Читать книгу Diagnostics and Therapy in Veterinary Dermatology - Группа авторов - Страница 50
Enzyme‐Linked Immunosorbent Assay
ОглавлениеELISA is a plate‐based assay that allows detection and quantification of antigens such as hormones, antibodies, enzymes, proteins, and peptides. This test is used frequently in veterinary medicine due to its diagnostic accuracy, affordability, accessibility, rapid turnaround time, and ability to be modified into a SNAPR assay for in‐clinic use. Detection strategies used include IF, chemiluminescence, and colorimetric (chromogenic) techniques.
Table 3.1 Common diagnostic tests available in veterinary dermatology. This table includes some of the common diagnostic testing modalities for dermatologic conditions, including hypersensitivities, infectious diseases, and genetic abnormalities. This table is not inclusive of every possible diagnostic test available, and the authors recommend that readers contact commercial and research laboratories for a more complete list of available diagnostic tests. Many research and university laboratories have the ability to develop new molecular assays, and represent an invaluable resource, especially when newly describing uncommon dermatologic disorders.
| Serologic allergy testing | |||||
|---|---|---|---|---|---|
| Test | Company | Type of test | Sample required | Information obtained | Website |
| SPOT Platinum+ | ACTT® and Spectrum Veterinary | ELISA | Serum | Quantitative assay to measure allergen‐specific IgE | www.acttallergy.com |
| Allercept | Heska | ELISA using high‐affinity receptor (FcꞓRIα) for IgE, which prevents non‐IgE antibody binding | Serum | Quantitative assay to measure allergen‐specific IgE. 86–92% agreement with Greer® Aller‐g‐complete | www.heska.com |
| Aller‐g‐detect™ Allergen Preliminary Panel | IDEXX | ELISA | Serum | Qualitative assay that tests for presence of environmental allergen‐specific IgE. If positive, follow up with quantitative analysis using Greer Aller‐g‐complete | www.idexx.com |
| Greer Aller‐g‐ complete® | IDEXX | Non‐competitive solid‐phase ELISA using macELISA technology (cocktail of biotinylated canine monoclonal antibodies specific to Fc region of IgE) | Serum | Quantitative assay to measure allergen‐specific IgE with Heska Allercept | www.idexx.com |
| VARL Liquid Gold | VARL | Liquid‐phase matrix enzyme immunometric system using avidin‐biotin interaction to capture allergen‐specific IgE combined with solid‐phase ELISA methodology to quantify IgE | Serum | Quantitative assay to measure allergen‐specific IgE | www.varlallergy.com |
| Molecular diagnostics for infectious organisms | |||||
| Test | Company | Sample required | Information obtained | Website | |
| Ringworm (Dermatophyte) RealPCR™ Panel | IDEXX | Hair, skin scrapings (from active border of lesion) | Dermatophyte (Microsporum spp. or Trichophyton spp.) Positive results indicate infection when clinical lesions are present, but indicate nonclinical carriage state without clinical lesions. Negative results indicate no or few Microsporum spp. or Trichophyton sp. organisms in sample. Because positive PCR results can be due to active infection, fomite carriage or nonviable fungal organisms from a successfully treated infection, PCR for dermatophyte may be most useful for initial diagnosis, while fungal culture may be a better indicator of mycologic cure. Note: False negatives have been seen with this test, so culture is still recommended | www.idexx.com | |
| Sarcoptes Antibody ELISA | IDEXX | Serum | Measures presence of anti‐Sarcoptes antibodies in serum. Positive results indicate possible Sarcoptes infection, or false positive due to house dust mite hypersensitivity (these antigens cross react). Dogs may remain positive for up to 6 months after successful treatment. Negative results indicate lack of exposure, early infection, or immunosuppressive medication interactions. Sensitivity is 84.2%, and specificity is 89.5% | http://www.idexx.com | |
| Tick PCR Panels | Antech: Tick Borne PCR Panel | Whole blood (EDTA) | Indicates the presence of DNA from various tick‐borne organisms | www.antechdiagnostics.com | |
| Cornell University College of Veterinary Medicine, Animal Health Diagnostic Center PCR | Indicates the presence of DNA from Ehrlichia canis, Neorickkettsia risticii, Anaplasma phagocytophilum, Bartonella spp., hemotropic Mycoplasma spp. Many additional individual PCR tests available on website | http://www.vet.cornell.edu/animal‐health‐diagnostic‐center/testing/protocols/blood‐parasite | |||
| IDEXX: Tick/Vector Comprehensive RealPCR Panel | Indicates the presence of DNA from Anaplasma spp., Babesia spp., Bartonella spp., canine hemotropic Mycoplasma, Ehrlichia spp., Hepatozoon spp., Leishmania spp., Neorickettsia risticii, and Rickettsia rickettsii. Quantifies DNA of Leishmania spp. | www.idexx.com | |||
| North Carolina State University Vector Borne Disease Diagnostic Laboratory | Indicates the presence of DNA from Bartonella spp., Anaplasma spp., Ehrlichia spp., Babesia spp., Cytauxzoon felis, Mycoplasma spp., Leishmania spp., Rickettsia spp. | http://cvm.ncsu.edu/research/labs/clinical‐sciences/vector‐borne‐disease | |||
| Tick Serology Panels | Antech: AccuPlex™ 4 Fastpanel PCR Canine Tick Borne Profile w/Lyme | Serum Serum Whole blood (EDTA) | Measures antibodies to Borrelia burgdorferi, Anaplasma sp., and Ehrilichia sp., and measures Dirofilaria immitis antigen Measures presence of DNA from several tick‐borne organisms, and measures Borrelia burgdorferi antibodies. Also offers B. burgdorferi PCR and Western Blot | www.antechdiagnostics.com | |
| Cornell University College of Veterinary Medicine, Animal Health Diagnostic Center | Serum | Measures antibodies to Rickettsia rickettsii, Neorickettsia risticii, Ehrlichia canis, Brucella canis, Bartonella spp. by IFA; measures antibodies to Borrelia burgdorferi, Ehrlichia canis, and Anaplasma spp. by ELISA; measures Leishmania infantum by indirect ELISA | http://www.vet.cornell.edu/animal‐health‐diagnostic‐center/testing/protocols/blood‐parasite | ||
| IDEXX: Tick Panel – immunofluorescence antibody (IFA) test Lab 4Dx® Plus Test (ELISA) Lyme Quant C6® | Serum Serum Serum Urine | Quantifies Ehrlichia canis, Rickettsia rickettsii, and Borrelia burgdorferi antibody Measures antibodies to Anaplasma phagocytophilum, Anaplasma platys, Ehrlichia canis, Ehrlichia ewingii, and Borrelia burgdorferi, and presence of Dirofilaria immitis antigen Measures Borrelia burgdorferi antibody, limited chemistry profile, limited complete blood count, SDMA, urinalysis, and urine protein:creatinine ratio. Lyme Quant C6 testing is available with the RealPCR panel. | www.idexx.com | ||
| North Carolina State University Vector Borne Disease Diagnostic Laboratory | Serum | Measures antibodies to Bartonella hensalae, Bartonella vinsonii, Bartonella koehlerae, Babesia canis, Babesia gibsonii, Ehrlichia canis, Rickettsia rickettsii, Leishmania infantum, Anaplasma sp., Borrelia burgdorferi | http://cvm.ncsu.edu/research/labs/clinical‐sciences/vector‐borne‐disease | ||
| Bacterial PCR | Provided by many research institutions and universities. Polymerase chain reaction (PCR) tests are available for common dermatologically relevant bacteria, including Staphylococcus spp., Pseudomonas sp., Streptococcus sp., E. coli, Bordetella sp., Mycobacteria spp., Bartonella sp., etc. Refer to diagnostic laboratories at large research institutions nearest you for more information, especially in regard to each test's sensitivity and specificity. While most PCRs are highly sensitive and specific, remember that the presence of DNA can represent contamination of the sample or the presence of nonviable bacteria. Results should be correlated with clinical findings, cytology, histopathology, and even culture when possible | ||||
| Fungal serology | IDEXX | Serum | Measures antibodies to Coccidioides sp., Blastomyces sp., Histoplasma sp., Aspergillus sp.; measures Cryptococcus sp. antigen (latex agglutination) | www.idexx.com | |
| Antech | Serum Urine | Measures antibodies to various fungi, including Coccidioides sp., Histoplasma sp., and others. Measures presence of Cryptococcus sp. antigen (latex agglutination) and Blastomyces sp. antigen (urine) | www.antechdiagnostics.com | ||
| Provided by many research institutions and universities. PCRs are available for common dermatologically relevant fungi. Refer to diagnostic laboratories at large research institutions nearest you for more information, especially in regard to each test's sensitivity and specificity | |||||
| Fungal PCR | IDEXX: Blastomyces dermatitidis RealPCR Test Systemic Mycoses RealPCR Test | Whole blood (EDTA) or deep pharyngeal swab Aspirated material or fresh tissue | Positive indicates the presence of Blastomyces dermatitidis DNA in sample and must be considered with presence of clinical signs consistent with infection. False negative is possible if sample is inadequate or due to antifungal therapy administration Positive indicates presence of DNA from Blastomyces dermatitidis, Coccidioides spp., Cryptococcus spp., or Histoplasma capsulatum. False negative possible if sample inadequate or due to antifungal therapy administration | www.idexx.com | |
| Provided by many research institutions and universities. PCRs are available for common dermatologically relevant fungi. Panfungal PCR for identification and sequencing of numerous fungi is available through some laboratories such as the Texas A&M Veterinary Medical Diagnostic Laboratory. Refer to diagnostic laboratories at large research institutions nearest you for more information, especially in regard to each test's sensitivity and specificity. While most PCRs are highly sensitive and specific, remember that presence of DNA can represent contamination of the sample or the presence of nonviable fungi. Results should be correlated with clinical findings, cytology, histopathology, and even culture when possible | |||||
| Oomycete ELISA | Auburn University Infectious Disease Laboratory Pan American Veterinary Laboratories | Serum | Percent positivity correlates with quantity of anti‐Pythium sp. antibodies in sample | http://www.vetmed.auburn.edu/academic‐departments/dept‐of‐pathobiology/diagnostic‐services www.pavlab.com | |
| Oomycete PCR | Texas A&M Veterinary Diagnostic Laboratory: Panfungal PCR | Tissue | Formalin‐fixed paraffin‐embedded tissue and unstained slides are used to detect and identify oomycete DNA | http://tvmdl.tamu.edu | |
| Viral PCR | IDEXX: RealPCR Test | Whole blood (EDTA) | Positive indicates presence of DNA from canine and feline herpesvirus, canine distemper virus, feline calicivirus, feline leukemia virus, feline infectious peritonitis virus, or feline coronavirus | www.idexx.com | |
| Provided by many research institutions and universities. PCRs are available for common dermatologically relevant viruses. Refer to diagnostic laboratories at large research institutions nearest you for more information. | |||||
| Additional molecular diagnostics | |||||
| IHC | Commonly used for diagnostic purposes. Provided by many research institutions and universities. Immunohistochemistry (IHC) stains to distinguish different cellular markers as well as different infectious organisms are available. Refer to commercial and university diagnostic laboratories nearest you for more information | ||||
| IF | Not commonly used or provided for diagnostic purposes. Immunofluorescence (IF) is primarily used by research institutions to distinguish different cellular markers, autoantibodies, proteins, receptors, and infectious organisms | ||||
| Flow cytometry | Commonly utilized for diagnostic and research purposes. Provided by many research institutions and universities. Most commonly used for cell sorting of immune cells, particularly when describing and diagnosing certain neoplastic and autoimmune conditions. Refer to diagnostic laboratories at large research institutions nearest you for more information | ||||
| TEM | Not commonly used or provided for diagnostic purposes. Transmission electron microscopy (TEM) is primarily used by research institutions to distinguish different cellular markers, autoantibodies, proteins, receptors, and infectious organisms | ||||
| Molecular diagnostics for genetic mutations – USA Most of these tests use a combination of techniques such as PCR, whole‐genome sequencing, whole‐exome sequencing, DNA profiling, etc. Genetic testing available outside of the USA can be found here: https://www.vet.upenn.edu/research/academic‐departments/clinical‐sciences‐advanced‐medicine/research‐labs‐centers/penngen/tests‐worldwide | |||||
| Laboratory | Tests available | Sample required | Information obtained | Website | |
| Animal Genetics | Acral mutilation syndrome, canine leukocyte adhesion deficiency, cyclic neutropenia/gray collie syndrome, dermatomyositis, hereditary nasal parakeratosis, ichthyosis (American bulldog and golden retriever), multidrug resistance (MDR1), Musladin‐Lueke syndrome, oculocutaneous albinism (Doberman pinscher) | Many sample types are acceptable, including cheek swab, blood card, and blood swab. Contact laboratory regarding specific sample depending on test | Test results will determine if the animal is a carrier for the known mutation causing the disorder | www.animalgenetics.us | |
| DNA Diagnostics Center | Hereditary nasal parakeratosis, multidrug resistance (MDR1) | Cheek swab | Test results will determine if the animal is a carrier for the known mutation causing the disorder | http://dnacenter.com/testing‐pets‐vets | |
| Embark (partnership with Cornell University College of Veterinary Medicine) | Multidisease screening. Dystrophic epidermolysis bullosa, ectodermal dysplasia (skin fragility syndrome), ichthyosis, epidermolytic hyperkeratotic ichythyosis, focal non‐epidermolytic ichthyosis, palmoplantar keratoderma, pachyonychia congenita, hereditary footpad hyperkeratosis, hereditary nasal parakeratosis, Musladin‐Lueke syndrome, and bald thigh syndrome. Additional unlisted genetic tests are available by contacting the laboratory | Cheek swab | Whole‐genomic sequencing with microarray SNP chip technology. Test results will determine if the animal is a carrier for the known mutation causing the disorder | http://embarkvet.com | |
| IDEXX | Hereditary equine regional dermal asthenia (HERDA) in US laboratory; junctional epidermolysis bullosa (JEB1 and JEB2), hereditary nasal parakeratosis, dry eye curly coat syndrome, ichthyosis, and Musladin‐Lueke syndrome (beagle) in European laboratory | Whole blood (EDTA) or cheek swab | PCR test results will determine if the animal is a carrier for the known mutation causing the disorder | www.idexx.com | |
| Institute of Genetics, University of Bern, Switzerland | Genomic sequencing and DNA analysis | Many sample types permitted. Contact laboratory regarding specific sample depending on test | Research laboratory specializing in discovery and characterization of genetic diseases in animals, with an interest in dermatologic disorders | https://www.genetics.unibe.ch/about_us/person/prof_dr_leeb_tosso/index_eng.html | |
| My Dog DNA | Multidisease screening | Cheek swab | Test results will determine if the animal is a carrier for the known mutation causing the disorder | www.mydogdna.com | |
| Optimal Selection™ | Multidisease screening | Cheek swab | Test results will determine if the animal is a carrier for the known mutation causing the disorder | www.optimal‐selection.com | |
| Orivet Genetic Pet Care | Congenital hypothyroidism, dermatomyositis, dry eye curly coat syndrome, ectodermal dysplasia, dystrophic epidermolysis bullosa, hereditary footpad hyperkeratosis, hereditary nasal parakeratosis, ichthyosis, (golden retriever, American bulldog, Norfolk terrier, great Dane, German shepherd), lethal acrodermatitis, multidrug resistance (MDR1), Musladin‐Lueke syndrome, renal cystadenocarcinoma and nodular dermatofibrosis | Cheek swab | Test results will determine if the animal is a carrier for the known mutation causing the disorder | www.orivet.com | |
| Paw Print Genetics of Genetic Veterinary Sciences and Canine Health Check (multidisease screening) | Anhidrotic ectodermal dysplasia, ectodermal dysplasia, epidermolytic hyperkeratosis, ichythyosis, dystrophic epidermolysis bullosa, Musladin‐Lueke syndrome, dry eye curly coat syndrome, D locus (dilute), leukocyte adhesion deficiency type I, severe combined immunodeficiency disease (terrier type), renal cystadenocarcinoma and nodular dermatofibrosis, Glanzmann's thrombasthenia | Many sample types are acceptable, including whole blood in EDTA and cheek swabs. Contact laboratory regarding specific sample depending on test | Test results will determine if the animal is a carrier for the known mutation causing the disorder | www.pawprintgenetics.com www.caninehealthcheck.com | |
| PennGen, University of Pennsylvania | Congenital hypothyroidism, ectodermal dysplasia (skin fragility syndrome – Chesapeake Bay retriever), epidermolysis bullosa (Eurasier), ichthyosis (American bulldog), lethal acrodermatosis (bull terrier), leukocyte adhesion deficiency (type I), exfoliative cutaneous lupus erythematosus (German shorthaired pointer) | Cheek swab or whole blood (EDTA) | PCR test results will determine if the animal is a carrier for the known mutation causing the disorder. Comprehensive list of genetic tests available worldwide at the PennGen website | http://www.vet.upenn.edu/research/academic‐departments/clinical‐sciences‐advanced‐medicine/research‐labs‐centers/penngen | |
| UC Davis Veterinary Genetics Laboratory | Hereditary nasal parakeratosis | Many sample types are acceptable, including cheek swab, whole blood in EDTA, and hair samples. Contact laboratory regarding specific sample depending on test | Test results will determine if the animal is a carrier for the known mutation causing the disorder | http://vgl.ucdavis.edu | |
| vetGen™ Veterinary Genetic Services | Albinism in Doberman pinscher, canine cyclic neutropenia, dry eye curly coat, hereditary footpad hyperkeratosis, heredity nasal parakeratosis, Musladin‐Lueke syndrome, renal cystadenocarcinoma nodular dermatofibrosis | Cheek swab | Test results will determine if the animal is a carrier for the known mutation causing the disorder | www.vetgen.com | |
| Wisdom Health, Wisdom Panel™, formerly Optigen | Dermatomyositis, hereditary nasal parakeratosis, ichthyosis (golden retriever), multidisease screening | Many sample types are acceptable, including cheek swab, whole blood in EDTA, semen, and tissue. Contact laboratory regarding specific sample depending on test | Test results will determine if the animal is a carrier for the known mutation causing the disorder | http://breeder.wisdompanel.com |
There are three basic types of ELISA techniques: direct, indirect, and capture (sandwich) techniques (Tizard 2013). The direct method (Figure 3.1) is used primarily to detect and quantify antigen in a sample. This technique requires fewer steps than the others and is easier to perform since it only involves use of a single primary antibody labeled with a reporter enzyme. Substrate added to the sample interacts with the reporter enzyme to produce a product (e.g. color, fluorescence, or luminescence) that is measured with a plate reader. The direct method's use is limited to IHC, since it requires that the sample antigen is immobilized. The indirect technique (Figure 3.2) is the most popular type of assay and is used primarily to detect antibodies in a sample. This requires binding of antigen to the assay plate, adding the animal's serum (containing the antibody), and then adding detection antibody that is linked to a reporter enzyme along with substrate to be measured with the plate reader. The capture technique (Figure 3.3), sometimes called the “sandwich assay,” is a modified version that detects antigen in the sample and has superior sensitivity and specificity compared to the other techniques.
Figure 3.1 Direct ELISA. In the first stage the antigen (orange triangle) from the patient's serum is added to the plate where it is absorbed. This antigen can be a protein, hormone, etc. Next the labeled antibody (blue antibody) is added. The plate is washed between steps to remove unattached antibodies and antigens. Finally the substrate (yellow star) for the enzyme is added to produce a color change that can be identified and quantitated.
Figure 3.2 Indirect ELISA. The antigen (orange triangle) is adhered to the plate. The patient's serum with the patient's antibodies (yellow) is added. The labeled antibody (blue) is then added. The plates are washed between each step to remove unattached antibodies. Finally the substrate (yellow star) for the enzyme is added to produce a color change that can be identified and quantitated.
Other types of ELISAs exist, including a competitive ELISA for detecting very small antigens (haptens), an enzyme‐linked immunospot assay (ELISPOT) for quantifying proteins, and an in‐cell ELISA to detect proteins within living cells (Ji 2016).
ELISA tests are highly variable in their sensitivity and specificity, which is influenced by the use of polyclonal versus monoclonal detection antibodies, different plate types, washing techniques, reporter enzymes, incubation times, and much more. In veterinary dermatology, ELISA tests are used to measure antibodies to various infectious organisms (e.g. Sarcoptes sp., Blastomyces spp., and numerous tick‐borne parasites). ELISA technology is also the primary modality used for measuring serum levels of allergen‐specific IgE (Diesel and Deboer 2011; Lee et al. 2009, 2012, 2015; Plant et al. 2014). Intradermal testing is still considered the gold standard for identification of relevant allergens for inclusion in allergen‐specific immunotherapy, due to its ability to demonstrate an immunologic response (wheal and flare) to the injected allergen in the skin, the target organ in our veterinary species. However, ELISA testing does have some advantages. Notably, ELISA technology makes allergy testing possible for pets without access to a veterinary dermatologist, pets unable to stop medications such as steroids that interfere with intradermal testing, and pets with comorbidities that prohibit sedation required for intradermal testing. The first commercially available ELISA tests for allergen‐specific IgE used polyclonal antibodies and suffered from a low specificity. More specialized ELISA technology has since been developed. Heska Allercept® testing uses a recombinant form of the human mast cell high‐affinity receptor for IgE (Fc€RIα) for improved specificity to allergen‐specific IgE. Greer’s Aller‐g‐complete® ELISA test uses monoclonal antibody cocktails recognizing multiple epitopes on IgE for improved sensitivity, and it is currently investigating the use of inhibitor antibodies to prevent nonspecific IgE binding to cross‐reactive carbohydrate modalities on the surface of plant and insect antigens (Gedon et al. 2019; Levy and Deboer 2018; Piccione and Deboer 2019).
Figure 3.3 Sandwich ELISA. Monoclonal antibody (yellow) for the antigen that is being tested is attached to a plate. The patient's serum with possible antigen (orange triangle) is then added to the plate. Next enzyme‐labeled monoclonal antibodies (blue) are added. The plate is washed between each step to remove unattached antigen and antibody. Finally the substrate (yellow star) is added to produce the color change.
For now, intradermal allergy testing (skin testing) remains the most reliable method for identifying allergens for use in immunotherapy. Clinicians must remember that neither skin testing nor serologic testing is used to make the diagnosis of atopy. These tests are used to identify indoor allergens such as house dust mite that might be avoided, or to identify allergens for inclusion in allergen‐specific immunotherapy.
