Contents of Kit
Each Test System contains the following components in sufficient quantities to perform the number of tests indicated on the packaging label. NOTE: The following components contain Sodium Azide as a preservative at a concentration of <0.1% (w/v): Controls, Calibrator, and SAVe Diluent.
1. PLATE, Plate: 96 wells configured in twelve, 1x8-well, strips coated with a heat-inactivated preparation of L. pneumophila Groups 1-6 antigens. The strips are packaged in a strip holder and sealed in an envelope with desiccant.
2. CONJ, Conjugate: Conjugated (horseradish peroxidase) goat anti-human IgG/IgM/IgA. One, 15mL, white-capped bottle. Ready to use.
3. CONTROL +, Positive Control (Human Serum): One, 0.35mL, red-capped vial.
4. CAL, Calibrator (Human Serum): One, 0.5mL, blue-capped vial.
5. CONTROL -, Negative Control (Human Serum): One, 0.35mL, green-capped vial.
6. DIL SPE, SAVe Diluent: One, 30mL, green-capped, bottle containing Tween-20, bovine serum albumin and phosphate-buffered-saline. Ready to use. NOTE: The SAVe Diluent will change color when combined with serum.
7. SOLN TMB, TMB: One, 15mL, amber-capped, amber bottle containing 3, 3', 5, 5' - tetramethylbenzidine (TMB). Ready to use.
8. SOLN STOP, Stop Solution: One, 15mL, red-capped, bottle containing 1M H2SO4, 0.7M HCl. Ready to use.
9. WASHBUF 10×, Wash Buffer Concentrate (10×): Dilute 1 part concentrate + 9 parts deionized or distilled water. One, 100mL, clear-capped, bottle containing a 10× concentrated phosphate-buffered-saline and Tween-20 solution (blue solution). NOTE: 1× solution will have a pH of 7.2 ± 0.2.
Test System also contains a Component Label containing lot specific information inside the Test System box.
Storage
Coated Microwell Strips: 2-8°C. Immediately reseal extra strips with desiccant and return to proper storage. After opening - strips are stable for 60 days, as long as the indicator strips on the desiccant pouch remains blue.
Conjugate: 2-8°C. DO NOT FREEZE.
Unopened Test System, Calibrator, Positive Control, Negative Control, TMB, SAVe Diluent : 2-8°C
Stop Solution: 2 - 25°C
Wash Buffer (1X): 20 - 25°C for up to 7 days, 2 - 8°C for 30 days.
Wash Buffer (10X): 2 - 25°C
Performance Characteristics
1. Comparative Studies Performance of a comparative study demonstrated the substantial equivalence of the ELISA L. pneumophila IgG/IgM/IgA Test System to another commercially available ELISA test system and to an IFA Legionella Test System. A three-site clinical investigation evaluated the performance of the ELISA Legionella IgG/IgM/IgA Test System. One clinical site compared the performance of the ELISA Legionella IgG/IgM/IgA Test System to another commercially available ELISA test system. A second clinical site compared the ELISA L. pneumophila IgG/IgM/IgA Test System to the IFA Legionella Test System. The third clinical site compared the ELISA L. pneumophila IgG/IgM/IgA Test System to a comercially available IFA Legionella Test System. The study tested a total of 240 specimens. Clinical specimens tested at sites one and two consisted primarily of routine specimens from a reference laboratory in Northeastern United States that tested for normal Legionella serological analysis. Some repository specimens were included which had been previously tested and were found to be positive for antibody to Legionella. Specimens tested at the third clinical site consisted of 22 paired specimens (acute and convalescent) from confirmed cases of Legionella infection. Tables 1, 2, and 3 show a summary of these comparative investigations. Analysis excluded any equivocal specimens.
With respect to Table 3 above; of the 22 pairs of acute and convalescent specimens, 17/22 were ELISA negative for the acute, and positive for the convalescent. Of the remaining five pairs, 3/22 were negative for both acute and convalescent, and 2/22 were positive for both the acute and convalescent.
NOTE: Be advised that relative refers to the comparison of this assay' s results to that of a similar assay. There was not an attempt to correlate either assays' results with disease presence or absence.
2. Precision and Reproducibility:To demonstrate inter-laboratory reproducibility of the assay, six specimens were evaluated; Two with an IFA titer of <1:128, two with an IFA titer of 1:512, and two with an IFA titer of ≥1:1024. Five vials of each specimen were prepared for a total of 30 vials. The 30 vials were randomized and simply numbered one through 30. Testing of the panel occurred in-house and at the two clinical sites. The study demonstrated excellent inter-laboratory reproducibility, with 100% agreement between all three sites. Precision was evaluated as outlined in CLSI/NCCLS document number EP5-T2: Evaluation of Precision Performance of Clinical Chemistry Devices - Second Edition. Both clinical sites performed reproducibility studies using the same eight specimens: two relatively strong positive specimens, two specimens near the cutoff, two that were clearly negative and the kit's negative control and positive control. On each day of testing, each specimen was assayed in duplicate, at two time points, morning and afternoon, for a total of four replicates per specimen. This reproducibility study ran for a 20-day period, for a total of 80 observations, for each of the eight panel members. A summary appears in Table 4 below:
NOTE: Table 4 depicts the reproducibility results only as an example of those results obtained during the clinical study, using ideal conditions of environment, equipment, and technique. Each laboratory should evaluate reproducibility as it may vary depending upon the conditions at the laboratory.
General Description
Scientists identified L. pneumophila as the causative agent for Legionellosis (Legionella pneumonia, or Legionnaire's Disease) in 1977. Presently, there are more than 25 species and 33 serogroups in the family Legionellaceae, with at least 18 species associated with pneumonia, accounting for roughly one to five percent of all cases of pneumonia. L. pneumophila displays a multitude of morphologies including the bacillus, coccobacillus, and elongated fusiform. Although often difficult to perform, the Gram stain will be Gram-negative.
The antibody response to L. pneumophila may be both specific and nonspecific, since the patient may have antibodies to similar antigens from other Gram-negative bacteria. Optimum times for specimen collection appear to be within the first week of illness, or as soon as possible after the onset (acute specimen), and at least three weeks after the onset (convalescent specimen). By the IFA method, considering a single result of greater than or equal to 1:256 is presumptive evidence of legionella infection. Diagnostic titers have been reported to be absent in as many as 25% of patients, but the use of multiple Legionella species as the antigen source and a polyvalent conjugate directed against IgG, IgM, and IgA maximize the accuracy of serological procedures.
COA
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