LEGIONELLA IGG/IGM ELISA TEST SYSTEM

K963318 · Armkel, LLC · LHL · Mar 3, 1997 · Microbiology

Device Facts

• Record ID: K963318
• Device Name: LEGIONELLA IGG/IGM ELISA TEST SYSTEM
• Applicant: Armkel, LLC
• Product Code: LHL · Microbiology
• Decision Date: Mar 3, 1997
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3300
• Device Class: Class 2

Intended Use

The Legionella pneumophila IgG/IgM ELISA kit is an Enzyme-Linked Immunosorbent Assay (ELISA) for the qualitative detection of total antibodies (IgG and IgM) to Legionella pneumophila serogroups 1-6 in serum from patients with clinical suspicion of Legionella Disease.

Device Story

Device is an ELISA test kit for qualitative detection of total IgG/IgM antibodies to Legionella pneumophila (serogroups 1-6). Input is patient serum; antigen-coated microtiter wells capture specific antibodies; enzyme-labeled anti-human IgG/IgM conjugate binds captured antibodies; substrate addition causes color change proportional to antibody concentration. Photometric measurement provides indirect quantification. Used in clinical laboratories by trained personnel. Output is color intensity measured photometrically, interpreted to determine presence of antibodies. Assists clinicians in diagnosing Legionella Disease in patients with clinical suspicion.

Clinical Evidence

Clinical performance evaluated via comparison to IFA. Study 1 (n=33) showed 90% relative sensitivity. Study 2 (n=72) showed 98.53% relative specificity and 98.57% relative agreement. CDC masked serum panel (n=31 pairs) demonstrated 93.5% sensitivity in detecting seroconversions. Precision studies (intra-assay, inter-assay, inter-site) performed per NCCLS EP5 guidelines, with CVs reported across various serum samples.

Technological Characteristics

Enzyme-Linked Immunosorbent Assay (ELISA) using purified Legionella pneumophila antigen (serogroups 1-6) immobilized on solid-phase microtiter wells. Detection via enzyme-labeled anti-human IgG/IgM conjugate and substrate-induced colorimetric reaction. Photometric measurement of optical density. Manual or automated plate processing.

Indications for Use

Indicated for patients with clinical suspicion of Legionella Disease to qualitatively detect total IgG/IgM antibodies to Legionella pneumophila serogroups 1-6 in serum.

Regulatory Classification

Identification

Haemophilus spp. serological reagents are devices that consist of antigens and antisera, including antisera conjugated with a fluorescent dye, that are used in serological tests to identify Haemophilus spp. directly from clinical specimens or tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria belonging to the genus Haemophilus and provides epidemiological information on diseases cause by these microorganisms. Diseases most often caused by Haemophilus spp. include pneumonia, pharyngitis, sinusitis, vaginitis, chancroid venereal disease, and a contagious form of conjunctivitis (inflammation of eyelid membranes).

Special Controls

*Classification.* Class II (special controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 866.9.

Predicate Devices

• BioWhittaker’s Legionella STAT test

Submission Summary (Full Text)

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K963318

# Summary of Safety and Effectiveness Information
*Legionella pneumophila* IgG/IgM ELISA Test Kit

MAR - 3 1997

## I. Immuno Probe Inc.
1306 Bailes Lane, Suite F
Frederick, Maryland 21701
Contact person: William Boteler
Telephone: [phone]
Date of preparation: Feb 13, 1997

## II. Description of Device
The *Legionella pneumophila* IgG/IgM ELISA kit is an Enzyme-Linked Immunosorbent Assay (ELISA) for the qualitative detection of total antibodies (IgG and IgM) to *Legionella pneumophila* serogroups 1-6 in serum from patients with clinical suspicion of Legionella Disease.

The *Legionella pneumophila* IgG/IgM ELISA test is an enzyme linked immunosorbent assay to detect IgG/IgM antibodies to Legionella. Purified *Legionella pneumophila* antigen (serogroups 1, 2, 3, 4, 5, 6) is attached to a solid phase microtiter well. Diluted test sera is added to each well. If the antibodies are present that recognize the antigen, they will bind to the antigen in the well. After incubation the wells are washed to remove unbound antibody. An enzyme labeled anti-human IgG/IgM is added to each well. If antibody is present it will bind to the antibody attached to the antigen on the well. After incubation the wells are washed to remove unbound conjugate. A substrate solution is added to each well. If enzyme is present the substrate will undergo a color change. After an incubation period the reaction is stopped and the color intensity is measured photometrically, producing an indirect measurement of specific antibody in the patient specimen.

## III. Predicate Device
The *Legionella pneumophila* IgG/IgM ELISA test is substantially equivalent to BioWhittaker’s Legionella STAT test. Equivalence is demonstrated by the following comparative results:

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# Performance Characteristics

1. Relative sensitivity and specificity. The *Legionella pneumophila* IgG/ IgM ELISA was evaluated relative to Legionella Stat at two different sites. The first site was a commercial R&D lab located in Maryland. Thirty three single IFA positive sera from an outbreak and samples routinely submitted for Legionella testing were tested. The results of the study are summarized in Table 3.

## Table 3
Comparison of *Legionella pneumophila* IgG/IgM ELISA and Legionella IFA

| IFA | Wampole Legionella pneumophila IgG/IgM ELISA | | | | |
| --- | --- | --- | --- | --- | --- |
| | + | + | eq | - | Total |
| IFA | + ≥256 | 27 | 3 | 3 | 33 |
| | - >256 | 0 | 0 | 0 | 0 |
| | Total | 27 | 3 | 3 | 33 |

Relative Sensitivity = 27/30 = 90.00% 95% Confidence interval = 79.0% - 100%
Relative Specificity = NA 95% Confidence interval = NA

Equivocals were not included in the above calculations.
The 95% confidence intervals were calculated using the normal method.

Please be advised that ‘relative’ refers to the comparison of this assay’s results to that of similar assay. There was not an attempt to correlate the assay’s results with disease presence or absence. No judgment can be made on the comparison assay’s accuracy to predict disease.

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The second site was a clinical laboratory located in Pennsylvania. Seventy two prospective serum for Legionella testing were tested. The results of the study are summarized in Table 4.

## Table 4
Comparison of *Legionella pneumophila* IgG/IgM ELISA and Legionella IFA

| Wampole *Legionella pneumophila* IgG/IgM ELISA | | | | | |
| --- | --- | --- | --- | --- | --- |
| | | + | eq | - | Total |
| IFA | + ≥256 | 2 | 0 | 0 | 2 |
| | - <256 | 1 | 2 | 67 | 70 |
| | Total | 3 | 2 | 67 | 72 |

Relative Specificity = 67/68 = 98.53% 95% Confidence interval = 95.6% - 100%
Relative Agreement = 69/70 = 98.57% 95% Confidence interval = 95.7% - 100%

Equivocals were not included in the above calculations.
The 95% confidence intervals were calculated using the normal method.

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2. Precision. Seven different sera were assayed at two different sites to determine the precision of the assay. An additional three sera were tested at site 1. Each sera was tested ten times each, on three different days at each of the two study sites. The intra and inter assay precision for each site is presented in Tables 5 and 6. The inter-site coefficient of variation (CV) for each serum is presented in table 7.

Table 5
*Legionella pneumophila* IgG/IgM ELISA Intra and Inter Assay Precision Study 1

| Assay(n=30) | Assay 1 (n=10) | | | Assay 2 (n=10) | | | Assay 3 (n=10) | | | Inter- | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | X | SD | CV | X | SD | CV | X | SD | CV | X | SD | CV |
| 1 | 3.17 | 0.138 | 4.35% | 3.55 | 0.235 | 6.62% | 3.41 | 0.349 | 10.2% | 3.42 | 0.305 | 8.92% |
| 2 | 2.44 | 0.244 | 10.0% | 2.66 | 0.267 | 10.0% | 2.41 | 0.127 | 5.27% | 2.50 | 0.247 | 9.88% |
| 3 | 2.49 | 0.322 | 12.9% | 2.78 | 0.240 | 8.63% | 2.81 | 0.332 | 11.8% | 2.70 | 0.327 | 12.1% |
| 4 | 1.22 | 0.180 | 14.8% | 1.36 | 0.131 | 9.63% | 1.16 | 0.125 | 10.8% | 1.25 | 0.164 | 13.1% |
| 5 | 0.50 | 0.051 | 10.2% | 0.56 | 0.042 | 7.50% | 0.53 | 0.041 | 7.74% | 0.53 | 0.050 | 9.43% |
| 6 | 0.18 | 0.025 | 13.9% | 0.21 | 0.023 | 11.0% | 0.20 | 0.031 | 15.5% | 0.20 | 0.030 | 15.0% |
| 7 | 0.28 | 0.039 | 13.9% | 0.34 | 0.046 | 13.5% | 0.33 | 0.048 | 14.6% | 0.32 | 0.051 | 15.9% |
| 8 | 1.02 | 0.051 | 5.00% | 1.13 | 0.039 | 3.45% | 1.19 | 0.044 | 3.70% | 1.11 | 0.084 | 7.57% |
| 9 | 0.85 | 0.053 | 6.24% | 0.92 | 0.025 | 2.72% | 0.99 | 0.043 | 4.34% | 0.92 | 0.069 | 7.50% |
| 10 | 0.96 | 0.067 | 6.98% | 1.05 | 0.056 | 5.33% | 1.11 | 0.094 | 8.47% | 1.03 | 0.122 | 11.20% |
| HPC* | | | | | | | | | | 3.64 | .0402 | 11.05% |
| CAL** | | | | | | | | | | 1.44 | 0.122 | 8.44% |
| LPC* | | | | | | | | | | 1.49 | 0.195 | 13.11% |
| NC* | | | | | | | | | | 0.18 | 0.052 | 28.97% |

* n = 17
** n = 51

Table 6
*Legionella pneumophila* IgG/IgM ELISA Intra and Inter Assay Precision Study 2

| Sera# | Assay 1 (n=10) | | | Assay 2 (n=10) | | | Assay 3 (n=10) | | | Inter-Assay(n=30) | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | X | SD | CV | X | SD | CV | X | SD | CV | X | SD | CV |
| 1 | 2.80 | 0.246 | 8.79% | 2.66 | 0.165 | 6.20% | 3.08 | 0.245 | 7.95% | 2.85 | 0.272 | 9.54% |
| 2 | 3.10 | 0.343 | 11.1% | 3.05 | 0.276 | 9.05% | 3.14 | 0.259 | 8.25% | 3.10 | 0.293 | 9.45% |
| 3 | 3.31 | 0.392 | 11.8% | 3.17 | 0.220 | 6.94% | 3.38 | 0.214 | 6.33% | 3.31 | 0.289 | 8.73% |
| 4 | 1.10 | 0.135 | 12.3% | 1.15 | 0.131 | 11.4% | 1.18 | 0.142 | 12.0% | 1.16 | 0.138 | 11.9% |
| 5 | 0.56 | 0.060 | 10.7% | 0.58 | 0.053 | 9.14% | 0.59 | 0.036 | 6.10% | 0.58 | 0.050 | 8.62% |
| 6 | 0.28 | 0.016 | 5.71% | 0.26 | 0.013 | 5.00% | 0.29 | 0.020 | 6.90% | 0.28 | 0.020 | 7.14% |
| 7 | 0.29 | 0.018 | 6.21% | 0.28 | 0.020 | 7.14% | 0.31 | 0.023 | 7.42% | 0.29 | 0.023 | 7.93% |
| HPC* | | | | | | | | | | 3.16 | 0.092 | 2.91% |
| CAL** | | | | | | | | | | 1.45 | 0.060 | 4.11% |
| LPC* | | | | | | | | | | 1.68 | 0.190 | 11.29% |
| NC* | | | | | | | | | | 0.35 | 0.121 | 34.44% |

* n = 5
** n = 15

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# Table 7 Legionella pneumophila IgG/IgM ELISA Inter Site Precision Study

| Inter Site (n=60) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Sera # | X | SD | CV | # positive | #equ | #negative |
| 1. | 3.13 | 0.406 | 13.0% | 60 | 0 | 0 |
| 2. | 2.80 | 0.403 | 14.4% | 60 | 0 | 0 |
| 3. | 3.00 | 0.431 | 14.4% | 60 | 0 | 0 |
| 4. | 1.21 | 0.158 | 13.1% | 43 | 16 | 1 |
| 5. | 0.56 | 0.055 | 9.82% | 0 | 0 | 60 |
| 6. | 0.24 | 0.046 | 19.2% | 0 | 0 | 60 |
| 7. | 0.30 | 0.040 | 13.3% | 0 | 0 | 60 |
| 8.* | 1.11 | 0.084 | 7.57% | 19 | 11 | 0 |
| 9.* | 0.92 | 0.069 | 7.50% | 0 | 20 | 10 |
| 10.* | 1.03 | 0.122 | 11.20% | 8 | 20 | 2 |
| HPC** | 3.42 | 0.401 | 11.75% | 9 | 0 | 0 |
| CAL*** | 1.45 | 0.069 | 4.77% | 27 | 0 | 0 |
| LPC** | 1.56 | 0.240 | 15.46% | 9 | 0 | 0 |
| NC** | 0.27 | 0.124 | 45.68% | 0 | 0 | 9 |

* n = 30
** n = 9
*** n = 27

X = Mean
SD = standard deviation
CV = coefficient of variation = SD/X x 100
The methods in NCCLS EP5 were utilized for precision parameters.

## 4. IFA Paired Serum Analysis (CDC Panel).

The following information is from a serum panel tested at the CDC by IFA and confirmed to be serologically positive for an increase in titer from &lt;1:256 to &gt;1:256. The sera were submitted to CDC for titer conformation. The results are presented as a means to convey further information on the performance of this assay with a masked serum panel. This does not imply an endorsement of the assay by the CDC.

The panel consists of 31 serum pairs showing a greater than 4 fold increase in IFA titer. Each serum pair was evaluated on the *Legionella pneumophila* IgG/IgM ELISA assay to determine an seroconversion in antibody. Twenty nine pairs had a seroconversion thus giving a sensitivity of 29/31 = 93.5% in detecting seroconversions.