MONOLISA ANTI-HBS EIA

P050048 · Bio-Rad Laboratories · SEI · Aug 25, 2006 · Microbiology

Device Facts

• Record ID: P050048
• Device Name: MONOLISA ANTI-HBS EIA
• Applicant: Bio-Rad Laboratories
• Product Code: SEI · Microbiology
• Decision Date: Aug 25, 2006
• Decision: APRL
• Regulation: 21 CFR 866.3173
• Device Class: Class 2

Intended Use

The Bio-Rad MONOLISA™ Anti-HBs EIA is a qualitative and quantitative enzyme immunoassay for the detection of antibody to hepatitis B surface antigen in human serum and EDTA or citrated plasma. The assay results may be used as an aid in the determination of susceptibility to hepatitis B virus (HBV) infection in individuals prior to or following HBV vaccination or where vaccination status is unknown. Assay results may be used with other HBV serological markers for the laboratory diagnosis of HBV disease associated with HBV infection. A reactive assay result will allow a differential diagnosis in individuals displaying signs and symptoms of hepatitis in whom etiology is unknown. The MONOLISA™ Anti-HBs Calibrator Kit is intended for quantitative determination of anti-HBs in human serum and EDTA or citrated plasma when used in conjunction with the MONOLISA™ Anti-HBs EIA.

Device Story

Direct antibody sandwich enzyme immunoassay (EIA) for detection of anti-HBs antibodies in human serum or EDTA/citrated plasma. Microwell plates coated with purified native ad and ay hepatitis B surface antigens capture patient antibodies; peroxidase-labeled HBsAg conjugate binds captured antibodies; TMB substrate added for colorimetric reaction. Optical absorbance measured at 450 nm (or 405 nm for high concentrations) via spectrophotometer. Used in clinical laboratories by technicians. Results aid in determining HBV susceptibility, vaccination response, or diagnosis of HBV infection. Quantitative determination requires use of MONOLISA™ Anti-HBs Calibrator Kit to plot calibration curve against WHO standard. Benefits include accurate assessment of immunity status, guiding vaccination decisions, and differential diagnosis of hepatitis.

Clinical Evidence

Multi-center clinical trial of 1452 prospective subjects (high-risk and symptomatic). Compared to reference anti-HBs EIAs. Overall positive percent agreement 94.3% (95% CI: 92.2–95.9%); negative percent agreement 93.5% (95% CI: 91.6–95.0%). Performance evaluated in vaccinated populations (99.3% positive agreement) and seroconversion panels. Analytical sensitivity (LOD 4.14 mIU/mL) and precision (total CV ≤13.2%) established. No significant interference from hemolyzed, lipemic, icteric, or proteinemic samples.

Technological Characteristics

Enzyme immunoassay (EIA) using direct antibody sandwich principle. Components: microwell strip plates coated with purified native ad and ay HBsAg, peroxidase-labeled HBsAg conjugate, TMB chromogen, H2SO4 stop solution. Quantitative range 0-1000 mIU/mL. Calibrated against WHO standard. Preservatives: ProClin 300, ProClin 950, Gentamicin Sulfate. Storage: 2-8°C. Standalone assay requiring spectrophotometer.

Indications for Use

Indicated for individuals requiring assessment of susceptibility to HBV infection, including those prior to or following HBV vaccination, individuals with unknown vaccination status, and patients displaying signs/symptoms of hepatitis of unknown etiology. Not for screening blood, plasma, or tissue donors. Performance not established for immunocompromised or immunosuppressed patients.

Regulatory Classification

Identification

A hepatitis B virus (HBV) antibody assay is identified as an in vitro diagnostic device intended for prescription use in the detection of antibodies to HBV in human serum, plasma, or other matrices, and as a device that aids in the diagnosis of HBV infection in persons with signs and symptoms of hepatitis and in persons at risk for hepatitis B infection. Results from assays may be qualitative or quantitative, such as quantitative anti-HBs. In addition, results from an anti-HBc IgM (IgM antibodies to core antigen) assay indicating the presence of anti-HBc IgM are indicative of recent HBV infection. Anti-HBs (antibodies to surface antigen) assay results may be used as an aid in the determination of susceptibility to HBV infection in individuals prior to or following HBV vaccination or when vaccination status is unknown. The assay is not intended for screening of blood, plasma, cells, or tissue donors. The assay is intended as an aid in diagnosis in conjunction with clinical findings and other diagnostic procedures.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the assay is not intended for the screening of blood, plasma, cells, or tissue donors. (ii) A detailed explanation of the principles of operation and procedures for performing the assay. (iii) A detailed explanation of the interpretation of results. (iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and management. The limitations must include statements that indicate: (A) When appropriate, performance characteristics of the assay have not been established in populations of immunocompromised or immunosuppressed patients or other special populations where assay performance may be affected. (B) Detection of HBV antibodies to a single viral antigen indicates a present or past infection with hepatitis B virus, but does not differentiate between acute, chronic, or resolved infection. (C) The specimen types for which the device has been cleared, and that use of the assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results. (D) Diagnosis of hepatitis B infection should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures. (E) A non-reactive assay result may occur early during acute infection, prior to development of a host antibody response to infection, or when analyte levels are below the limit of detection of the assay. (F) Results obtained with this assay may not be used interchangeably with results obtained with a different manufacturer's assay. (v) For devices intended for the quantitative detection of HBV antibodies (anti-HBs), in addition to the special controls listed in paragraphs (b)(1) and (2) of this section, labeling required under § 809.10(b) of this chapter must include: (A) The assay calibrators' traceability to a standardized reference material that FDA has determined is appropriate ( *e.g.,* a recognized consensus standard) and the limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), linearity, and precision to define the analytical measuring interval.(B) Performance results of the analytical sensitivity study testing a standardized reference material that FDA has determined is appropriate ( *e.g.,* a recognized consensus standard).(2) Design verification and validation must include the following: (i) Detailed device description, including all parts that make up the device, ancillary reagents required but not provided, an explanation of the device methodology, and design of the antigen(s) and capture antibody(ies) sequences, rationale for the selected epitope(s), degree of amino acid sequence conservation of the target, and the design and composition of all primary, secondary and subsequent standards used for calibration. (ii) Documentation and characterization ( *e.g.,* supplier, determination of identity, and stability) of all critical reagents (including description of the antigen(s) and capture antibody(ies)), and protocols for maintaining product integrity throughout its labeled shelf life.(iii) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance. (iv) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released at the extremes of the specifications will meet the identified analytical and clinical performance characteristics as well as stability. (v) Stability studies for reagents must include documentation of an assessment of real-time stability for multiple reagent lots using the indicated specimen types and must use acceptance criteria that ensure that analytical and clinical performance characteristics are met when stability is assigned based on the extremes of the acceptance range. (vi) All stability protocols, including acceptance criteria. (vii) When applicable, analytical sensitivity of the assay that is the same or better than that of other cleared or approved assays. (viii) Analytical performance studies and results for determining the limit of blank (LoB), limit of detection (LoD), cutoff, precision (reproducibility), including lot-to-lot and/or instrument-to-instrument precision, interference, cross reactivity, carryover, hook effect, seroconversion panel testing, matrix equivalency, specimen stability, reagent stability, and cross-genotype antibody detection sensitivity, when appropriate. (ix) For devices intended for the detection of antibodies for which a standardized reference material (that FDA has determined is appropriate) is available, the analytical sensitivity study and results testing the standardized reference material. Detailed documentation of that study and its results must be provided, including the study protocol, study report, testing results, and all statistical analyses. (x) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review. (xi) Detailed documentation of clinical performance testing from a clinical study with an appropriate number of HBV reactive and non-reactive samples in applicable risk categories and conducted in the appropriate settings by the intended users. Performance must be analyzed relative to an FDA cleared or approved HBV antibody assay or a comparator that FDA has determined is appropriate. Additional relevant patient groups must be validated as appropriate. The samples must include prospective (sequential) samples for each identified specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas. (3) For any HBV antibody assay intended for quantitative detection of anti-HBV antibodies, the following special controls, in addition to those special controls listed in paragraphs (b)(1) and (2) of this section, also apply: (i) Detailed documentation of the metrological calibration traceability hierarchy to a standardized reference material that FDA has determined is appropriate. (ii) Detailed documentation of the following analytical performance studies conducted, as appropriate to the technology, specimen types tested, and intended use of the device, including upper and lower limits of quantitation (UloQ and LloQ, respectively), linearity using clinical samples, and an accuracy study using the recognized international standard material.

Submission Summary (Full Text)

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# SUMMARY OF SAFETY AND EFFECTIVENESS

## I. General Information

Device Generic Name: Antibody to Hepatitis B Surface Antigen (Anti-HBs) assay

Device Trade Name: MONOLISA™ Anti-HBs EIA
MONOLISA™ Anti-HBs Calibrator Kit

Applicant: Bio-Rad Laboratories
6565 185th Avenue NE
Redmond, WA 98052
Phone: [phone]
Fax: [phone]

Date of Panel Recommendation: None

PMA Number: P050048

Date of Notice of Approval to the Applicant: August 25, 2006

## II. Indications For Use

### MONOLISA™ Anti-HBs EIA

The Bio-Rad MONOLISA™ Anti-HBs EIA is a qualitative and quantitative enzyme immunoassay for the detection of antibody to hepatitis B surface antigen in human serum and EDTA or citrated plasma. The assay results may be used as an aid in the determination of susceptibility to hepatitis B virus (HBV) infection in individuals prior to or following HBV vaccination or where vaccination status is unknown. Assay results may be used with other HBV serological markers for the laboratory diagnosis of HBV disease associated with HBV infection. A reactive assay result will allow a differential diagnosis in individuals displaying signs and symptoms of hepatitis in whom etiology is unknown. The MONOLISA™ Anti-HBs Calibrator Kit is intended for quantitative determination of anti-HBs in human serum and EDTA or citrated plasma when used in conjunction with the MONOLISA™ Anti-HBs EIA.

### MONOLISA™ Anti-HBs Calibrator Kit

The MONOLISA™ Anti-HBs Calibrator Kit is intended for quantitative determination of anti-HBs in human serum and EDTA or citrated plasma. The MONOLISA™ Anti-HBs Calibrator Kit is to be used only with the MONOLISA™ Anti-HBs EIA (Catalog # 25220).

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Summary of Safety and Effectiveness Data

## III. CONTRAINDICATIONS: None known.

## IV. WARNINGS AND PRECAUTIONS:

Warnings and precautions for the MONOLISA™ Anti-HBs EIA, and The MONOLISA™ Anti-HBs Calibrator Kit are stated in the respective product labeling.

Both the MONOLISA™ Anti-HBs EIA and The MONOLISA™ Anti-HBs Calibrator Kit are for in vitro diagnostic use only. Both kits are not intended for use in screening blood, plasma, or tissue donors. The effectiveness of the MONOLISA™ Anti-HBs EIA and the MONOLISA™ Anti-HBs Calibrator Kit for use in screening blood, plasma, or tissue donors has not been established. Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients. The user is responsible for establishing their own assay performance characteristics in these populations.

## V. DEVICE DESCRIPTION:

### Components of the MONOLISA™ Anti-HBs EIA:

- Anti-HBs Microwell Strip Plate: Plates containing 96 wells coated with human HBsAg (ad and ay sybtypes). Preservative: ProClin (trace).
- Wash Solution (30X): Contains sodium chloride and Tween 20.
- Specimen Diluent: Buffer with protein stabilizers, fetal calf serum, and sample indicator dye. Preservative: 0.1% ProClin 300™.
- Negative Control: Human serum non-reactive for HBsAg and antibodies to HBs, HIV-1, HIV-2, and HCV. Preservatives: 0.005% Gentamicin and 0.16% ProClin 950™ and Gentamicin Sulfate.
- Positive Control: Human serum with anti-HBs immunoglobulin; Non-reactive for HBsAg and for antibodies to HIV-1, HIV-2, and HCV. Preservative: 0.16% ProClin 950™ and 0.005% Gentamicin Sulfate.
- Cutoff Calibrator: Fetal calf serum, buffer with protein stabilizers, bovine serum albumin, anti-HBs immunoglobulin and dye. Preservative: 0.16% ProClin 950™.
- Conjugate Concentrate: Purified HBsAg (human) labeled with peroxidase in a buffer containing protein stabilizers and dye. Preservative: 0.5% ProClin 300™ and 0.005% Gentamicin Sulfate.
- Conjugate Diluent: Buffer with protein stabilizers. Preservative: 0.1% ProClin 300™.
- Chromogen: Contains tetramethylbenzidine (TMB).
- Substrate Buffer: Contains hydrogen peroxide, citric acid, and dimethylsulfoxide (DMSO).
- Stopping Solution: Contains 1N H₂SO₄
- Plate Sealers: Used to cover the plates during testing.

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Summary of Safety and Effectiveness Data

## Components of the MONOLISA™ Anti-HBs Calibrator Kit:

- 0 mIU/mL Calibrator: Fetal calf serum, buffer with protein stabilizers, bovine serum albumin, anti-HBs immunoglobulin, and dye. Preservative: 0.16% ProClin 950™.
- 100 mIU/mL Calibrator: Fetal calf serum, buffer with protein stabilizers, bovine serum albumin, anti-HBs immunoglobulin, and dye. Preservative: 0.16% ProClin 950™.
- 400 mIU/mL Calibrator: Fetal calf serum, buffer with protein stabilizers, bovine serum albumin, anti-HBs immunoglobulin, and dye. Preservative: 0.16% ProClin 950™.
- 1000 mIU/mL Calibrator: Fetal calf serum, buffer with protein stabilizers, bovine serum albumin, anti-HBs immunoglobulin, and dye. Preservative: 0.16% ProClin 950™.

## Assay Principal and Format

The MONOLISA™ Anti-HBs EIA is a quantitative enzyme immunoassay for the detection of antibody to hepatitis B surface antigen in human serum and EDTA or citrated plasma. The assay is based on the direct antibody sandwich principle, in which microwell strip plates (the solid phase) are coated with purified native ad and ay antigens of the hepatitis B virus.

Patient samples are evaluated for the presence of anti-HBs by interaction with the adsorbed antigen on the wells. After incubation of the samples in the microwells, the plates are washed. If antibodies to the virus are present, they bind to the antigen and are not removed by washing. The Conjugate reagent, containing peroxidase-labeled purified hepatitis B ad and ay antigens, is then added to the wells and will bind to the IgG, IgM, or IgA captured from the patient samples and bound on the solid phase. Unbound conjugate is removed by a wash step. Next, Working TMB Solution is added to the plate and allowed to incubate. A blue or blue-green color develops in proportion to the amount of antibody that has been bound to the antigen-coated plate. The enzyme reaction is stopped by the addition of acid, which results in a color change to yellow. The optical absorbance of controls and specimens is determined with a spectrophotometer with wavelength set at 450 nm.

The kit contains a Negative Control, prepared from human serum negative for hepatitis B antibody, and a Positive Control, prepared from human serum negative for hepatitis B antibody spiked with purified anti-HBs. Also included in the kit is a cutoff calibrator (10 mIU/ml), prepared in a PBS-based buffer with protein stabilizers and spiked with purified anti-HBs, and it is calibrated against the WHO standard.

The MONOLISA™ Anti-HBs Calibrator Kit contains 4 levels of purified anti-HBs, prepared in a PBS-based buffer with protein stabilizers, and they are calibrated against the WHO standard. The kit is used to plot a calibration curve from the optical density value of each calibrator, for the determination of anti-HBs concentration in samples.

For more concentrated samples, plates are read at both 450nm and 405nm wavelengths. When read at 405nm, all absorbance values are reduced so that samples which are above the reader maximum at 450nm will often be within the linear range of the reader at 405nm. By using the second wavelength, the dynamic range of the assay can be extended to calibrate samples from 400-1000 mIU/mL, without diluting and testing them again.

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Summary of Safety and Effectiveness Data

## VI. Alternative Practices and Procedures

Determination of the presence of anti-HBs in patients may be attained by using a number of commercially available, FDA licensed/approved, serological tests. When these test results are evaluated in conjunction with a physician's assessment and biochemical test results, susceptibility to HBV can be excluded.

## VII. Marketing History

The MONOLISA™ Anti-HBs EIA and MONOLISA™ Anti-HBs Calibrator Kit have not been commercially marketed in countries outside of the U.S.

## VIII. Potential Adverse Effects of the Device on Health

Failure of the product to perform as intended, or errors in the use of the product, may lead to a false result. A false nonreactive result is not considered a public health risk, as the individual would either be vaccinated unnecessarily, or would be erroneously diagnosed as having not recovered from an acute HBV infection. A false reactive result can be considered a public health risk because an individual would be considered either to have been previously vaccinated or to have been previously exposed to the virus, and would therefore be assumed to be immune to HBV. As such, the risk is that the individual would not be vaccinated and would be at a higher risk of infection if exposed to HBV, and would then also be at a subsequent risk of spreading infection to other non-immune individuals.

## IX. Summary of Preclinical Studies

### A. Analytical Sensitivity

The MONOLISA™ Anti-HBs EIA may be utilized as a quantitative assay when run in conjunction with the calibrators from the MONOLISA™ Anti-HBs Calibrator Kit. Studies that have been performed to test the analytical sensitivity of the assay used dilutions of the WHO anti-HBs Standard to demonstrate correlation with concentrations of anti-HBs ranging from 0 mIU/mL to 1000 mIU/mL. The dilutional linearity was evaluated using high-titer anti-HBs specimens to prepare a series of dilutions in negative plasma. The data provide assurance that the assay, when run with the Anti-HBs Calibrator Kit, yields valid quantitative results.

The limit of detection, the lowest amount of anti-HBs that can be detected with a 95% probability, was determined for the MONOLISA™ Anti-HBs EIA. The calculation of the limit was based on NCCLS/CLSI EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline. The limit of detection was 4.14 mIU/mL at an OD of 0.034.

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Summary of Safety and Effectiveness Data

## B. Cross-Reactivity

The specificity of the MONOLISA™ Anti-HBs EIA assay was evaluated during the analysis of 393 serum samples from individuals with unrelated medical conditions, representing 20 potentially cross-reacting conditions. All of the samples were anti-HBs negative on another commercially available Anti-HBs assay. The results of each sample tested on the MONOLISA™ Anti-HBs EIA are summarized in Table 1.

Table 1
Potentially Cross-Reactive Medical Conditions

| Clinical Condition | Nonreactive | Reactive | Borderline | Total |
| --- | --- | --- | --- | --- |
| Autoimmune Diseases^{1} | 20 | 0 | 0 | 20 |
| Cytomegalovirus (CMV) | 19 | 1^{2} | 0 | 20 |
| Elevated liver enzymes | 3 | 0 | 0 | 3 |
| Epstein Barr Virus (EBV) | 20 | 0 | 0 | 20 |
| H. pylori positive | 10 | 0 | 0 | 10 |
| Hepatic Cancer | 4 | 0 | 0 | 4 |
| Hepatitis A Infection (HAV) | 19 | 1^{2} | 0 | 20 |
| Hepatitis C Infection (HCV) | 18 | 2^{2} | 0 | 20 |
| Hepatitis D Infection (HDV) | 6 | 0 | 0 | 6 |
| Herpes Simplex Virus (HSV) | 20 | 0 | 0 | 20 |
| HIV-1 | 19 | 1^{2} | 0 | 20 |
| HIV-2 | 15 | 4^{3} | 1^{4} | 20 |
| HTLV-I/II | 19 | 1^{2} | 0 | 20 |
| Influenza Vaccine Recipients | 18 | 2^{2} | 0 | 20 |
| Parvovirus B19 | 20 | 0 | 0 | 20 |
| Pregnant (bHCG positive) | 50 | 0 | 0 | 50 |
| Rheumatoid Factor (RF) | 20 | 0 | 0 | 20 |
| Rubella | 19 | 1^{2} | 0 | 20 |
| SLE / ANA Positive | 19 | 1^{2} | 0 | 20 |
| Syphilis | 20 | 0 | 0 | 20 |
| Toxoplasmosis | 20 | 0 | 0 | 20 |
| TOTAL | 378 | 14 | 1 | 393 |

1 Scleroderma, Sjögren’s, MCTD etc.
2 Of the 10 medical condition specimens that were reactive on MONOLISA™ Anti-HBs EIA (excluding HIV-2 positive samples), 3 were reactive on the reference anti-HBs EIA, 5 were nonreactive, and 2 were QNS for additional testing.
3 Two specimens were reactive when tested with a reference anti-HBs EIA; 2 were QNS for additional testing.
4 Specimen was reactive when tested with a reference anti-HBs EIA.

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Summary of Safety and Effectiveness Data

Of the 393 specimens from 20 unrelated medical conditions that were tested, 378/393 (96.2%) were nonreactive on the MONOLISA™ Anti-HBs EIA. Fourteen (14) specimens were reactive: 4 HIV-2 reactive specimens, 2 influenza vaccine specimens, 2 HCV positive specimens and 1 each of 8 other conditions (CMV, HAV, HIV-1, HTLV, Rubella, and SLE).

## C. Interfering substances

The MONOLISA™ Anti-HBs EIA was evaluated to determine if there were any effects on assay performance when tested with specimens containing potentially interfering substances, according to CLSI Document EP7. None of the interferents at the levels tested below produced a change in clinical interpretation or a significant ≥ 10% change of the assay.

- Hemolyzed: 500 mg/dL of hemoglobin
- Lipemic: 1000 mg/dL of triglycerides
- Icteric: 20 mg/dL of bilirubin
- Proteinemic: 15 g/dL of protein

The MONOLISA™ Anti-HBs assay did not exhibit a high-dose hook effect in patient samples with levels of antibodies to HBsAg as high as 175,000 mIU/mL. The MONOLISA™ Anti-HBs EIA is designed using a two-step format, where a high-dose hook effect is not normally observed.

## D. Stability Studies

### 1. Kit Stability

A functional stability study of the MONOLISA™ Anti-HBs EIA test kit and the MONOLISA™ Anti-HBs Calibrator Kit has demonstrated that kits which are stored at 2-8°C are stable for the intended shelf-life of the kits. Real-time studies were performed on three kit lots of both the EIA and Calibrator kits, at multiple time points throughout the shelf life of the kits. Data from these studies support a 12-month dating period for the MONOLISA™ Anti-HBs EIA test kit and the MONOLISA™ Anti-HBs Calibrator Kit, with the expiration of the assembled kit based on the component with the shortest dating period.

### 2. Interchangeability of Common Reagents

The MONOLISA™ Anti-HBs EIA contains four common reagents that may be used interchangeably with the same components in other lots of the Anti-HBs EIA: Wash Solution Concentrate, Chromogen, Substrate Buffer, and Stopping Solution. Matrix studies performed with the MONOLISA™ Anti-HBs EIA have evaluated different lots of each of these components in the kit, and demonstrated equivalent results. Therefore, any lot number of these reagents may be used with this assay provided they are not used beyond their labeled expiration date.

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Summary of Safety and Effectiveness Data

## E. Microbiology Studies

Antimicrobial preservatives have been added to the components in the Anti-HBs EIA kit and Anti-HBs Calibrator Kit to protect the product from degradation and performance failure due to the presence of microbial contamination. Preservative effectiveness studies have been conducted in accordance with the protocol specified in the United States Pharmacopoeia (Microbiological Tests, <51> Antimicrobial Preservatives/Effectiveness) to assess the efficacy of these preservatives in suppressing microbial growth. These studies have demonstrated that the antimicrobial agents are present in concentrations required to inhibit the growth of adventitious agents.

A Microbial Challenge study has been performed to evaluate the functional stability of the Anti-HBs EIA kit and Anti-HBs Calibrator Kit components in the presence of microbial organisms. One set of Anti-HBs EIA and Anti-HBs Calibrator kit components that were inoculated with microorganisms was tested in comparison to a reference second set of Anti-HBs EIA and Anti-HBs Calibrator kit components that had not been inoculated. A variety of organisms from the environment were used in this challenge study. Each kit was stored at the recommended product storage of 2-8°C after inoculation and tested at multiple time points throughout kit expiration. These studies have demonstrated that the functionalities of the EIA and Calibrator products are not impaired and the reagents are stable for the 12-month shelf life when microbial contamination is present.

## F. Reproducibility

A 7-member panel consisting of diluted patient samples in various matrices (serum and EDTA) was tested in duplicate, once a day for 10 days, on 3 lots of the MONOLISA™ Anti-HBs EIA.

The data from all 3 reagent lots were combined to obtain standard deviation (SD) and percent coefficient of variation (CV) for within run, between run, and total variance. The data were analyzed according to the principles described in CLSI EP5-A2 and ISO/TR 22971:2005. The data summary for this study is shown in Tables 2 and 3.

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Summary of Safety and Effectiveness Data

Table 2
MONOLISA™ Anti-HBs EIA Reproducibility Results
by Panel Member Signal to Cutoff (S/CO)

| Test Site | Panel Member | N | Mean S/CO | Within Run^{1} | | Between Run^{2} | | Total^{3} | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Site #1 | 1 Pos Serum | 60 | 8.265 | 0.309 | 3.7 | 0.579 | 7.0 | 0.487 | 5.9 |
| | 2 ~12 mIU/mL (Serum) | 60 | 1.349 | 0.051 | 3.8 | 0.104 | 7.7 | 0.085 | 6.3 |
| | 3 ~8 mIU/mL (Serum) | 60 | 0.955 | 0.047 | 4.9 | 0.072 | 7.5 | 0.064 | 6.7 |
| | 4 Neg (Serum) | 60 | 0.288 | 0.012 | 4.3 | 0.073 | 25.3 | 0.054 | NA |
| | 5 ~12 mIU/mL (EDTA) | 60 | 1.435 | 0.032 | 2.2 | 0.090 | 6.3 | 0.078 | 5.5 |
| | 6 ~8 mIU/mL (EDTA) | 60 | 1.044 | 0.033 | 3.2 | 0.070 | 6.7 | 0.063 | 6.1 |
| | 7 Neg (EDTA) | 60 | 0.329 | 0.018 | 5.5 | 0.090 | 27.3 | 0.065 | NA |
| Site #2 | 1 Pos Serum | 60 | 8.008 | 0.259 | 3.2 | 0.984 | 12.3 | 0.696 | 8.7 |
| | 2 ~12 mIU/mL (Serum) | 60 | 1.289 | 0.078 | 6.1 | 0.136 | 10.5 | 0.109 | 8.5 |
| | 3 ~8 mIU/mL (Serum) | 60 | 0.950 | 0.089 | 9.3 | 0.225 | 23.6 | 0.165 | 17.4 |
| | 4 Neg (Serum) | 60 | 0.285 | 0.093 | 32.7 | 0.197 | 69.1 | 0.149 | NA |
| | 5 ~12 mIU/mL (EDTA) | 60 | 1.384 | 0.098 | 7.0 | 0.205 | 14.8 | 0.156 | 11.3 |
| | 6 ~8 mIU/mL (EDTA) | 60 | 0.971 | 0.037 | 3.8 | 0.085 | 8.7 | 0.068 | 7.0 |
| | 7 Neg (EDTA) | 60 | 0.292 | 0.058 | 19.9 | 0.149 | 50.9 | 0.117 | NA |
| Site #3 | 1 Pos Serum | 60 | 6.707 | 0.373 | 5.6 | 1.560 | 23.3 | 1.150 | 17.1 |
| | 2 ~12 mIU/mL (Serum) | 60 | 1.034 | 0.055 | 5.3 | 0.243 | 23.5 | 0.176 | 17.0 |
| | 3 ~8 mIU/mL (Serum) | 60 | 0.705 | 0.050 | 7.1 | 0.146 | 20.7 | 0.111 | 15.7 |
| | 4 Neg (Serum) | 60 | 0.249 | 0.061 | 24.3 | 0.130 | 52.0 | 0.098 | NA |
| | 5 ~12 mIU/mL (EDTA) | 60 | 1.124 | 0.093 | 8.2 | 0.299 | 26.6 | 0.218 | 19.4 |
| | 6 ~8 mIU/mL (EDTA) | 60 | 0.763 | 0.078 | 10.2 | 0.181 | 23.7 | 0.139 | 18.2 |
| | 7 Neg (EDTA) | 60 | 0.200 | 0.042 | 21.0 | 0.062 | 30.8 | 0.054 | NA |

NA = Not Applicable.
1 Within Run: variability of the assay performance from replicate to replicate.
2 Between Run: variability of the assay performance from run to run.
3 Total variability of the assay performance includes within run, between run and between lot.

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Summary of Safety and Effectiveness Data

Table 3
MONOLISA™ Anti-HBs EIA Reproducibility Results (Positive, Low Positive, and High Negative)
by Panel Member S/CO

| Summary of Panel Members | N= | Mean S/CO | Between Lot | | Between Site^{1} | | Total^{2} | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | CV | SD | CV | SD | CV |
| Positive Serum | 180 | 7.660 | 1.334 | 17.4 | 6.473 | 84.5 | 1.069 | 14.0 |
| ~12 mIU/mL (Serum) | 180 | 1.223 | 0.194 | 15.9 | 1.295 | 105.9 | 0.188 | 15.3 |
| ~8 mIU/mL (Serum) | 180 | 0.870 | 0.145 | 16.7 | 1.107 | 127.2 | 0.168 | 19.3 |
| ~12 mIU/mL (EDTA) | 180 | 1.314 | 0.207 | 15.7 | 1.292 | 98.3 | 0.211 | 16.0 |
| ~8 mIU/mL (EDTA) | 180 | 0.926 | 0.175 | 18.9 | 1.131 | 122.2 | 0.153 | 16.6 |

1 Sites were nested within lots
2 Total variability includes within run, between run, between lot, and between site.

G. Quantitative Precision

A precision study was performed with the MONOLISA Anti-HBs EIA using quantitative panels prepared in serum and EDTA plasma. Each 7-member panel spanned the linear range of the assay: The 14 specimens were tested in triplicate for 20 days, and results are summarized in Table 4.

Table 4
MONOLISA™ Anti-HBs EIA 20-Day Precision Results in mIU/mL

| Panel Member | N | mIU/mL
Mean | Within-Run | | Between-Day | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | CV% | SD | CV% | SD | CV% |
| Serum ~10 mIU/mL | 60 | 11.1 | 0.259 | 2.34 | 1.361 | 12.30 | 1.386 | 12.52 |
| Serum ~25 mIU/mL | 60 | 26.6 | 1.163 | 4.37 | 1.589 | 5.97 | 1.969 | 7.40 |
| Serum ~85 mIU/mL | 60 | 89.1 | 1.372 | 1.54 | 2.383 | 2.67 | 2.750 | 3.09 |
| Serum ~350 mIU/mL | 60 | 363.3 | 3.372 | 0.93 | 5.202 | 1.43 | 6.199 | 1.71 |
| Serum ~500 mIU/mL | 60 | 492.2 | 19.882 | 4.04 | 17.794 | 3.62 | 26.682 | 5.42 |
| Serum ~750 mIU/mL | 60 | 726.9 | 13.088 | 1.80 | 17.429 | 2.40 | 21.796 | 3.00 |
| Serum ~950 mIU/mL | 60 | 946.3 | 11.786 | 1.25 | 14.777 | 1.56 | 18.901 | 2.00 |
| EDTA Plasma ~10 mIU/mL | 60 | 12.4 | 0.596 | 4.80 | 1.263 | 10.17 | 1.396 | 11.24 |
| EDTA Plasma ~25 mIU/mL | 60 | 27.9 | 0.462 | 1.66 | 1.111 | 3.99 | 1.204 | 4.32 |
| EDTA Plasma ~85 mIU/mL | 60 | 92.5 | 1.055 | 1.14 | 2.281 | 2.47 | 2.513 | 2.72 |
| EDTA Plasma ~350 mIU/mL | 60 | 367.5 | 8.216 | 2.24 | 7.953 | 2.16 | 11.435 | 3.11 |
| EDTA Plasma ~500 mIU/mL | 60 | 496.0 | 11.173 | 2.25 | 19.743 | 3.98 | 22.685 | 4.57 |
| EDTA Plasma ~750 mIU/mL | 60 | 738.5 | 10.457 | 1.42 | 16.504 | 2.23 | 19.538 | 2.65 |
| EDTA Plasma ~950 mIU/mL | 60 | 940.1 | 20.127 | 2.14 | 13.425 | 1.43 | 24.193 | 2.57 |

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Summary of Safety and Effectiveness Data

## IX. Summary of Clinical Studies

A multi-center clinical trial was conducted to evaluate the performance of the MONOLISA™ Anti-HBs EIA and the MONOLISA™ Anti-HBs Calibrator Kit in human serum and plasma. A total of 1452 prospective subjects at high risk for viral hepatitis and/or showing signs/symptoms of HBV were included in the study. Of these 1452, 1373 were from the asymptomatic high risk population and 79 reported signs or symptoms of HBV.

## Expected Values

The expected values that can be seen with the MONOLISA™ Anti-HBs EIA, by gender and age range, were determined during the evaluation of 1373 prospective asymptomatic subjects. All subjects (100%) were at high risk for viral hepatitis including intravenous drug users (N = 476), homosexual males (N = 144), sex workers (N = 171), prison history (N = 340), high risk sex partners (167), high risk occupation/health care workers (N = 85), hemodialysis (N = 58), hemophiliacs (3), and other (N = 470). Many had more than 1 high risk behavior or risk factor. One hundred seventy six (12.8%) of these high risk subjects also reported having received a full course of injections of an HBV vaccine. Subjects in the asymptomatic prospective population were from the following geographic locations: 459 from Los Angeles, CA, (33.4%), 57 from Santa Ana, CA (4.1%), 72 from Miami, FL (5.2%), 345 from Cocoa, FL (25.1%), 273 from San Francisco, CA (19.9%), and 167 from Seattle, WA (12.2%). The group was Caucasian (36.5%), Black or African American (41.1%), Hispanic or Latino (13.3%), Asian (4.2%), Native Hawaiian or other Pacific Islander (0.7%), and American Indian or Alaska Native (2.2%), with the remaining 2.0% represented by multiple ethnic groups or was unknown. The subjects were male (70.1%) and female (29.9%) and ranged in age from 18 to 81 years.

The MONOLISA™ Anti-HBs EIA results for the asymptomatic prospective population, by gender and age range, are presented in Table 5.

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Summary of Safety and Effectiveness Data

Table 5 Expected Values by Gender and Age - MONOLISA™ Anti-HBs EIA

| Age Range | Gender | MONOLISA™ Anti-HBs EIA Result | | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Reactive | | Borderline | | Non-reactive | | |
| | | N | % | N | % | N | % | N |
| 10-19 | F | 6 | 100.0% | 0 | 0.0% | 0 | NA | 6 |
| | M | 7 | 70.0% | 0 | 0.0% | 3 | 30.0% | 10 |
| 20-29 | F | 44 | 42.3% | 1 | 1.0% | 59 | 56.7% | 104 |
| | M | 48 | 39.0% | 0 | 0.0% | 75 | 61.0% | 123 |
| 30-39 | F | 42 | 37.5% | 4 | 3.6% | 66 | 58.9% | 112 |
| | M | 75 | 35.0% | 1 | 0.5% | 138 | 64.5% | 214 |
| 40-49 | F | 40 | 37.4% | 3 | 2.8% | 64 | 59.8% | 107 |
| | M | 162 | 45.8% | 7 | 2.0% | 185 | 52.3% | 354 |
| 50-59 | F | 33 | 51.6% | 4 | 6.3% | 27 | 42.2% | 64 |
| | M | 108 | 51.2% | 8 | 3.8% | 95 | 45.0% | 211 |
| 60-69 | F | 5 | 41.7% | 0 | 0.0% | 7 | 58.3% | 12 |
| | M | 23 | 57.5% | 0 | 0.0% | 17 | 42.5% | 40 |
| 70-79 | F | 1 | 50.0% | 0 | 0.0% | 1 | 50.0% | 2 |
| | M | 3 | 60.0% | 0 | 0.0% | 2 | 40.0% | 5 |
| 80-89 | F | 0 | NA | 0 | NA | 0 | NA | 0 |
| | M | 0 | NA | 0 | 0.0% | 1 | 100.0% | 1 |
| Unknown | F | 2 | 66.7% | 0 | 0.0% | 1 | 33.3% | 3 |
| | M | 1 | 20.0% | 1 | 20.0% | 3 | 60.0% | 5 |
| Totals | | 600 | 43.7% | 29 | 2.1% | 744 | 54.2% | 1373 |

## Reference Markers

The HBV disease classification for each subject in the total prospective population (N = 1452) was determined by a serological assessment using a hepatitis marker profile consisting of commercially available reference EIAs. The six HBV reference marker assays included HBsAg, hepatitis B virus e antigen (HBeAg), total antibody to hepatitis B virus core antigen (Anti-HBc, Total), IgM antibody to hepatitis B virus core antigen (Anti-HBc IgM), total antibody to HBe Ag (Anti-HBe), and total antibody to hepatitis B virus surface antigen (anti-HBs, qualitative or quantitative). All reference EIAs were tested according to the manufacturer's package insert instructions. Agreement of the

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Summary of Safety and Effectiveness Data

MONOLISA™ Anti-HBs EIA was assessed relative to the reference anti-HBs result and to HBV classification.

In the MONOLISA™ Anti-HBs EIA clinical study, across 3 clinical sites, there were 38 unique reference marker patterns observed. Table 6 summarizes the patterns and their associated classifications. No other laboratory or clinical information was used in the disease classification process.

Table 6 Characterization of Prospective Samples

| FDA Characterization (single point specimen) | HBsAg | HBeAg | Anti-HBc IgM | Total HBc | Anti-HBe | Anti-HBs |
| --- | --- | --- | --- | --- | --- | --- |
| Acute infection | + | + | + | + | - | - |
| Acute infection | + | + | - | - | - | - |
| Acute infection | + | - | + | - | - | + |
| Acute infection | + | - | - | - | - | - |
| Acute infection | + | - | + | - | - | - |
| Acute infection | + | + | I | + | - | - |
| Chronic infection | + | + | - | + | - | - |
| Chronic infection | + | + | - | + | - | + |
| Chronic infection | + | + | - | + | + | - |
| Chronic infection | + | + | + | + | - | + |
| Chronic infection | + | - | - | + | - | - |
| Chronic infection | + | - | - | + | - | + |
| Chronic infection | + | - | - | + | + | - |
| Chronic infection | + | - | - | + | + | + |
| Chronic infection | + | - | - | + | I | - |
| Chronic infection | + | + | - | + | + | + |
| Early recovery | - | - | - | + | - | - |
| Early recovery | - | - | - | + | + | - |
| Early recovery | - | - | I | + | + | + |
| Early recovery | - | - | - | + | I | - |
| Early recovery | - | - | + | + | - | + |
| Early recovery | - | - | + | + | + | - |
| Recovery | - | - | - | - | + | + |
| Recovery | - | - | - | + | + | + |
| Recovery | - | - | - | + | + | I |
| Recovery | - | - | - | + | I | + |
| Recovered | - | - | - | + | - | I |
| Recovered | - | - | - | + | I | I |
| Recovered or Immune due to natural infection | - | - | - | + | - | + |

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Summary of Safety and Effectiveness Data

| HBV vaccine response | - | - | - | - | - | + |
| --- | --- | --- | --- | --- | --- | --- |
| HBV vaccine response (?) | - | - | - | - | - | I |
| Not previously infected with HBV | - | - | - | - | - | - |
| Uninterpretable | + | - | - | - | + | - |
| Uninterpretable | - | + | - | - | - | + |
| Uninterpretable | - | + | - | + | - | - |
| Uninterpretable | - | + | - | - | - | - |
| Uninterpretable | - | - | - | - | + | - |

(-) = Negative / Non-reactive: (+) = Positive / Reactive: (I) = Indeterminate

## Comparison of Results

A comparison of the MONOLISA™ Anti-HBs EIA results with the reference anti-HBs assay for each specimen classification is shown in Table 7. In clinical studies, specimens that had indeterminate results on the reference test were retested in duplicate per the manufacturer’s instructions for use. Any specimens with 2/3 or 3/3 results within the indeterminate range were classified as indeterminate.

Table 7 Comparison of Results - High Risk Prospective Samples MONOLISA™ Anti-HBs EIA versus Reference Anti-HBs EIAs

| Reference HBV Classification | Reference Anti-HBs Result | | | | | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Positive | | | Indeterminate | | | Negative | | | |
| | MONOLISA Anti-HBs | | | MONOLISA Anti-HBs | | | MONOLISA Anti-HBs | | | |
| | R | BRD² | NR | R | BRD² | NR | R | BRD² | NR | |
| Acute infection | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 13 | 14 |
| Chronic infection | 4 | 1 | 1 | 0 | 0 | 0 | 4¹ | 0 | 71 | 81 |
| Early Recovery | 3 | 1 | 0 | 0 | 0 | 0 | 10 | 5 | 88 | 107 |
| Recovery | 160 | 3 | 4 | 3 | 4 | 2 | 0 | 0 | 0 | 176 |
| Recovered | 0 | 0 | 0 | 5 | 6 | 1 | 0 | 0 | 0 | 12 |
| Recovered or Immune due to natural infection | 91 | 1 | 4 | 0 | 0 | 0 | 0 | 0 | 0 | 96 |
| HBV vaccine response | 307 | 1 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 316 |
| HBV vaccine response (?) | 0 | 0 | 0 | 16 | 8 | 7 | 0 | 0 | 1 | 32 |
| Not previously infected with HBV | 0 | 0 | 0 | 0 | 0 | 0 | 11 | 0 | 598¹ | 609 |
| Uninterpretable | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 9 |
| Total | 567 | 7 | 17 | 24 | 18 | 10 | 25 | 5 | 779 | 1452 |

¹ Includes specimens that were NRR (not repeatedly reactive)
² BRD = Borderline (± 10% of cutoff value)

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Summary of Safety and Effectiveness Data

Overall, 567 samples were positive on both assays, 18 samples were indeterminate/borderline on both assays, and 779 samples were negative on both assays.

## Percent Agreement

The percent agreement between the MONOLISA™ Anti-HBs EIA and the reference anti-HBs assays was evaluated for each specimen classification, including the upper and lower 95% Wilson confidence bounds. A summary of this analysis for the prospective population is presented for each HBV classification in Table 8.

Table 8 Percent Agreement MONOLISA™ Anti-HBs EIA versus Reference Anti-HBs EIA

| HBV Classification | N^{1} | Positive Percent Agreement^{2} | 95% Confidence Interval | Negative Percent Agreement^{3} | 95% Confidence Interval |
| --- | --- | --- | --- | --- | --- |
| Acute Infection | 14 | 100.0% (1/1) | 20.7%, 100.0% | 100.0% (13/13) | 77.2%, 100.0% |
| Chronic Infection | 81 | 66.7% (4/6) | 30.0%, 90.3% | 94.7% (71/75) | 87.1%, 97.9% |
| Early Recovery | 107 | 75.0% (3/4) | 30.1%, 95.4% | 85.4% (88/103) | 77.4%, 91.0% |
| Recovery | 176 | 94.7% (160/169) | 90.2%, 97.2% | 0.0% (0/3) | NA |
| Recovered | 12 | 0.0% (0/1) | NA | 0.0% (0/5) | NA |
| Past Infection | 96 | 94.8% (91/96) | 88.4%, 97.8% | NA (0/0) | NA |
| HBV Vaccine response | 316 | 97.2% (307/316) | 94.7%, 98.5% | NA (0/0) | NA |
| HBV vaccine response (?) | 32 | 0.0% (0/7) | NA | 5.9% (1/17) | 1.0%, 27.0% |
| Not previously infected | 609 | NA (0/0) | NA | 98.2% (598/609) | 96.8%, 99.0% |
| Uninterpretable | 9 | 100.0% (1/1) | 20.7%, 100.0% | 100.0% (8/8) | 67.6%, 100.0% |
| Total | 1452 | 94.3% (567/601) | 92.2%, 95.9% | 93.5% (779/833) | 91.6%, 95.0% |

1 N=Total number of samples; refer to Table 3 for correlation of borderline samples. The eighteen specimens that were indeterminate by both assays were not included in the percent agreement calculations. Positive or negative results from the MONOLISA™ Anti-HBs EIA were considered as non-agreements in the calculation of percent positive agreement and percent negative agreement when the corresponding reference assay result was indeterminate/borderline.
2 Compares number of samples positive on both assays to sum of all positive samples on the reference assay + samples indeterminate on the reference assay and negative on MONOLISA™ Anti-HBs EIA.

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Summary of Safety and Effectiveness Data

3 Compares number of samples negative on both assays to sum of all negative samples on the reference assay + samples indeterminate on the reference assay and positive on MONOLISA™ Anti-HBs EIA.

The positive percent agreement with the reference method is 94.3% (567/601) with a 95% confidence interval of 92.2 – 95.9%. The negative percent agreement with the reference method is 93.5% (779/833) with a 95% confidence interval of 91.6 – 95.0%.

## Seroconversion Panels

The comparative sensitivity of the MONOLISA™ Anti-HBs EIA was determined by testing 4 commercially available Anti-HBV seroconversion panels and comparing the results to those in the associated certificates of analysis. Comparative results for only panel members near the point of seroconversion are presented in Table 9.

Table 9 HBV Seroconversion Panel Results

| Panel ID | Day since
1^{st} bleed | Total
#
Members | MONOLISA™ Anti-HBs | | Reference Anti-HBs EIA^{1} | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | S/CO | Result | S/CO | Result |
| RP016-08 | 60 | 20 | 0.26 | NR | 0.50 | NR |
| RP016-09 | 74 | | 1.02 | BRD^{2} | 1.47 | R |
| RP016-10 | 79 | | 1.95 | R | 2.43 | R |
| RP016-11 | 81 | | 3.72 | R | 2.43 | R |
| 6506-06 | 69 | 14 | 0.74 | NR | 0.8 | NR |
| 6506-07 | 83 | | 1.75 | R | 1.1 | R |
| 6506-08 | 97 | | 3.00 | R | 2.0 | R |
| 6514-09 | 112 | 17 | 0.57 | NR | 0.7 | NR |
| 6514-10 | 126 | | 1.20 | R | 1.3 | R |
| 6514-11 | 140 | | 1.91 | R | 1.4 | R |
| 6536-06 | 70 | 12 | 0.85 | NR | 0.7 | NR |
| 6536-07 | 84 | | 1.15 | R | 0.9 | NR |
| 6536-08 | 98 | | 1.42 | R | 1.3 | R |

1 From Certificates of Analysis.
2 BRD = Borderline

In 2 of the 4 seroconversion panels, the MONOLISA™ Anti-HBs EIA detected reactive levels of hepatitis B surface antibody at the same member as the reference anti-HBs EIA. In 1 of the 4 seroconversion panels the MONOLISA™ Anti-HBs EIA detected reactive levels of hepatitis B surface antibody 1 member before the reference anti-HBs EIA. One panel was borderline (S/CO = 1.02) on the MONOLISA™ Anti-HBs EIA at the first reactive bleed on the reference test.

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Summary of Safety and Effectiveness Data

# Clinical Performance with Individuals Who Received a Full Course of Hepatitis B Vaccine

Retrospective studies were conducted to evaluate a total of 197 serum specimens from 197 subjects who had received a full course of 3 HBV vaccinations (SmithKline-Beecham Biologicals Engerix-B® HBV vaccine or Merck & Co., Inc. Recombivax HB® vaccine). Testing was compared to reference anti-HBs EIA. The MONOLISA™ Anti-HBs EIA demonstrated immunity in 141/197 specimens (71.6%; 95% confidence interval of 64.9% to 77.4%). The reference method demonstrated immunity in 134/197 specimens (68.0%; 95% confidence interval of 61.2% - 74.1%).

Table 10 Post-HBV Vaccination Results

| MONOLISA™ Anti-HBs Result | Reference Anti-HBs Result | | | |
| --- | --- | --- | --- | --- |
| | Immune | Indeterminate | Not-Immune | Totals |
| Immune | 134 | 3 | 4 | 141 |
| Borderline | 0 | 2* | 1 | 3 |
| Not-Immune | 0 | 1 | 52 | 53 |
| Totals | 134 | 6 | 57 | 197 |

* Two specimens that were indeterminate by both assays were not included in percent agreement calculations.

The positive percent agreement with the reference method is 99.3% (134/135) with a 95% confidence interval of 95.9 – 99.9%. The negative percent agreement with the reference method is 86.7% (52/60) with a 95% confidence interval of 75.8 – 93.1%.

# Clinical Performance with Matched Pre- and Post-HBV Vaccination Specimens

In another study, matched sets of pre- and post-vaccination specimens from thirty-eight individuals who had received recombinant HBV vaccine (either SmithKline-Beecham Biologicals Engerix-B® HBV vaccine or Merck & Co., Inc. Recombivax HB® vaccine) were tested with the MONOLISA™ Anti-HBs EIA. The matched sets from each subject included four specimens. One specimen was a pre-vaccination specimen collected before receiving the first vaccination dose of HBV vaccine. The second and third specimens were collected right before the second vaccination dose and third vaccination dose respectively. A post vaccination specimen was collected a minimum of 2 weeks after receiving the full course of 3 injections.

# Pre-Vaccination Samples

In pre-vaccination samples, one sample was reactive (immune) on the MONOLISA™ Anti-HBs EIA but nonreactive (not immune) on the reference assay. The negative percent agreement with the reference method is 97.4% (37/38) with a 95% confidence interval of 86.5 - 99.5%. Results are presented in Table 11 below.

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Summary of Safety and Effectiveness Data

## Table 11
### Pre-Vaccination Specimen Results
#### MONOLISA™ Anti-HBs EIA versus Reference EIA

| MONOLISA™ Anti-HBs Result | Reference Anti-HBs Result | | |
| --- | --- | --- | --- |
| | I | NI | Total |
| Immune | 0 | 1 | 1 |
| Borderline | 0 | 0 | 0 |
| Not Immune | 0 | 37 | 37 |
| Total | 0 | 38 | 38 |

I = Immune, NI = Not Immune

## Pre-Second Vaccination Samples

In samples drawn just prior to the second vaccination in the series, the MONOLISA™ Anti-HBs EIA demonstrated immunity in 4/38 (10.5%) of the samples. The reference method demonstrated immunity in 1/38 (2.6%) of the samples.

The positive percent agreement with the reference method is 100% (1/1) with a 95% confidence interval of 20.7 – 100%. The negative percent agreement with the reference method is 89.2% (33/37) with a 95% confidence interval of 75.3 – 95.7%. One sample was borderline on the MONOLISA™ Anti-HBs EIA assay. Results are presented in Table 12 below.

## Table 12
### Pre-Second Vaccination Specimen Results
#### MONOLISA™ Anti-HBs EIA versus Reference EIA

| MONOLISA™ Anti-HBs Result | Reference Anti-HBs Result | | |
| --- | --- | --- | --- |
| | I | NI | Total |
| Immune | 1 | 3 | 4 |
| Borderline | 0 | 1 | 1 |
| Not Immune | 0 | 33 | 33 |
| Total | 1 | 37 | 38 |

I = Immune, NI = Not Immune

## Pre-Third Vaccination Samples

The positive percent agreement with the reference method is 100% (15/15) with a 95% confidence interval of 79.6 - 100%. The negative percent agreement with the reference method is 82.6% (19/23) with a 95% confidence interval of 62.9 – 93.0%. One sample was borderline on the MONOLISA™ Anti-HBs EIA assay. Results are presented in Table 13 below.

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Summary of Safety and Effectiveness Data

## Table 13
Pre-Third Vaccination Specimen Results
MONOLISA™ Anti-HBs EIA versus Reference EIA

| MONOLISA™ Anti-HBs Result | Reference Anti-HBs Result | | |
| --- | --- | --- | --- |
| | I | NI | Total |
| Immune | 15 | 3 | 18 |
| Borderline | 0 | 1 | 1 |
| Not Immune | 0 | 19 | 19 |
| Total | 15 | 23 | 38 |

I = Immune, NI = Not Immune

## Post Vaccination Samples

In samples drawn after the complete vaccination series (post vaccination), the positive percent agreement with the reference method is 97.0% (32/33) with a 95% confidence interval of 84.7 – 99.5%. The negative percent agreement with the reference method is 60% (3/5) with a 95% confidence interval of 23.1 – 88.2%. One sample that was immune and two that were not immune with the reference assay were borderline with the MONOLISA™ Anti-HBs EIA. Results are presented in Table 14 below.

## Table 14
Post-Vaccination Results
MONOLISA™ Anti-HBs EIA versus Reference EIA

| MONOLISA™ Anti-HBs Result | Reference Anti-HBs Result | | |
| --- | --- | --- | --- |
| | I | NI | Total |
| Immune | 32 | 0 | 32 |
| Borderline | 1 | 2 | 3 |
| Not Immune | 0 | 3 | 3 |
| Total | 33 | 5 | 38 |

I = Immune, NI = Not Immune

## X. Conclusions Drawn from the Studies

Multi-centered clinical and non-clinical were conducted in the US to evaluate the MONOLISA™ Anti-HBs EIA. A method comparison was performed with a commercially available licensed assay to detect anti-HBs antibodies in specimens from an intended use diagnostic population.

The performance characteristics of the assay are not affected by potential cross-reacting substances that may be present in clinical samples, or by interfering substances (hemoglobin, lipemia, bilirubin, or elevated protein levels).

Stability studies have demonstrated that Anti-HBs EIA kits and Anti-HBs Calibrator Kits which are stored as indicated (2-8°C) are stable for the intended shelf-life of the kits.

18
25

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Summary of Safety and Effectiveness Data

Multi-centered clinical studies and in-house analytical studies were conducted in the US to evaluate the MONOLISA™ Anti-HBs EIA. A method comparison was performed with a commercially available licensed assay to detect anti-HBs antibodies in specimens from an intended use diagnostic population.

Hepatitis B virus classification using the prospective population showed 38 unique reference marker patterns. The overall positive percent agreement between the MONOLISA™ Anti-HBs EIA and the reference assay was 94.3% (567/601) in the high risk, signs and symptoms, and vaccinated populations. The overall negative percent agreement between the MONOLISA™ Anti-HBs EIA and the reference assay was 93.5% (779/833) in the same population.

In HBV vaccinated individuals, the positive agreement was 99.3% (134/135) and the negative percent agreement was 86.7% (52/60) with the comparison method. In another study, the Pre-vaccination HBV vaccine group recipients, the negative agreement with the comparison method was 97.4 (37/38) and in the post vaccination group the positive agreement rate with the comparison method was 97.0% (32/33).

The ability of the MONOLISA™ Anti-HBs EIA to detect the anti-HBS antibodies was demonstrated in pediatric specimen testing.

Precision and reproducibility of the MONOLISA™ Anti-HBs EIA was established for within-run (≤8.9%), within-day (≤9.1%), within-lab (≤11.4%), and between sites (≤13.2%).

Tube Type Interference study results support the use of human serum and EDTA or citrated specimens in the MONOLISA™ Anti-HBs EIA.

The results from both the non-clinical and clinical studies indicate that the MONOLISA™ Anti-HBs EIA can be used safely and effectively for the qualitative and quantitative determination of anti-HBs antibodies in human serum and plasma. The assay may be used with other HBV serological markers to define the clinical status of patients known to be infected with HBV.

## RISK BENEFIT ANALYSIS

As a diagnostic test, the MONOLISA™ Anti-HBs EIA involves the removal of blood from an individual for testing purposes. This test presents no more of a safety hazard to an individual than is presented to an individual who is having their blood drawn for any other diagnostic evaluation. The benefits to HBV-infected individuals tested by the assay outweigh any potential adverse event or risk to the patient or user due to assay malfunction or operator error.

The potential risks encountered with this in vitro diagnostic test are not unusual in the clinical laboratory setting. Appropriate warnings for these risks are contained in the labeling and package inserts for the device. Standard good laboratory practices are considered sufficient to mitigate the risks to the end user.

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Summary of Safety and Effectiveness Data

## SAFETY

Based on the results of the preclinical and clinical laboratory studies, the MONOLISA™ Anti-HBs EIA, when used according to the provided directions and in conjunction with other serological and clinical information, should be safe and effective and pose minimal risk to the patient due to false test results.

## EFFECTIVENESS

The effectiveness of the MONOLISA™ Anti-HBs EIA has been demonstrated for use in determining if antibodies to the HBs antigen of the hepatitis B virus are present in an individual’s serum or plasma. A reasonable determination of effectiveness of the MONOLISA™ Anti-HBs EIA assay for aiding in the diagnosis of immunity and status of HBV infection in suspected individuals has been demonstrated.

## XI. Panel Recommendations

In accordance with the provisions of section 515(c)(2) of the act as amended by the Safe Medical Devices Act of 1990, this PMA was not referred to the Microbiology Advisory Panel, an FDA advisory committee, for review and recommendation because the information in the PMA substantially duplicates information previously reviewed by this panel.

## XI. CDRH Decision

FDA issued an approval order on August 25, 2006.

The applicant's manufacturing facilities were inspected on May 17 – June 1, 2006 and found to be in substantial compliance with the Quality Systems Regulation as defined in 21 CFR 820.

## XII. Approval Specifications

Directions for Use: See labeling.

Hazards to Health from Use of the Device: Refer to the Warnings, Precautions, and Contraindications in the device labeling.

Postapproval Requirements and Restrictions: See approval order.