4.13 Immunostaining with B. cereus antiserum showing the widest cross-reactivity among four

In certain lesions, B. cereus antiserum was reactive to the pathogen, while the other three antisera were poorly reactive. It is plausible that antiserum against B. cereus, a Gram-positive spore-forming rod, shows the widest cross-reactivity including Gram-positive bacteria. Bacterial microbes positive for B. cereus-related antigens were demonstrated in brain abscess, placental chorioamnionitis, and Propionibacterium acnes-induced folliculitis of the skin (Figure 22) (refer also to Figure 45 for P. acnes folliculitis). Positive signals of B. cereus antigens in a lethal adult case of Pseudomonas aeruginosa-induced pneumonia/septicemia are demonstrated in Figure 23 [19].

#### Figure 21.

Rhinoscleroma caused by Klebsiella rhinoscleromatis (left upper: H&E, right upper: reactivity with monoclonal antibody 70-2 to Klebsiella spp., left lower: B. cereus antigens, right lower: E. coli antigens, inset: BCG antigens). Foamy macrophages with intracytoplasmic vacuoles containing short rod-like material (Mickulicz cells) are immunoreactive with monoclonal antibody 70-2 to Klebsiella spp. and B. cereus antiserum. Fewer cells are labeled with BCG antiserum, while E. coli antiserum is unreactive.

paraffin-embedded sections were scarcely detected by the peroxidase-labeled secondary antihuman immunoglobulins. High-sensitivity detection sequence such as the labeled polymer method naturally leads to high-background staining, because of the detection of endogenous immunoglobulins in sections. The method is simple, economic, useful, and beautiful for the histopathologic diagnosis of infectious diseases, and it is particularly suitable for the developing countries, since the patient's serum is free in charge. This approach is especially effective for detecting protozoa and helminth [22], because the specific antibodies are often commercially unavailable.

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens…

DOI: http://dx.doi.org/10.5772/intechopen.85055

There are two situations. In some cases, the causative pathogen has been identified by clinical, laboratory, and/or histopathological analysis, and thus the specificity of the patient's serum can be expected before immunostaining. In other cases, the causative pathogen is unsettled yet or unknown. In the latter situation, the patient's serum functions as a low-specificity and high-sensitivity probe in immunostaining. Table 4 summarizes patients'sera applicable to immunostaining in paraffin sections.

Human tissues and organs, obtained either by biopsy, during surgery or at autopsy, were routinely fixed in 10% unbuffered or buffered formalin for 1 day to 4 weeks. The indirect immunoperoxidase technique was applied to deparaffinized sections [4, 7, 8, 20–23]. The serum of patient, principally not in an immunocompromised state, was diluted at 1:500 or 1:1000 and was incubated for 30 min or overnight. In case of protozoan infection, the presence of high-titer IgG antibodies in the patients'serum was commonly confirmed by immunofluorescence titration, as shown in Table 4. The serum from patients positive for human immunodeficiency virus (HIV) or hepatitis virus markers must not be utilized, in order to avoid biohazard. The serum of immunocompromised, non-HIV patients may be utilized after dilution at 1:5 or 1:10. The second layer reagent was horseradish peroxidase-labeled goat IgG to human immunoglobulins (Dako/Agilent) at a 1:50 dilution. Endogenous peroxidase activity was quenched in methanol containing 0.3% hydrogen peroxide for 20 min. No other pretreatment procedures such as proteinase digestion or heat-induced antigen retrieval were needed, but heating pretreatment was infrequently necessary for certain instances (e.g., the detection of free-living amoeba, Balamuthia; see below). Therefore, the author recommends introducing a heat-induced antigen retrieval procedure in 10 mM citrate buffer, pH 6, for immunostaining using silane (3-aminopropyltrimethoxysilane)-coated glass slides. After the diaminobenzidine coloring reaction, the nuclei were counterstained with 5% methyl green or Mayer's hematoxylin. When necessary, paraffin sections of related or unrelated infectious lesions were immunostained to confirm the cross-reactivity or specificity of the patients'serum. It is of notice that IgG in the patients'serum may show cross-reactivity to related

pathogens to certain or considerable degrees [4, 20, 21]. In bacterial and fungal infections, the sera often serve as pan-bacterial or pan-fungal probes. The crossreactivity may result from the naturally acquired antibodies in healthy individuals [20]. In viral, protozoan, and helminthic infections, in contrast, high-grade specificity with limited cross-reactivity can be expected [22]. When the specificity of the sera of patients with parasitic infestation is known, it is sufficiently satisfactory to enable them to be employed as specific antibody reagents for the following new cases.

5.2 Immunostaining using sera of patients with established diagnosis

or fixed diagnosis is shown, including bacterial, fungal, viral, protozoan, and

Here, the immunohistochemical application of sera of patients with established

5.1 Methodology

helminthic infections.

89

#### Figure 22.

The widest cross-reactivity shown by Bacillus cereus antiserum (left upper, brain abscess; right upper, ascending chorioamnionitis of placenta; lower panels, Propionibacterium acnes-induced folliculitis of the chest, combination of H&E, and B. cereus immunostaining). Gram-positive bacterial microbes are visualized by immunostaining with B. cereus antiserum. No positivity is seen with BCG, T. pallidum, and E. coli antisera.

#### Figure 23.

Lethal Pseudomonas aeruginosa pneumonia/septicemia (left, H&E; center, immunostaining using monoclonal antibody B11; right, immunostaining using B. cereus antiserum). Gram-negative bacilli grow along the alveolar septum, and no cellular reactions are noted. Immunoreactive signals are observed with the monoclonal antibody, as well as with B. cereus antiserum.
