QUICKELISA ANTHRAX-PA KIT

K040407 · Immunetics, Inc. · NRL · Jun 3, 2004 · Microbiology

Device Facts

• Record ID: K040407
• Device Name: QUICKELISA ANTHRAX-PA KIT
• Applicant: Immunetics, Inc.
• Product Code: NRL · Microbiology
• Decision Date: Jun 3, 2004
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.3045
• Device Class: Class 2

Indications for Use

The Immunetics® QuickELISA™ Anthrax-PA Kit is intended for use in the qualitative detection of antibodies to the Protective Antigen (PA) protein of B. anthracis in human serum. The assay should be used only on serum samples from individuals with a clinical history, signs or symptoms consistent with anthrax infection as an aid in the diagnosis of anthrax, or from recipients of anthrax vaccine.

Device Story

The QuickELISA Anthrax-PA Kit is an in vitro diagnostic ELISA for detecting total antibodies (IgG/IgM) against B. anthracis Protective Antigen (PA) in human serum. The assay uses a single-step binding process where serum antibodies form ternary complexes with biotinylated streptavidin-rPA and rPA-horseradish peroxidase (HRP) conjugates. These complexes bind to a biotin-coated microplate. After washing, a chromogenic TMB substrate is added; the resulting color change is measured via optical absorbance at 450nm (with 620-650nm background subtraction) using an ELISA microplate reader. The test is performed in a laboratory setting by trained personnel. Results are qualitative (or semi-quantitative with serial dilutions) and serve as an aid in diagnosis alongside clinical history and other laboratory data. The device benefits patients by providing rapid presumptive detection of anthrax-specific antibodies, supporting clinical decision-making for infection management or vaccine response assessment.

Clinical Evidence

Performance evaluated using serum panels. Specificity: 99.14% (n=578) in normal human sera. Cross-reactivity: 99.56% (n=225) across various disease conditions (e.g., HIV, SLE, Influenza, H. pylori). Sensitivity: 100% in AVA vaccinees (n=49), cutaneous anthrax patients (n=18), and inhalational anthrax patients (n=16). Analytical sensitivity established at ≤31.3 ng/mL. Reproducibility demonstrated across intra-assay, inter-assay, and inter-lot studies.

Technological Characteristics

Microwell ELISA format; utilizes biotin-coated microplate, streptavidin-rPA, and rPA-HRP conjugates. Detection via chromogenic TMB substrate and optical absorbance measurement at 450nm. Requires ELISA microplate reader. No specific materials standards cited. Standalone diagnostic kit.

Indications for Use

Indicated for qualitative detection of IgG and IgM antibodies to B. anthracis Protective Antigen (PA) in human serum. Patient population includes individuals with clinical history, signs, or symptoms consistent with anthrax infection, anthrax vaccine recipients, or those with presumed contact with anthrax spores/bacilli. Not evaluated for asymptomatic individuals with suspected exposure; results for gastrointestinal anthrax unknown.

Regulatory Classification

Identification

An in vitro diagnostic device for Bacillus species (spp.) detection is a prescription device used to detect and differentiate among Bacillus spp. and presumptively identify B. anthracis and other Bacillus spp. from cultured isolates or clinical specimens as an aid in the diagnosis of anthrax and other diseases caused by Bacillus spp. This device may consist of Bacillus spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to presumptively identify bacillus-like organisms in clinical specimens; bacteriophage used for differentiating B. anthracis from other Bacillus spp. based on susceptibility to lysis by the phage; or antigens used to identify antibodies to B. anthracis (anti-toxin and anti-capsular) in serum. Bacillus infections include anthrax (cutaneous, inhalational, or gastrointestinal) caused by B. anthracis, and gastrointestinal disease and non-gastrointestinal infections caused by B. cereus.

Special Controls

*Classification.* Class II (special controls). The special controls are set forth in FDA's special controls guideline document entitled “In Vitro Diagnostic Devices for*Bacillus* spp. Detection; Class II Special Controls Guideline for Industry and Food and Drug Administration Staff.” For availability of the guideline document, see § 866.1(e).(c) *Restriction on Distribution.* The distribution of these devices is limited to laboratories that follow public health guidelines that address appropriate biosafety conditions, interpretation of test results, and coordination of findings with public health authorities.(d) *Restriction on Use.* The use of this device is restricted to prescription use and must comply with the following:(1) The device must be in the possession of: (i)(A) A person, or his agents or employees, regularly and lawfully engaged in the manufacture, transportation, storage, or wholesale or retail distribution of such device; or (B) A practitioner, such as a physician, licensed by law to use or order the use of such device; and (ii) The device must be sold only to or on the prescription or other order of such practitioner for use in the course of his professional practice. (2) The label of the device shall bear the statement “Caution: Federal law restricts this device to sale by or on the order of a ____”, the blank to be filled with the word “physician” or with the descriptive designation of any other practitioner licensed by the law of the State in which he practices to use or order the use of the device. (3) Any labeling, as defined in section 201(m) of the Federal Food, Drug, and Cosmetic Act, whether or not it is on or within a package from which the device is to be dispensed, distributed by, or on behalf of the manufacturer, packer, or distributor of the device, that furnishes or purports to furnish information for use of the device contains adequate information for such use, including indications, effects, routes, methods, and frequency and duration of administration and any relevant hazards, contraindications, side effects, and precautions, under which practitioners licensed by law to employ the device can use the device safely and for the purposes for which it is intended, including all purposes for which it is advertised or represented. This information will not be required on so-called reminder-piece labeling which calls attention to the name of the device but does not include indications or other use information. (4) All labeling, except labels and cartons, bearing information for use of the device also bears the date of the issuance or the date of the latest revision of such labeling.

Predicate Devices

• US Army Biological Laboratories reagents for modified agar diffusion assay

Submission Summary (Full Text)

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# 510(k) SUBMISSION TEMPLATE

A. 510(k) Number: K040407
B. Analyte: Antibodies to Protective Antigen (PA) protein, *Bacillus anthracis*
C. Type of Test: ELISA
D. Applicant: Immunetics, Inc.
E. Proprietary and Established Names of the Product: QuickELISA™ Anthrax-PA Kit

F. Regulatory Information:
1. Regulation section: Unclassified
2. Classification: Unclassified
3. Product code: NRL - ENZYME LINKED IMMUNOABSORBENT ASSAY, ANTIBODY, B. ANTHRACIS
4. Panel: 83

G. Intended Use and Indication for Use:
1. Intended use(s): The Immunetics® QuickELISA™ Anthrax-PA Kit is intended for use in the qualitative detection of antibodies to the Protective Antigen (PA) protein of *B. anthracis* in human serum. The assay should be used only on serum samples from individuals with a clinical history, signs or symptoms consistent with anthrax infection as an aid in the diagnosis of anthrax, or from recipients of anthrax vaccine.
2. Indication(s) for use: The assay should be used only on serum samples from individuals with a clinical history, signs or symptoms consistent with anthrax infection as an aid in the diagnosis of anthrax, or from recipients of anthrax vaccine.

3. Special conditions for use statement(s):
- The diagnosis of anthrax infection must be made based on history, signs, symptoms, exposure likelihood, and when possible other laboratory data, in addition to the presence of antibodies to *B. anthracis* PA. Negative results in ELISA should not be used in isolation from other evidence to exclude anthrax.
- The assay has not been evaluated in individuals without clinical signs or symptoms who were presumed exposed and who subsequently developed cutaneous or inhalation anthrax. There is no information available to interpret the meaning of a positive or negative test result for such individuals.
- The assay has not been evaluated with specimens from patients infected by the gastrointestinal route; expected results with such infections are unknown.
- The minimum level of anti-PA antibodies that confers protection following vaccination is not known. The QuickELISA measures total antibody and the

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relationship between this value and protective immunity has not been established.

- The affinity and/or avidity of anti-PA IgG and IgM for the rPA antigen have not been determined with this assay.
This assay is for Rx use.

4. Special instrument requirements: ELISA reader, dual wavelength; ELISA plate washer; microplate shaker

## H. Device Description:

The QuickELISA assay kit includes necessary reagents to perform laboratory testing, using immunochemical reactions in an ELISA plate format. Conjugates are manufactured from a recombinant protein product produced by an avirulent plasmidless B. anthracis strain. Kit reagents include positive controls (sera from rabbits immunized with recombinant PA), Conjugate A (streptavidin-rPA), Conjugate B (rPA-horseradish peroxidase), a negative control (normal human serum), microwell strips (precoated with biotinylated bovine serum albumin, sample diluent, TMB substrate, wash buffer, and stop reagent. An ELISA reader (dual wavelength), ELISA plate washer and microplate shaker are needed to perform the testing. The cutoff was determined based on preclinical testing to be above the highest negative sample and validated to detect approximately 300 ng/mL of PA-specific antibody (pool of vaccinee sera).

## I. Substantial Equivalence Information (if known):

1. Predicate device name(s): preamendments device, US Army Biological Laboratories reagents for modified agar diffusion assay
2. Predicate 510(k) number(s): NA
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Detection of anti-PA antibodies | Same |
| Specimen Type | Serum | Same |
| Negative Control | Normal human serum | Same |
| Differences | | |
| Item | Device | Predicate |
| Antigen | Recombinant PA (Protective Antigen) | Purified PA from Sterne strain culture |
| Assay Endpoint | Spectrophotometric measure (Net OD-450) | Visualized band of identity |
| Positive Control | Pooled sera from rabbits immunized with rPA | Serum dilution from human vaccinee |
| Testing Time | Overnight (18-24 h) | 35 min (with agitation); 80 min (without agitation) |
| | | |

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J. Standard/Guidance Document Referenced (if applicable): NA
K. Test Principle: Immunochemical using ELISA microwell plate format.

L. Performance Characteristics:

1. Analytical performance:
a. Precision(Repeatability/Reproducibility):

| Reproducibility Panel Sample | Agitated Incubations: Intra-Assay | | Agitated Inter-Assay CV % | Agitated Inter-Lot CV % | Agitated Inter-Site CV % | Stationary Intra-Assay CV % | Stationary vs. Agitated CV % |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | Net OD-450 | CV % | | | | | |
| PC | 2.233 | 4.4 | 2.0 | 11.2 | 35.4 | 10.8 | 15.1 |
| Low PC | 0.860 | 4.1 | 1.5 | 10.2 | 13.8 | 13.2 | 11.6 |
| NC | 0.018 | 7.2 | 6.2 | 5.4 | 18.1 | 10.8 | 27.9 |
| HiP1 | 3.809 | 6.2 | 0.4 | 2.2 | 3.9 | 6.6 | 1.3 |
| HiP2 | 3.830 | 5.6 | 0.5 | 0.9 | 3.5 | 7.6 | 1.2 |
| HiP3 | 3.806 | 5.2 | 2.0 | 1.7 | 1.9 | 6.9 | 0.8 |
| LoP1 | 2.225 | 6.1 | 2.5 | 22.6 | 15.3 | 3.2 | 19.0 |
| LoP2 | 0.609 | 7.9 | 3.0 | 26.3 | 4.0 | 4.5 | 27.1 |
| LoP3 | 0.442 | 8.3 | 6.4 | 20.5 | 10.0 | 7.9 | 22.9 |
| N1 | 0.019 | 9.5 | 21.5 | 19.2 | 7.9 | 10.9 | 12.8 |
| N2 | 0.021 | 8.0 | 23.1 | 14.9 | 23.9 | 9.9 | 12.8 |
| N3 | 0.021 | 5.9 | 9.5 | 18.5 | 19.8 | 3.0 | 37.3 |
| N4 | 0.021 | 12.2 | 6.4 | 13.6 | 20.9 | 2.3 | 33.5 |
| N5 | 0.023 | 14.3 | 8.6 | 24.2 | 20.2 | 6.1 | 32.4 |
| REF-1 | 0.357 | 8.6 | 8.4 | 15.7 | 13.7 | 7.6 | 15.4 |
| MeP1 | 2.575 | 6.7 | 7.8 | 16.0 | 15.9 | 5.0 | 5.2 |

b. Linearity/Assay measuring (reportable) range: NA
c. Calibrators, and Controls: Pooled sera from rabbits immunized with rPA
d. Traceability of the assay: NA
e. Analytical limits at low levels: approx. 300 ng of PA-specific antibody
f. Analytical specificity:

Sera from 225 individuals with other antibody responses associated with various disease conditions, and 583 sera from healthy blood donors were tested with QuickELISA. Positive results were obtained for 5/583 (0.9%) blood donors and 1/225 (0.4%) of patients with other disease conditions.

g. Analytical characterization of cut-off:

Titrations of pooled human sera from AVA vaccinees (standardized using radial immunodiffusion and nephelometry with US National Reference Preparation for Specific Human Serum Proteins, and mass value using USFDA reference serum, 1983 H. influenzae type b)

2. Comparison studies using clinical specimens:

a. Method comparison: NA
b. Matrix description and comparison: NA

3. Clinical Studies :

a. Clinical sensitivity and clinical specificity: NA – anthrax infection would not be expected in a US population.

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b. Other clinical supportive data, statistical analysis and results if applicable: Archived sera from patients diagnosed with inhalational or cutaneous anthrax (n=13 US patients and 6 non-US patients), along with 49 AVA vaccine recipients were tested with QuickELISA. The anthrax sera were collected 17 days to 15 months post-infection and after development of symptoms. All sera from patients with anthrax were positive when tested with the QuickELISA.

4. Clinical cut-off: The cutoff is determined by adding 0.1 to the average of duplicate Negative Control Net OD-450. This level was determined to be higher than readings obtained from sera from unvaccinated/uninfected individuals in preclinical testing.

5. Reference interval (Expected values): Anthrax is virtually non-existent in the US. All AVA-vaccinated individuals that have been evaluated are reported to develop an anti-PA immune response. The magnitude of the Net OD-450 measurement is not correlated with antibody level or an endpoint titer.

N. Conclusion: The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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