2. Commercially available antibodies against pathogens: the author's experience

In Table 1, commercially available antibodies against pathogens are listed up, simply for the convenience of the readers. The catalog is solely based on the author's experience, so that the antibodies may not be most suitable for detecting pathogens in routinely prepared sections [8, 19]. Some antibodies may be no longer available, simply because of limited market. The specificity of the antibodies is categorized


increasing the sensitivity of detection have been developed, in order to localize the antigens under highly specific and highly sensitive conditions in routinely processed (formalin-fixed, paraffin-embedded) sections [11–13]. Immunohistochemical approach is quite fitted to the histopathological diagnosis of infectious diseases, since the antigens of the pathogen are absent from the human tissue

pear from the market because of a simple reason: the dead stock [16].

In the routine practice of histopathological services using hematoxylin and eosin (H&E) staining, it is often the situation that the pathologists are requested to judge if the lesion is infective or not. Namely, the presence of some sort of pathogenic organisms within the lesion should be shown with the maximal priority in making the diagnosis, and the detailed identification of the species name should be analyzed using different technologies afterward. For example, if a lesion showing massive necrosis is experienced, we must comment that the necrosis is infective in origin or tumor-related. In such a situation, an antibody widely cross-reactive to bacteria but unreactive to human tissue is valuable. A monoclonal antibody H9 (a gift of Prof. Shigeru Kamiya, Department of Microbiology, Kyorin University School of Medicine, Mitaka, Tokyo) against bacteria-specific heat shock protein (HSP)-60, not cross-reactive animal mitochondrial HSP-60, is quite valuable [7, 17], but it is not commercially available. Lipopolysaccharide (LPS) or endotoxin, located in the outer layer of the bacterial wall, should be the good marker of Gram-negative bacteria (refer to Figure 11) [18], but it is practically difficult for us to get a monoclonal or polyclonal antibody detecting widely

In order to visualize pathogens in formalin-fixed, paraffin-embedded sections, we do not necessarily need to prepare antibodies with high specificity [4, 7, 19]. The author routinely uses four kinds of commercially available rabbit antisera raised against Bacillus Calmette-Guérin (BCG; Mycobacterium bovis), Bacillus cereus (B. cereus),Treponema pallidum (T. pallidum), and Escherichia coli (E. coli) [16, 19]. For immunoperoxidase staining, the amino acid polymer technique or indirect immunoperoxidase method is employed. Immunostaining using these lowspecificity antimicrobial antisera commonly yields clear high-sensitivity signals with low background, because of poor cross-reactivity of bacterial antigens to

histopathological sections is the use of patient's serum [4, 7, 8, 20–23]. Patients'sera

embedded sections, particularly when cellular tissue reactions have been confirmed under the microscope. Serum antibody titer should be high in the recovery or chronic stage of illness. The existence of inflammatory tissue reaction, such as an abscess or granuloma, indicates that immune cells in the patient have been activated against the causative pathogen. The second approach is of high value for protozoan

The second approach for detecting unknown pathogens in the

immunoperoxidase localization of pathogens in formalin-fixed, paraffin-

diluted at 1:500 or 1:1000 become convenient probes for indirect

However, pathogens express multiple antigens, and the antigens are often crossreactive among different pathogens [14]. The pathogen in a single category further reveals a variety of serum types [15]. It is more difficult for us pathologists than expected to detect a certain pathogen using a single antibody [4, 7]. It is next to impossible to prepare and keep specific antibodies in hand for immunohistochemical diagnosis in a single institution simply because there are too many species of microbes pathogenic to humans. We have a limited number of commercially available antibodies against pathogens. Useful commercial antibodies may soon disap-

specimens [4, 7–10].

Immunohistochemistry - The Ageless Biotechnology

cross-reactive LPS.

human cells and tissues.

and helminthic disorders.

70


into three grades: high, moderate, and low. Even when we use the high-specificity

Pathogen Type (clone) Company Dilution Pretreatment Specificity SV40 (T-antigen) Mo (PAb416) Millipore 1:100 EDTA High VZV Mo (C90.2.8) Novocastra 1:100 None High

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

Toxoplasma gondii Rabbit Abcam 1:200 CB6 High Abbreviations: Mo, monoclonal antibody; PK, proteinase K; CB6, citrate buffer at pH 6 (heating); CB7, citrate buffer at pH 7 (heating); EDTA, ethylenediaminetetraacetic acid at pH 8 (heating); DSHB, Developmental Studies Hybridoma Bank; BCG, Bacillus Calmette-Guérin; EB, Epstein-Barr; HB, hepatitis B; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; RS, respiratory syncytial; SV40, simian virus 40;

3. Application of commercially available antisera against BCG, B. cereus,

Immunoperoxidase application using four kinds of commercially available rabbit antisera raised against BCG, B. cereus,T. pallidum, and E. coli is described [4, 7, 8, 16, 19]. In this section, immunostaining application to mycobacterial infection, B. cereus pneumonia, syphilitic lesions, and E. coli infections is described. Although the specificity of the antisera is low, as described below, the respective

antibody for immunostaining, careful judgment is requested for the final

Antimicrobial antibodies the author is using are listed up for the readers' convenience.

List of commercially available antibodies against pathogens: the author's experience.

causative pathogens were clearly demonstrated within the lesions.

3.1 Immunostaining for mycobacterial infections using BCG antiserum

skin lesion. No positivity was seen in tuberculoid (paucibacillary) leprosy.

The BCG antigens were extremely stable after prolonged fixation in formalin for a long period of time [19, 26]. Figure 3 displays dense-positive signals in an exudative pulmonary tuberculosis lesion fixed in formalin for nearly 70 years.

Indirect immunoperoxidase staining using BCG rabbit antiserum (Dako/Agilent Technologies) diluted at a 1:5000 was highly sensitive for detecting mycobacteria in histopathological sections [4, 7, 8, 24, 25]. No pretreatment for antigen retrieval was given. Mycobacterial antigens were clearly demonstrable not only in caseous necrosis in active tuberculosis but also in a fibrous nodule of old calcified tuberculosis (Figure 1). BCG antigens were scarcely detectable in epithelioid granulomas. The BCG immunostaining was much more sensitive to detect mycobacteria than conventional acid-fast (Ziehl-Neelsen) staining, as indicated in Table 2 [4, 24]. In BCG immunostaining, the judgment of positivity can be done easily and quickly, while it takes minutes or longer in case of conventional acid-fast staining. It is evident that the antiserum is reactive with mycobacterial antigenic substances on destroyed bacterial fragments. The BCG immunostaining was also applicable to demonstrating non-tuberculous mycobacteria and Mycobacterium leprae. Figure 2 illustrates positive findings in opportunistic Mycobacterium avium-intracellulare (MAI) infection in AIDS and in lepromatous (multibacillary) leprosy in a biopsied

identification of the causative microbe.

T. pallidum, and E. coli

Antiprotozoan antibody

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

VZV, varicella-zoster virus

Table 1.

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Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.85055


Abbreviations: Mo, monoclonal antibody; PK, proteinase K; CB6, citrate buffer at pH 6 (heating); CB7, citrate buffer at pH 7 (heating); EDTA, ethylenediaminetetraacetic acid at pH 8 (heating); DSHB, Developmental Studies Hybridoma Bank; BCG, Bacillus Calmette-Guérin; EB, Epstein-Barr; HB, hepatitis B; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; RS, respiratory syncytial; SV40, simian virus 40; VZV, varicella-zoster virus

Antimicrobial antibodies the author is using are listed up for the readers' convenience.

#### Table 1.

Pathogen Type (clone) Company Dilution Pretreatment Specificity Aspergillus Mo (WF-AF-1) Thermo 1:50 EDTA High Candida Rabbit Unitika 1:8000 PK Moderate Candida Mo (MaB806) Chemicon 1:400 Pepsin High Cryptococcus Rabbit Dako 1:500 None Moderate Pneumocystis Mo (3F6) Dako 1:100 CB6 High

Adenovirus Mo (M58 + M73) Abcam 1:400 PK High BK virus Mo (5E6) Abnova 1:500 CB6 High

EB virus (LMP1) Mo (CS1–CS4) Dako 1:50 PK High EB virus (EBNA2) Mo (PE2) Dako 1:100 EDTA High Influenza virus A Mo (1331) AbD 1:100 PK High Influenza virus A Mo (1A52.9) Acris 1:100 PK High Influenza virus B Guinea pig Denka Seiken 1:100 EDTA High HBs antigen Goat Bioss 1:2000 PK High HBs antigen Mo (HB-024) Japan Biotest 1:500 None High HBc antigen Rabbit Dako 1:15,000 EDTA High

Immunology

Rabbit Dako 1:500 None Moderate

Rabbit Dako 1:1500 None Moderate

Mo (CM2B4) Santa Cruz 1:100 EDTA High

HCV (NS3–NS4) Mo (Tordji-22) Signet 1:200 EDTA High HCV (NS3) Mo (MMM33) Novocastra 1:100 CB7 High HCV (NS4) Mo (5D4-10E7) Abcam 1:300 EDTA High HCV (NS5a) Mo (7-D4) Fitzgerald 1:100 EDTA High HCV (core) Mo (Aa70–Aa90) Chemicon 1:100 EDTA High

HIV (p24) Mo (kal-1) Dako 1:1000 EDTA High HPV Mo (K1H8) Dako 1:500 CB6 High HPV16 Mo (CamVir-1) BioGenex 1:5000 EDTA High JC virus Rabbit Dako 1:300 CB7 High

Measles virus Rabbit Novus 1:1000 EDTA High Mumps virus Mo (MAB846) Chemicon 1:400 EDTA High Norovirus (GII/4) Rabbit Denka Seiken 1:500 None High Parvovirus B19 Mo (R92.2.8) Novocastra 1:1000 EDTA High RS virus Mo (603705) Novocastra 1:200 EDTA High

Thermo 1:50 CB6 High

Dako 1:200 EDTA High

1:50 PK High

Rhizopus Mo (WSSA-RA-

Immunohistochemistry - The Ageless Biotechnology

Cytomegalovirus Mo

Antiviral antibody

Herpes simplex virus-1

Herpes simplex virus-2

Merkel cell polyomavirus

72

1)

(CCH2 + DDG9)

HBe antigen Mo (BE-05) Institute of

List of commercially available antibodies against pathogens: the author's experience.

into three grades: high, moderate, and low. Even when we use the high-specificity antibody for immunostaining, careful judgment is requested for the final identification of the causative microbe.
