum filtration. Wash the wells by filtration, three times with TBS-T and once with TBS. Each wash lasts 5 min. Remove any drops on the plate bottom.

  5. 5.

    Dispense HRP labeled antirabbit IgG diluted in TBS-B in 200-μL aliquots/well. After 30 min of incubation with slow agitation, remove the well contents by filtration. Wash the wells by filtration, three times with TBS-T and once with TBS. Each wash lasts 5 min. Remove any drops on the plate bottom.

  6. 6.

    Add the developing solution in 200 μL aliquots/well. A reddish coloration appearing on the membrane filter within 1 min is the sign of a positive reaction. When the reaction is complete, wash the plate with distilled water (without filtration), and examine before drying (see Note 4 ).

4 Notes

  1. 1.

    MF dot is specific, but not very sensitive. Sensitivity varies from 104–107 mycoplasmas/well depending on the hyperimmune serum used and mycoplasma species tested (14). To avoid false-negative reactions, only cultures in which growth turbidity can be visually detected should be used. A 0.1-μm durapore filtration membrane must be used for ureaplasma serotyping because of the smaller cell size of this species.

  2. 2.

    The blocking of well filters occasionally occurs during the first step of vacuum filtration. There are two main causes: (a) the high density of the cultures of certain fast growing mollicutes (e.g., Acholeplasma laidlawii). These cultures should be diluted before use from 1/2 to 1/10; (b) precipitate in the broth medium or cell remains from the sample. These must to be eliminated by filtration through a 0.80-μm filter before use. A clear broth medium should always be used for culture, and vacuum depression should never exceed −40 to −60 kPa for more than 2 min, or the filtration membranes may be distorted.

  3. 3.

    The quality of the hyperimmune sera strongly affects the results. Many preparative techniques have been proposed, and the the following procedure has proven to be reliable. Hyperimmune sera are produced in rabbits, because the absence of a natural mycoplasma flora makes the rabbit highly suitable for the production of specific mycoplasma antisera. The mycoplasma culture in late log phase is centrifuged at 10,000g for 45 min, washed, and sedimented three times in PBS, pH 7.4, and resuspended in PBS to obtain a final concentration close to 1010 mycoplasmas/mL. Aluminum hydroxide gel is used as adjuvant. Immunization is performed as follows: at d 0 and 2, 105 mycoplasmas are inoculated intravenously and 105 intraperitonealy with adjuvant; at d 4, 108 mycoplasmas are inoculated subcutaneously with adjuvant; at d 6 and 8, 1010 are inoculated intramuscularly with adjuvant at six sites. Blood samples are obtained regularly from d 15 onward to test specificity and sensitivity of the sera by MF dot. As soon as working titres of 1/1500 to 1/2500 are obtained, the rabbits are bled. Higher titers have to be avoided, since problems of background staining may occur beyond dilutions of 1/5000.

  4. 4.

    Interpretation: always include a reference strain as a technical control. Deterioration of the color reagents or enzyme substrate is the most common cause of failure (see conservation of these solutions). MF dot immunobinding, like all serological identification tests, is only qualitative. Owing to the high rate of variability of the surface antigens in many species, reaction intensity varies from strain to strain, even with equal numbers of mycoplasmas in the broths. A positive reaction toward two or more hyperimmune sera may occur with some field isolates. In most cases, this does not result from technical problems, but from crossreactions or species mixtures. The causes may be:

    1. a.

      “Classical” crossreactions between reference strains. The homologous reaction is usually stronger than the heterologous one; in this case, MAb must be used;

    2. b.

      “Occasional” crossreactions. A few field strains appear to be antigenically intermediate between different reference strains. This presents a real problem in certain mycoplasma groups, such as in the “Mycoplasma mycoides cluster” (19);

    3. c.

      Mixed cultures of mycoplasma species frequently occur, especially in samples from respiratory tract. Mixed cultures cannot be distinguished from crossreactions in theory. In practice, however, crossreactions and mixtures do not involve the same species, but only immunobinding on colonies obtained according to appropriate procedures (14) will allow definitive conclusions to be drawn.