Direct fluorescent antibody. A direct fluorescent antibody ( DFA or dFA ), also known as " direct immunofluorescence ", is an antibody that has been tagged in a direct fluorescent antibody test. Its name derives from the fact that it directly tests the presence of an antigen with the tagged antibody, unlike western blotting,...
What is the sensitivity of direct fluorescent antibody test?
The initial antigen detection test was the direct fluorescent antibody (DFA) test, which in experienced laboratories had a specificity of 99.6%, but its sensitivity was only 61% when compared with culture. 182,185,186,190–192 The development of highly sensitive and specific PCR assays have made the DFA test obsolete.
What is direct fluorescent antibody (DFA)?
Direct fluorescent antibody. Commercial DFA testing kits are available, which contain fluorescently labelled antibodies, designed to specifically target unique antigens present in the bacteria or virus, but not present in mammals ( Eukaryotes ). This technique can be used to quickly determine if a subject has a specific viral or bacterial...
How to test for tissue antigen?
Test for tissue antigen using either a direct method, by conjugation of antibody with fluorescent dye ( fluorescent antibody technique, direct) or an indirect method, by formation of antigen-antibody complex which is then labeled with ( fluorescent antibody technique, indirect ).
How is a DFA test done?
The most important part of a dFA test is flouresecently-labeled anti-rabies antibody. When labeled antibody is incubated with rabies-suspect brain tissue, it will bind to rabies antigen. Unbound antibody can be washed away and areas where antigen is present can be visualized as fluorescent-apple-green areas using a fluorescence microscope.
What is the difference between a direct fluorescence assay and an indirect fluorescence assay?
Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore. Indirect IF uses two antibodies. The primary antibody is unconjugated and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.
How is direct fluorescent antibody test performed?
The sample is sent to a lab. There, a fluorescent dye is added to the sample. If micro-organisms are present, a bright glow (fluorescence) can be seen in the sputum sample using a special microscope.
What is fluorescent antibody technique?
In general, fluorescence antibody (FA) techniques will involve the use of cells or an antibody, whose constant region has been tagged with a fluorescent label otherwise known as a fluorophore. FA techniques are often categorized as either direct or indirect.
What is direct immunofluorescence test?
Direct immunofluorescence (DIF) is a technique used in the laboratory to diagnose diseases of the skin, kidney, and other organ systems. It is also called the direct immune fluorescent test or primary immunofluorescence.
What is IFA testing?
IFA is an assay which uses fluorescent microscopy to detect antibodies to specific antigenic material. This test is often used to confirm positive results obtained by ELISA (Enzyme Linked Immunosorbent Assay) or MFIA® (Multiplexed Fluorometric ImmunoAssay®).
What is the purpose of immunofluorescence?
Immunofluorescence (IF) is an important immunochemical technique that allows detection and localization of a wide variety of antigens in different types of tissues of various cell preparations.
What are the types of immunofluorescence assay?
Types of immunofluorescence There are two classes of immunofluorescence techniques, primary (or direct) and secondary (or indirect).
What's the difference between fluorescence and immunofluorescence?
Immunofluorescence indicates that a fluorescent tag was used to visualize the marker of interest but fluorescent markers can be used for immunocytochemistry (cells) or for immunohistochemsitry (tissues).
What is fluorescent antibody staining?
Immunofluorescence or fluorescent antibody staining is an antigen-detection test that is used primarily on frozen tissue sections, cell “smears,” or cultured cells; formalin-fixed tissue samples are generally not useful with this procedure.
What is direct and indirect assays?
Direct ELISAs use a conjugated primary antibody, while indirect ELISAs include an additional amplification step. In an indirect ELISA, an unconjugated primary antibody binds to the antigen, then a labeled secondary antibody directed against the host species of the primary antibody binds to the primary antibody.
What is indirect fluorescent antibody test?
The indirect fluorescent antibody test (IFA) is a semi-quantitative, sensitive, and rapid test for the detection of anti-rabies virus (RABV) immunoglobulin M (IgM) and G (IgG) antibodies in serum and cerebral spinal fluid (CSF) samples.
Why is indirect immunofluorescence more commonly used?
In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody.
What is fluorescence test?
The fluorescent eye test is useful in determining if there is a scratch or other problem with the surface of the cornea. It can also be used to detect foreign bodies on the surface of the eye, and determine if there is an injury to the eye or eye infection.
What is fluorescent antibody staining?
Immunofluorescence or fluorescent antibody staining is an antigen-detection test that is used primarily on frozen tissue sections, cell “smears,” or cultured cells; formalin-fixed tissue samples are generally not useful with this procedure.
What is FITC in flow cytometry?
Fluorescein isothiocyanate (FITC), for example, will absorb light from 400-530 nm but absorbs most efficiently at its peak or excitation maximum of 490 nm wavelength.
Which technique used to diagnose a disease is also called the double diffusion method?
Immunodiffusion is one of the methods used to diagnose fungal diseases. Complement fixation is also used to test for some of the fungal diseases. The specific type of immunodiffusion is called Double (Ouchterlony) microimmunodiffusion. The procedure involves adding antigen and antibody to wells in an agarose gel.
What is the DFA test?
The dFA test is based on the observation that animals infected by rabies virus have rabies virus proteins (antigen) present in their tissues. Because rabies is present in nervous tissue (and not blood like many other viruses), the ideal tissue to test for rabies antigen is brain. The most important part of a dFA test is flouresecently-labeled ...
Can antibody be washed away?
When labeled antibody is incubated with rabies-suspect brain tissue, it will bind to rabies antigen. Unbound antibody can be washed away and areas where antigen is present can be visualized as fluorescent-apple-green areas using a fluorescence microscope.
What is a DFA antibody?
A direct fluorescent antibody ( DFA or dFA ), also known as " direct immunofluorescence ", is an antibody that has been tagged in a direct fluorescent antibody test. Its name derives from the fact that it directly tests the presence of an antigen with the tagged antibody, unlike western blotting, which uses an indirect method of detection, ...
What is direct immunofluorescence?
Direct immunofluorescence can be used to detect deposits of immunoglobulins and complement proteins in biopsies of skin, kidney and other organs. Their presence is indicative of an autoimmune disease. When skin not exposed to the sun is tested, a positive direct IF (the so-called Lupus band test) is an evidence of systemic lupus erythematosus. ...
What is a DFA test kit?
Commercial DFA testing kits are available, which contain fluorescently labelled antibodies, designed to specifically target unique antigens present in the bacteria or virus, but not present in mammals ( Eukaryotes ). This technique can be used to quickly determine if a subject has a specific viral or bacterial infection.
What is a positive direct IF?
When skin not exposed to the sun is tested, a positive direct IF (the so-called Lupus band test) is an evidence of systemic lupus erythematosus. Direct fluorescent antibody can also be used to detect parasitic infections, as was pioneered by Sadun, et al. (1960).
Why is monoclonal antibody used in IFA?
selection of particularly well-suited monoclonal antibodies as the primary antibody in IFA tests, to improve sensitivity and specificity
What is a DFA test?
DFA tests are commonly performed on mucosal and skin lesions for herpes simplex virus and varicella-zoster virus, as well as on ocular specimens for identification of herpes simplex virus and adenovirus.
How long does a pertussis PCR assay last?
PCR assays identify unique gene sequences of B. pertussis in respiratory secretions. 153,160–165 Although bacteria can no longer be cultured after 5 days of antibiotic therapy, the PCR can remain positive for an additional week. 166 Although demanding of scrupulous technique to avoid cross-contamination, this rapid, highly sensitive, and specific diagnostic method is steadily becoming the gold standard. Two types of clinical samples have been tested in PCR assays: nasopharyngeal aspirates and nasopharyngeal swab specimens. During the investigation of pertussis epidemics, most studies have demonstrated that nasopharyngeal swabs provide adequate samples for analysis. Several PCR assays have been developed, the majority of which target one of four chromosomal regions of the organism for amplification: (1) the PT promoter region, (2) repeated insertion sequences, (3) a region upstream from the porin gene, and (4) the adenylate cyclase toxin gene. Some have suggested that assays with repeated insertion sequences as the target are more sensitive with a low number of amplification cycles, but there also is an increased risk for cross-reaction with other species. A comparative trial examined the nationwide use of a PCR assay in Finland and Switzerland for nearly 4,000 clinical samples and found that the sensitivity of the PT promoter–based PCR was higher than that of the insertion sequence–based PCR. 167,168 In these studies, the PCR remained positive longer than culture and offered results more rapidly. B. pertussis cultures typically take 3 to 7 days to become positive, whereas PCR can be completed more rapidly. 167
What is the definitive method for diagnosing syphilis?
Darkfield examinations and direct fluorescent antibody tests of lesion exudate or tissue are the definitive methods for diagnosing early syphilis. A presumptive diagnosis is possible with the use of two types of serologic tests for syphilis: (1) nontreponemal tests (e.g., Venereal Disease Research Laboratory [VDRL] and rapid plasma reagin [RPR]) and (2) treponemal tests (e.g., fluorescent treponemal antibody absorbed [FTA-ABS] and T. pallidum particle agglutination [TP-PA]). The use of only one type of serologic test is insufficient for diagnosis, because false-positive nontreponemal test results may occur secondary to various medical conditions.
What is the DFA test for chlamydia?
DFA tests have mostly used MAbs conjugated with fluorescent molecules for direct detection of C. trachomatis in cellular smears. MAbs against MOMP (species-specific) or LPS (may cross-react with other chlamydiae and also non-chlamydial bacterial species) are used. The MOMP MAbs display superior staining, more characteristic morphology of EBs, and less background fluorescence. Commonly used, currently available DFA tests such as MicroTrak C. trachomatis direct specimen test and Pathfinder Chlamydia DFA employ MOMP MAbs, conjugated with fluorescein isothiocyanate (FITC). Appropriate collection, application (by rolling of the swab) and fixation of the clinical specimen to the slide are necessary to ensure presence of columnar cells and non-distorted cell morphology. After application of the conjugated MAbs, these bind to chlamydial EBs and eventual RBs. A rinse step removes unbound antibody. Under UV fluorescence microscopic examination, C. trachomatis positive smears display apple-green fluorescence and morphologically characteristic EBs (examples of DFAs are illustrated in Figs 3.53 and 3.54 ). At least 10 fields of the slide, under magnification ×400, should be examined. If the preparation contains enough columnar epithelial cells and ideally ≥10 typical EBs are found, the result is considered positive.
How sensitive is DFA?
The sensitivity of DFA tests when compared to cell culture varies between studies, but averages ≥80% in women and symptomatic men. The test specificity can be high, i.e. ≥97%, using MOMP MAbs in experienced laboratories.
What are the advantages and disadvantages of DFA?
Moreover, the DFA test can be used to assess the adequacy of the clinical samples ( Figs 3.55–3.57 ), i.e. slides displaying insufficient columnar epithelial cells or disrupted cells should be discarded, the size and morphology of the fluorescing organisms can be determined visually, and the test is rapid. Disadvantages of DFA tests include subjectiveness (experienced microscopists are required), and the labor-intensive and time-consuming evaluation of each slide, which makes the method unsuitable for high-throughput diagnostics and screening of specimens. Artifacts may be noted due to cross-reactions with other micro-organisms ( Figs 3.58 and 3.59 ).
What is fluorescently labeled antibody?
Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using flow cytometry (Figure 4), an automated, cell-counting system that detects fluorescing cells as they pass through a narrow tube one cell at a time.
What is a DFA test?
DFA tests are particularly useful for the rapid diagnosis of bacterial diseases. For example, fluorescence-labeled antibodies against Streptococcus pyogenes ( group A strep) can be used to obtain a diagnosis of strep throat from a throat swab. The diagnosis is ready in a matter of minutes, and the patient can be started on antibiotics before even leaving the clinic. DFA techniques may also be used to diagnose pneumonia caused by Mycoplasma pneumoniae or Legionella pneumophila from sputum samples (Figure 1). The fluorescent antibodies bind to the bacteria on a microscope slide, allowing ready detection of the bacteria using a fluorescence microscope. Thus, the DFA technique is valuable for visualizing certain bacteria that are difficult to isolate or culture from patient samples.
Is the IFA test positive for syphilis?
A patient suspected of having syphilis is tested using both the VDRL test and IFA. The IFA test comes back positive, but the VDRL test is negative. What is the most likely reason for these results?
What antibodies can detect low abundance proteins?
Detection of low abundance proteins can be sometimes challenging even with indirect methods. Biotinylated antibodies offer an extra layer for increased signal amplification. Learn more about how methods based on the use of biotin-conjugated antibodies work here .
What is the purpose of fluorophores in IF?
The fluorophore allows visualization of the target distribution in the sample under a fluorescent microscope ( eg epifluorescence and confocal microscopes). We distinguish between two IF methods depending on whether the fluorophore is conjugated to the primary or ...
What is direct if?
Direct IF uses a single antibody directed against the target of interest. The primary antibody is directly conjugated to a fluorophore.
Is a direct IF protocol shorter than a conjugated secondary antibody?
Protocols for direct IF are usually shorter as they only require one labeling step. The fact that you have to use a conjugated secondary antibody to detect the primary antibody results in additional steps. Conjugated primary antibodies are usually more expensive than their unconjugated counterparts.
Do secondary antibodies bind to primary antibodies?
Several secondary antibodies will bind to the primary antibody resulting in an amplified signal. Species cross-reactivity is minimized in direct methods as the fluorophore is already conjugated to the primary antibody. Secondary antibodies may cross-react with species other than the target.
How long does it take to get results from an immunofluorescence assay?
Immunofluorescence assays are antigen detection assays that generally require use of a fluorescent microscope to produce results in approximately 2-4 hours with moderate sensitivity and high specificity. Both direct (DFA) and indirect fluorescent antibody (IFA) staining assays are available to detect influenza A and B viral antigens in respiratory tract specimens. Subtyping or further identification of influenza A viruses is not possible by immunofluorescence assays. One rapid immunofluorescence assay is an RIDT and utilizes an analyzer device to produce results in approximately 15 minutes.
How long does it take for a rapid molecular assay to produce results?
FDA-cleared rapid molecular assays are available that produce results in approximately 15-30 minutes. Some of these rapid molecular assays are CLIA-waived for point-of-care use.
How to detect influenza RNA?
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and other molecular assays can identify the presence of influenza viral RNA or nucleic acids in respiratory specimens with very high sensitivity and specificity. Some molecular assays are able to detect and discriminate between infections with influenza A and B viruses; other tests can identify specific seasonal influenza A virus subtypes [A (H1N1)pdm09, or A (H3N2)]. These assays can yield results in approximately 45 minutes to several hours depending upon the assay. Notably, the detection of influenza viral RNA or nucleic acids by these assays does not necessarily indicate detection of viable infectious virus or on-going influenza viral replication. It is important to note that not all assays have been cleared by the FDA for diagnostic use. Some multiplex molecular assays are available that can detect influenza viral nucleic acids and distinguish influenza virus infection from other respiratory pathogens and may also be useful for management of severely immunosuppressed patients, or for use in identifying the cause of an institutional outbreak of respiratory illness.
What is the IDSA recommendation for RT-PCR?
IDSA recommends use of RT-PCR or other molecular assays for detection of influenza viruses in respiratory specimens of hospitalized patients. Consult the IDSA Influenza Clinical Practice Guidelines. for recommendations on influenza testing and information on interpretation of testing results.
What tests are available for influenza?
Diagnostic tests available for detection of influenza viruses in respiratory specimens include molecular assays (including rapid molecular assays, reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests); and antigen detection tests (including rapid influenza diagnostic tests and immunofluorescence assays). Viral culture is important for public health purposes, but does not provide timely results to inform clinical management. Sensitivity and specificity of any test for influenza viruses in respiratory specimens might vary by the type of testing method and specific test used, the time from illness onset to specimen collection, the quality of the specimen collected, the respiratory source of the specimen, handling and processing of the specimen, and the time from specimen collection to testing. The post-test probability or predictive values (positive and negative predictive values) of an influenza virus test depend upon the prevalence of circulating seasonal influenza viruses in the patient population, and the specific test characteristics (sensitivity and specificity) compared to a “gold standard” comparison test (molecular assay or viral culture). As with any diagnostic test, results should be evaluated in the context of other clinical and epidemiologic information available to health care providers. Serological testing does not provide timely results to inform clinical management decisions.
How long does it take to detect influenza?
Rapid influenza diagnostic tests (RIDTs) are antigen detection assays that can detect influenza viral antigens in 10-15 minutes with moderate sensitivity (50-70%) and high specificity. Some tests are CLIA-waived and approved for use in any outpatient setting, whereas others must be used in a moderately complex clinical laboratory. Some RIDTs utilize an analyzer reader device to standardize results to improve sensitivity (75-80%). FDA now requires RIDTs to achieve 80% sensitivity. Detection of influenza virus antigen does not necessarily indicate detection of viable infectious virus or on-going influenza viral replication.
How long does it take to get viral culture results?
Shell-vial tissue culture results may take 1-3 days, while traditional tissue-cell viral culture results may take 3-10 days. However, viral culture allows for extensive antigenic and genetic characterization of influenza viruses. The collection of some respiratory samples for viral culture is essential for for surveillance and antigenic characterization of new seasonal influenza A and B virus strains that may need to be included in the next year’s influenza vaccine.
