ELISA blood test

As discussed in the introduction to Section IV, the enzyme-Zinked Zmmunarorbent assay (ELISA) has become a widely used and powerful technique in the field of medical diagnostics. If an individual has been exposed to a pathogenic organism (virus, bacteria), antibodies that recognize particular antigens specific to these organisms will be present in the blood plasma. Because of this, a blood test can often reveal the source of an infection and dictate a proper course of treatment. 

Diagnosis and Treatment Skin anthrax may be diagnosed from the biopsy of the sore and performing microscopic examination of the organism. Inhalation anthrax however, is difficult to diagnose. Chest x-ray, lab cultures and blood tests should be carried out. Rapid laboratory tests may be carried out to diagnose anthrax. Such tests include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and direct fluorescent antibody (DFA) methods. 

This test depends on attaching virus protein to a small laboratory dish. A serum sample is prepared from the blood of the individual to be tested, and it is placed in the dish containing bound HIV viral proteins. If HIV-specific antibodies are present in the serum, they will become tightly bound to the dish by way of the HIV proteins. The serum is then removed, and the dish is washed during this procedure, only antibodies specific for HIV will be retained. The dish is then reacted with a stain that will detect any human antibodies. Thus, dishes that were exposed to serum containing HIV-specific antibodies will be stained, while dishes from antibody-negative serum samples will be unstained. A modified ELISA test was developed in which the virus proteins are attached to small beads that can float in solution, instead of to the bottom of the dish. The test is carried out in a test tube and proceeds as before. 

Patients and blood donors are routinely screened for exposure to HIV by means of ElISA and Western blot assays of blood samples (F uie 1-7-15). The assays are designed to detect antibodies to HIV in the blood of the test subject The ELISA is used as the primary screening assay because it is very sensitive. Because the reference interval for the test is set to include everyone with antibodies to HIV, it also gives false positives and thus has a rather low positive predictive value, especially in low-risk populations. The Western blot (or immunoblot) is used as the confirmatory test for HIV exposure. In the Western blot technique, specific HIV proteins are separated by gel electrophoresis and blotted to a filter. The filter is incubated with the test sample. If the sample contains antibodies to HIV, they will bind to the proteins on the filter. The filter is next washed and incubated with a labeled goat anti-human IgG to visualize any bound human antibodies. The Western blot is highly specific. The combination of an ELISA and Western blot has a positive predictive value of greater than 99%,... 

IL-6 induction in human peripheral whole-blood cell culture [10,11] and determination of its level using ELISA and Limulus activity measured by means of Endospecy Test (Seikagaku Co., Tokyo, Japan) were performed as described [8]. lL-6 induction in THP-1 cells was performed as reported [12]. In brief, sample solution (25 p.L) and 100 p.L of THP-1 cells (1.0 X 10 cells/mL, preincubation with 50 ng/mL vitamin D3 at 37°C in CO2 for 72 h) were incubated in RPMl-1640 medium with or without 2% fetal calf serum... 

Organ weights (spleen, thymus, all animals) Immunotoxicity tests (1) functional tests (either a splenic plaque-forming cell (PEC) assay or an Enzyme-Linked Immunosorbent Assay (ELISA) to determine the response to antigen administration) (2) enumeration of splenic or peripheral blood total B cells, total T cells, and T-cell subpopulations Detailed clinical observations Functional observations (sensory reactivity to stimuli of different types, grip strength, motor activity, more specialized tests on indication)... 

Blood serum samples from 10 sick animals in which the titer of the specific Ab was 1 256 according to RID test, were analyzed. The sensitivities of both methods (the immune biosensor and ELISA) were similar. The limit of the serum dilution for detection of the specific antibodies was 1 15,000. 

Similar tests were carried out with the blood serum obtained from the National Reference Laboratory for FBI (Federal Research Institute for Animal Health, Germany). In this case a pool of 21 serum samples was analyzed. The results of three methods were in agreement except for two samples for which ELISA tests were low positive, whereas the immune biosensor analysis results were negative. 

The other approach is based largely on informatics. In such an approach, tumors would be profiled in contrast to normal tissue. Tumor-specific markers would be identified via microarrays, as has been done in many publications already. From here, the list of tumor-specific markers would be analyzed to determine if any of these markers represented proteins which were likely to be secreted out of the cell and which may be detected in the peripheral blood stream. Preferably multiple markers would be identified that could be tested using multiplex ELISA assays (antibody arrays). Such work will take time, however, because once the potential markers are identified, antibodies must be generated, validated, and tested for effectiveness as an early diagnostic tool. Such work is being done, but little has been published so far. 

Detecting exposure to HIV ELISA assays and western blots are commonly used to detect exposure to HIV by measuring the amount of anti-HIV antibodies present in a patient s blood sample. ELISA assays are used as the primary screening tool, because they are very sensitive. These assays sometimes give false-posi-tives, however, so western blots, which are more specific, are often used as a confirmatory test (Figure 32.23). [Note ELISA and western blots can only detect HIV exposure after anti-HIV antibodies appear in the bloodstream. PCR-based testing for HIV is more useful in the first few months after exposure.]... 

One week later the animals were bled, and serum was prepared from whole blood and tested in ELISA. (If the sera was negative in the direct ELISA, then the animals would have been reinjected for two additional weeks and then retested with ELISA.)... 

Several methods of detection of exposiue to ricin include use of enzyme-linked immunosorbent assay (ELISA) testing of blood or other body fluids or immunohistochemical techniques may be useful in confirming ricin intoxication (Poli et al, 1994). No widely available, reliable test exists to detect and confirm exposure to abrin. 

Laboratory testing for ricin is limited, especially for inhalational exposures. The two common methods that can detect ricin in blood or other body fluids are the radioimmunoassay and the enzyme-linked immunosorbent assay (ELISA). Because ricin binds quickly and the body metabolizes it efficiently before excretion, the length of time necessary for these tests limits their usefulness for inhalation exposures (35). Besides testing body fluids, the CDC and member LRN state public health laboratories have a time-resolved fluorescence immunoassay that can test preparations of suspected ricin-containing substances and environmental specimens for the presence of ricin (40). 

To assess the practical application of a-gal polymers, inhibition ELISA was used to test intact human serum (male, blood type AB) instead of pmified anti-Gal antibody (Table 4). The results also indicated the activity enhancement of the a-gal polymers in comparison to the monomer 11. The same trends were also observed in interaction of a-gal polymers with different isotypes of the antibody and with the varied densities of the a-gal epitope conjugated to the polymer. Interestingly, the IC50 results observed with the pmified antibodies were consistently lower than the IC50 results with human sera. One explanation is that the pmified antibodies were obtained from affinity colunm immobilized with a-gal trisaccharide similar in structure to the epitope on the polymer. Therefore, subsets of antibodies selected during the pmification would bind most tightly to the polymer. Since the purified antibodies... 

---