BIO RAD MONOLISA ANTI-HBC IGM EIA

P060034 · Bio-Rad Laboratories, Inc. · SEI · May 31, 2007 · Microbiology

Device Facts

• Record ID: P060034
• Device Name: BIO RAD MONOLISA ANTI-HBC IGM EIA
• Applicant: Bio-Rad Laboratories, Inc.
• Product Code: SEI · Microbiology
• Decision Date: May 31, 2007
• Decision: APRL
• Regulation: 21 CFR 866.3173
• Device Class: Class 2

Intended Use

The MONOLISA™ Anti-HBc IgM EIA is a qualitative enzyme immunoassay for the detection of IgM antibodies to hepatitis B core antigen in human serum and plasma. The detection of IgM antibodies to hepatitis B core antigen is a significant marker for the presence of recent infection by the hepatitis B virus (HBV): 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.

Device Story

Qualitative enzyme immunoassay (EIA) for detection of IgM antibodies to hepatitis B core antigen (Anti-HBc IgM) in human serum and plasma; utilizes IgM antibody capture format; patient specimens incubated in microwells coated with anti-human IgM; specific Anti-HBc IgM binds to capture antibody; HRP-conjugated recombinant HBc added; chromogen/substrate (TMB/H2O2) added; color change measured spectrophotometrically; intensity proportional to Anti-HBc IgM concentration; results compared to cutoff value; performed in clinical laboratories by trained personnel; aids in differential diagnosis of HBV infection when used with other serological markers; assists in identifying recent HBV infection; benefits include accurate diagnosis of acute HBV, guiding clinical management.

Clinical Evidence

Multi-center clinical trial (N=1430) of prospective subjects at high risk for HBV. Compared to FDA-approved reference assays. Overall positive percent agreement 91.7% (11/12); negative percent agreement 95.3% (1350/1417). Retrospective studies: 85 acute HBV samples (100% positive agreement); 120 chronic HBV samples (93.8% negative agreement). Analytical sensitivity evaluated using PEI reference standard. Cross-reactivity tested against 357 samples from 21 medical conditions. Reproducibility established across 3 sites, 3 lots, 10 days.

Technological Characteristics

Enzyme immunoassay (EIA) using IgM antibody capture. Components: microwell strip plates coated with anti-human IgM, HRP-conjugated recombinant HBc, TMB chromogen, H2O2 substrate, 1N H2SO4 stop solution. Preservatives: ProClin, Gentamicin, Thimerosal. Spectrophotometric detection. Compatible with serum, EDTA, sodium/lithium heparin, ACD, sodium citrate plasma. Manual or automated processing.

Indications for Use

Indicated for individuals displaying signs and symptoms of hepatitis or at risk for HBV infection to detect IgM antibodies to hepatitis B core antigen in human serum and plasma. 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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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

# Summary of Safety and Effectiveness

## I. General Information

Device Generic Name: IgM Antibody to Hepatitis B Core Antigen (Anti-HBc IgM) assay
Device Trade Name: MONOLISA™ Anti-HBc IgM EIA
Applicant: Bio-Rad Laboratories
6565 185th Avenue NE
Redmond, WA 98052
Phone: [phone]
Fax: [phone]
PMA Number: P060034
Date of Panel Recommendation: N/A
Date of Notice of Approval to the Applicant: May 31, 2007

## II. Indications for Use

The MONOLISA™ Anti-HBc IgM EIA is a qualitative enzyme immunoassay for the detection of IgM antibodies to hepatitis B core antigen in human serum and plasma. The detection of IgM antibodies to hepatitis B core antigen is a significant marker for the presence of recent infection by the hepatitis B virus (HBV): 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.

## III. Device Description

The MONOLISA™ Anti-HBc IgM EIA is a qualitative enzyme immunoassay for the detection of IgM antibody to hepatitis B core antigen in human serum and plasma. The assay is based on the IgM antibody capture format. In the assay procedure, patient specimens and controls are incubated with anti-human IgM antibodies coated on the microwells. If IgM antibodies to HBc are present in a specimen or control, they bind to the antibody. Excess sample is removed by a wash step. The conjugate is then added to the microwells and allowed to incubate. The conjugate binds to any antibody-IgM antibody complexes (specific for HBc) that are present in the microwells. Excess conjugate is removed by a wash step, and a chromogen/substrate solution is added to the microwells and allowed to incubate. If a sample contains anti-HBc IgM, the bound enzyme (HRP) causes the colorless tetramethylbenzidine (TMB) in the chromogen solution to change to blue. The blue color turns yellow after the addition of a stopping solution. If a sample does not contain anti-HBc IgM, the chromogen/substrate solution in the well remains colorless during the substrate incubation, and after addition of the stopping solution. The color intensity, measured spectrophotometrically, is proportional to the amount of anti-HBc IgM present in the specimen. Absorbance value readings for patient specimens are compared to a cutoff value.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

## Components of the MONOLISA™ Anti-HBc IgM EIA:

- **Anti-HBc IgM Microwell Strip Plate:** Plates containing 96 wells coated with anti-human IgM. Preservative: ProClin (trace).
- **Wash Solution (30X):** Contains sodium chloride and Tween 20.
- **Specimen Diluent:** Buffer with protein stabilizers and red dye. Preservative: 0.5% ProClin 300®.
- **Negative Control:** Human serum non-reactive for HBsAg and antibodies to HBc, HBs, HIV-1, HIV-2, and HCV. Preservatives: 0.005% Gentamicin and 0.16% ProClin 950®.
- **Positive Control:** Human serum prepared from infectious material positive for anti-HBc IgM and HBsAg, and negative for HIV and HCV antibodies; red dye. Preservative: 0.5% ProClin 300®.
- **Cutoff Calibrator:** Human serum non-reactive for HBsAg and antibodies to HBc, HBs, HIV-1, HIV-2, and HCV. Preservatives: 0.005% Gentamicin and 0.16% ProClin 950®.
- **Conjugate:** HBc recombinant conjugated to HRP, Lyophilized. Preservative: 0.1% ProClin 300®.
- **Conjugate Diluent:** Buffer with protein stabilizers and green dye. Preservatives: 0.5% ProClin® 300 and < 0.001% Thimerosal.
- **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.

## IV. Warnings, Precautions, and Contraindications

- For *in vitro* diagnostic use only.
- 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.
- Other warnings and precautions for the MONOLISA™ Anti-HBc IgM EIA can be found in the associated product labeling.

## V. Alternative Practices and Procedures

Determination of the presence of anti-HBc IgM antibodies 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, a diagnosis of infection with HBV can be determined.

## VI. Marketing History

The MONOLISA™ Anti-HBc IgM EIA has not been commercially marketed in countries outside of the U.S.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

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

The MONOLISA™ Anti-HBc IgM EIA is intended for *in vitro* diagnostic use, and therefore there is no direct adverse effect on the patient. Failure of the product to perform as intended, or errors in the use of the product, may lead to a false result.

For diagnostic purposes, results should be used in conjunction with patient history and other hepatitis markers for diagnosis of acute and chronic infection. A false nonreactive result may be considered a public health risk if it leads to inappropriate therapy, and individuals are not tested with other methodologies to determine the presence of HBV infection. A non-reactive test result does not exclude the possibility of exposure to hepatitis B virus, and may be due to antibody levels below the detection limits of this assay.

A false reactive result is not considered a public health risk, since the immune status of subjects should be evaluated based on a combination of their clinical status, related risk factors, and other diagnostic test results.

## VIII. Summary of Preclinical Studies

### A. Analytical Sensitivity

The sensitivity of the MONOLISA™ Anti-HBc IgM EIA was evaluated by preparing serial dilutions of the Paul Ehrlich Institute (PEI) anti-HBc IgM Reference Standard. In the testing of the this standard on three kit lots, the MONOLISA™ Anti-HBc IgM EIA detected 19, 47 and 54 PEI Units/mL respectfully.

In testing of a Low Titer and a Mixed Titer Anti-HBc IgM Performance Panel, all positive samples (37/37) were correctly identified as reactive by all three kit lots tested. The detection of anti-HBc IgM in 8 commercial seroconversion panels was equivalent or better than other approved assays.

### B. Cross-Reactivity

The specificity of the MONOLISA™ Anti-HBc IgM EIA assay was evaluated by testing 357 characterized serum and plasma samples from 21 potentially cross-reacting sub-groups. Each sample was tested once on the MONOLISA™ Anti-HBc IgM EIA. Any sample that was reactive on the MONOLISA™ Anti-HBc IgM EIA was also tested on a reference Anti-HBc IgM assay. A summary of the results is given in Table 1.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

Table 1
Potentially Cross-Reactive Medical Conditions

| Clinical Condition | N = | MONOLISA™ Anti-HBc IgM Result | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | NR | Borderline | | Reactive | | |
| | | | Reference Anti-HBc IgM | | | | |
| | | | NR | R | NR | | |
| Anti-CMV IgM Pos | 20 | 18 | 0 | 1 | 0 | 1 | 20 |
| Anti-EBV IgM Pos | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Anti-HAV Pos | 20 | 19 | 0 | 0 | 0 | 1 | 20 |
| Anti-HBc Pos/IgM Neg | 10 | 8 | 0 | 0 | 0 | 2 | 10 |
| Anti-HCV Pos | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Anti-HDV Pos | 10 | 10 | 0 | 0 | 0 | 0 | 10 |
| Anti-HIV-1 Pos | 20 | 19 | 0 | 0 | 0 | 1 | 20 |
| Anti-HIV-2 Pos | 20 | 18 | 0 | 0 | 1 | 1 | 20 |
| Anti-HSV-1 IgM Pos | 14 | 14 | 0 | 0 | 0 | 0 | 14 |
| Anti-HSV-2 IgM Pos | 6 | 6 | 0 | 0 | 0 | 0 | 6 |
| Anti-HTLV Pos | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Anti-Rubella IgM Pos | 20 | 19 | 1 | 0 | 0 | 0 | 20 |
| Anti-Toxo IgM Pos | 20 | 19 | 1 | 0 | 0 | 0 | 20 |
| Elevated Liver Enzymes | 3 | 3 | 0 | 0 | 0 | 0 | 3 |
| Flu Vaccine | 20 | 19 | 0 | 0 | 1 | 0 | 20 |
| Hepatic cancer | 4 | 4 | 0 | 0 | 0 | 0 | 4 |
| Heterophile Ab Pos | 10 | 10 | 0 | 0 | 0 | 0 | 10 |
| Non-Viral Liver Disease | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Parvovirus Pos | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Rheumatoid factor Pos | 20 | 16 | 2 | 1 | 0 | 1 | 20 |
| SLE | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Syphilis Pos | 20 | 20 | 0 | 0 | 0 | 0 | 20 |
| Total | 357 | 342 | 4 | 2 | 2 | 7 | 357 |

Of the 357 samples from 21 unrelated medical conditions tested, 342/357 (95.8%) were non-reactive on the MONOLISA™ Anti-HBc IgM EIA. Of the 9 samples that were reactive, 2/9 (22.2%) were also positive on the reference Anti-HBc IgM assay. Of the 7 samples that were negative on the reference Anti-HBc IgM and reactive on the MONOLISA™ Anti-HBc IgM EIA, 2 were from the Anti-HBc positive/Anti-HBc IgM negative group, 1 was CMV IgM positive, 1 was HAV positive, 1 was HIV-1 positive, 1 was HIV-2 positive and 1 was RF positive. Six (6) samples (1 CMV IgM, 3 Rheumatoid factor, 1 Rubella IgM and 1 Toxoplasmosis positive) were borderline on the MONOLISA™ Anti-HBc IgM EIA.

## C. Interfering substances

The MONOLISA™ Anti-HBc IgM EIA was evaluated for interference according to CLSI Document EP7. The following substances, and the upper levels that were tested, did not interfere with the performance of the assay.

- Hemolyzed: 500 mg/dL of hemoglobin
- Lipemic: 500 mg/dL of triglycerides
- Icteric: 20 mg/dL of bilirubin
- Proteinemic: 11 g/dL of protein

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

## D. Stability Studies

### 1. Kit Stability

A functional stability study of the MONOLISA™ Anti-HBc IgM EIA test 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 the Anti-HBc IgM EIA, at multiple time points throughout the shelf life of the kit. Data from these studies support a 7-month dating period for the MONOLISA™ Anti-HBc IgM EIA test 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-HBc IgM EIA contains four common reagents that may be used interchangeably with the same components in other lots of the Anti-HBc IgM EIA: Wash Solution Concentrate, Chromogen, Substrate Buffer, and Stopping Solution. Matrix studies performed with the MONOLISA™ Anti-HBc IgM 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.

## E. Microbiology Studies

Antimicrobial preservatives have been added to the components in the Anti-HBc IgM EIA 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-HBc IgM EIA kit components in the presence of microbial organisms. One set of Anti-HBc IgM EIA components that were inoculated with microorganisms was tested in comparison to a reference second set of Anti-HBc IgM EIA 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 functionality of the EIA is not impaired and the reagents are stable for the 7-month shelf life when microbial contamination is present.

## F. Reproducibility

A panel consisting of 13 diluted samples in various matrices (serum, EDTA, and lithium heparin) was tested in duplicate, once a day for 10 days on 3 lots of the MONOLISA™ Anti-HBc IgM EIA at each of the 3 trial sites. 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 & 3.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

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

| Test Site | Panel Member | N | Mean S/CO | Within Run¹ | | Between Day² | | Total³ | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | SD | CV (%) | SD | CV (%) | SD | CV (%) | |
| Site #1 | 1 | Pos Defibrinated Plasma | 60 | 6.01 | 0.289 | 4.8 | 0.281 | 4.7 | 0.781 | 13.0 |
| | 2 | Neg Defibrinated Plasma | 60 | 0.31 | 0.055 | 17.8 | 0.000 | 0.0 | 0.012 | 4.0 |
| | 3 | Neg Defibrinated Plasma | 60 | 0.30 | 0.045 | 14.8 | 0.000 | 0.0 | 0.000 | 0.0 |
| | 4 | Pos Serum | 60 | 2.80 | 0.090 | 3.2 | 0.061 | 2.2 | 0.677 | 24.2 |
| | 5 | CO+20%(Serum) | 60 | 1.65 | 0.025 | 1.5 | 0.071 | 4.3 | 0.474 | 28.8 |
| | 6 | CO-20%(Serum) | 60 | 1.02 | 0.062 | 6.1 | 0.035 | 3.5 | 0.289 | 28.4 |
| | 7 | Neg (Serum) | 60 | 0.44 | 0.047 | 10.6 | 0.048 | 10.8 | 0.075 | 16.9 |
| | 8 | CO+20%(EDTA) | 60 | 1.62 | 0.049 | 3.0 | 0.055 | 3.4 | 0.444 | 27.4 |
| | 9 | CO-20%(EDTA) | 60 | 1.05 | 0.026 | 2.5 | 0.045 | 4.3 | 0.269 | 25.7 |
| | 10 | Neg (EDTA) | 60 | 0.37 | 0.042 | 11.4 | 0.000 | 0.0 | 0.038 | 10.3 |
| | 11 | CO+20%(Li Heparin) | 60 | 1.50 | 0.025 | 1.6 | 0.059 | 3.9 | 0.361 | 24.2 |
| | 12 | CO-20%(Li Heparin) | 60 | 1.01 | 0.107 | 10.6 | 0.015 | 1.5 | 0.239 | 23.6 |
| | 13 | Neg (Li Heparin) | 60 | 0.39 | 0.022 | 5.5 | 0.017 | 4.4 | 0.052 | 13.4 |
| Site #2 | 1 | Pos Defibrinated Plasma | 60 | 5.94 | 0.390 | 6.6 | 0.375 | 6.3 | 0.420 | 7.1 |
| | 2 | Neg Defibrinated Plasma | 60 | 0.30 | 0.078 | 26.3 | 0.037 | 12.4 | 0.036 | 12.0 |
| | 3 | Neg Defibrinated Plasma | 60 | 0.30 | 0.030 | 10.1 | 0.038 | 12.9 | 0.017 | 5.6 |
| | 4 | Pos Serum | 60 | 2.84 | 0.049 | 1.7 | 0.137 | 4.8 | 0.637 | 22.5 |
| | 5 | CO+20%(Serum) | 60 | 1.67 | 0.058 | 3.5 | 0.089 | 5.3 | 0.462 | 27.6 |
| | 6 | CO-20%(Serum) | 60 | 1.07 | 0.050 | 4.6 | 0.060 | 5.6 | 0.282 | 26.3 |
| | 7 | Neg (Serum) | 60 | 0.50 | 0.030 | 6.1 | 0.062 | 12.4 | 0.063 | 12.7 |
| | 8 | CO+20%(EDTA) | 60 | 1.67 | 0.042 | 2.5 | 0.122 | 7.3 | 0.429 | 25.8 |
| | 9 | CO-20%(EDTA) | 60 | 1.05 | 0.031 | 3.0 | 0.084 | 8.0 | 0.229 | 21.8 |
| | 10 | Neg (EDTA) | 60 | 0.36 | 0.025 | 7.1 | 0.047 | 13.2 | 0.028 | 7.7 |
| | 11 | CO+20%(Li Heparin) | 60 | 1.54 | 0.032 | 2.1 | 0.157 | 10.2 | 0.316 | 20.5 |
| | 12 | CO-20%(Li Heparin) | 60 | 0.99 | 0.044 | 4.4 | 0.116 | 11.7 | 0.174 | 17.5 |
| | 13 | Neg (Li Heparin) | 60 | 0.38 | 0.037 | 9.9 | 0.049 | 12.9 | 0.027 | 7.2 |
| Site #3 | 1 | Pos Defibrinated Plasma | 60 | 6.11 | 0.405 | 6.6 | 0.649 | 10.6 | 0.686 | 11.2 |
| | 2 | Neg Defibrinated Plasma | 60 | 0.31 | 0.028 | 9.1 | 0.013 | 4.2 | 0.022 | 7.0 |
| | 3 | Neg Defibrinated Plasma | 59 | 0.30 | 0.020 | 6.9 | 0.012 | 4.0 | 0.017 | 5.8 |
| | 4 | Pos Serum | 60 | 2.89 | 0.077 | 2.6 | 0.135 | 4.7 | 0.710 | 24.5 |
| | 5 | CO+20%(Serum) | 60 | 1.67 | 0.031 | 1.9 | 0.084 | 5.0 | 0.476 | 28.6 |
| | 6 | CO-20%(Serum) | 60 | 1.07 | 0.029 | 2.7 | 0.051 | 4.7 | 0.301 | 28.2 |
| | 7 | Neg (Serum) | 60 | 0.49 | 0.015 | 3.0 | 0.027 | 5.6 | 0.097 | 19.9 |
| | 8 | CO+20%(EDTA) | 60 | 1.68 | 0.032 | 1.9 | 0.106 | 6.3 | 0.428 | 25.5 |
| | 9 | CO-20%(EDTA) | 60 | 1.07 | 0.025 | 2.3 | 0.080 | 7.5 | 0.237 | 22.2 |
| | 10 | Neg (EDTA) | 60 | 0.36 | 0.040 | 11.0 | 0.034 | 9.4 | 0.036 | 9.9 |
| | 11 | CO+20%(Li Heparin) | 60 | 1.51 | 0.042 | 2.8 | 0.069 | 4.6 | 0.344 | 22.8 |
| | 12 | CO-20%(Li Heparin) | 60 | 1.01 | 0.031 | 3.1 | 0.068 | 6.7 | 0.200 | 19.9 |
| | 13 | Neg (Li Heparin) | 59 | 0.38 | 0.028 | 7.5 | 0.040 | 10.7 | 0.045 | 11.9 |

¹ Within Run: variability of the assay performance from replicate to replicate.
² Between Day: 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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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

## Table 3
Summary of MONOLISA™ Anti-HBc IgM EIA Reproducibility Results
(Positive, Low Positive, and Negative) by Panel Member S/CO

| Panel Member | N | Mean S/CO | Within Run^{1} | | Between Day^{2} | | Between Lot^{3} | | Between Site | | Total^{4} | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| Pos Serum | 180 | 2.84 | 0.074 | 2.6 | 0.116 | 4.1 | 0.675 | 23.7 | 0.043 | 1.5 | 0.690 | 24.3 |
| CO+20%(Serum) | 180 | 1.66 | 0.041 | 2.4 | 0.082 | 4.9 | 0.471 | 28.3 | 0.000^{5} | 0.0 | 0.480 | 28.9 |
| CO-20%(Serum) | 180 | 1.05 | 0.049 | 4.6 | 0.050 | 4.7 | 0.291 | 27.6 | 0.026 | 2.5 | 0.300 | 28.5 |
| Neg(Serum) | 180 | 0.48 | 0.033 | 7.0 | 0.048 | 10.0 | 0.078 | 16.4 | 0.028 | 5.8 | 0.101 | 21.2 |
| CO+20%(EDTA) | 180 | 1.66 | 0.042 | 2.5 | 0.098 | 5.9 | 0.434 | 26.2 | 0.025 | 1.5 | 0.447 | 27.0 |
| CO-20%(EDTA) | 180 | 1.06 | 0.027 | 2.6 | 0.072 | 6.8 | 0.245 | 23.2 | 0.009 | 0.9 | 0.257 | 24.4 |
| Neg(EDTA) | 180 | 0.36 | 0.036 | 10.0 | 0.034 | 9.2 | 0.036 | 9.9 | 0.000^{5} | 0.0 | 0.061 | 16.9 |
| CO+20%(Li Heparin) | 180 | 1.52 | 0.034 | 2.2 | 0.105 | 6.9 | 0.341 | 22.5 | 0.014 | 0.9 | 0.359 | 23.7 |
| CO-20%(Li Heparin) | 180 | 1.00 | 0.069 | 6.9 | 0.078 | 7.8 | 0.205 | 20.5 | 0.005 | 0.5 | 0.231 | 23.0 |
| Neg(Li Heparin) | 179 | 0.38 | 0.030 | 7.8 | 0.038 | 9.9 | 0.042 | 11.1 | 0.005 | 1.3 | 0.064 | 16.8 |

1. Within Run: variability of the assay performance from replicate to replicate.
2. Between Day: variability of the assay performance from run to run.
3. Between Lot: variability of the assay performance from lot to lot.
4. Total variability of the assay performance includes within run, between run and between lot.
5. Negative variances were rounded to zero, per statistical convention.

## G. Precision

A precision study was performed with the MONOLISA™ Anti-HBc IgM EIA using panels prepared in serum, EDTA plasma, and sodium heparin. The 13 specimens were tested in triplicate, twice a day, for 20 days, with one lot, and results are summarized in Table 4.

## Table 4
MONOLISA™ Anti-HBc EIA 20-Day Precision Results in S/CO

| Panel Member | N | Mean S/CO | Within Run^{1} | | Between Day^{2} | | Between Run^{3} | | Total^{4} | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 1 Defibrinated Plasma | 120 | 4.80 | 0.052 | 1.1 | 0.091 | 1.9 | 0.103 | 2.1 | 0.147 | 3.1 |
| 2 Defibrinated Plasma | 120 | 0.33 | 0.058 | 17.8 | 0.034 | 10.3 | 0.000^{5} | 0.0 | 0.067 | 20.6 |
| 3 Defibrinated Plasma | 120 | 0.32 | 0.025 | 7.9 | 0.011 | 3.4 | 0.012 | 3.9 | 0.030 | 9.5 |
| 4 Serum | 120 | 2.28 | 0.041 | 1.8 | 0.050 | 2.2 | 0.028 | 1.2 | 0.070 | 3.1 |
| 5 Serum | 120 | 1.27 | 0.030 | 2.3 | 0.033 | 2.6 | 0.000^{5} | 0.0 | 0.044 | 3.5 |
| 6 Serum | 120 | 0.83 | 0.020 | 2.4 | 0.016 | 1.9 | 0.012 | 1.4 | 0.028 | 3.4 |
| 7 Serum | 120 | 0.41 | 0.014 | 3.5 | 0.018 | 4.5 | 0.012 | 3.0 | 0.026 | 6.5 |
| 8 EDTA Plasma | 120 | 1.38 | 0.030 | 2.2 | 0.036 | 2.6 | 0.005 | 0.4 | 0.047 | 3.4 |
| 9 EDTA Plasma | 120 | 0.93 | 0.023 | 2.5 | 0.016 | 1.7 | 0.011 | 1.2 | 0.030 | 3.2 |
| 10 EDTA Plasma | 120 | 0.39 | 0.017 | 4.3 | 0.025 | 6.3 | 0.000^{5} | 0.0 | 0.030 | 7.6 |
| 11 Na Heparin Plasma | 120 | 1.29 | 0.030 | 2.3 | 0.046 | 3.6 | 0.000^{5} | 0.0 | 0.055 | 4.3 |
| 12 Na Heparin Plasma | 120 | 0.94 | 0.025 | 2.7 | 0.033 | 3.5 | 0.000^{5} | 0.0 | 0.041 | 4.4 |
| 13 Na Heparin Plasma | 120 | 0.44 | 0.016 | 3.6 | 0.014 | 3.2 | 0.004 | 0.8 | 0.021 | 4.9 |

1. Within Run: variability of the assay performance from replicate to replicate.
2. Between Day: variability of the assay performance from day to day.
3. Between Run: variability of the assay performance from run to run.
4. Total variability of the assay performance includes within run, between day and between run.
5. Negative variances were rounded to zero, per statistical convention.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

## IX. Summary of Clinical Studies

A multi-center clinical trial was conducted to evaluate the performance of the MONOLISA™ Anti-HBc IgM Enzyme Immunoassay (EIA) in human serum and plasma. A total of 1430 prospective subjects at high risk for viral hepatitis and/or showing signs/symptoms of HBV were included in the study. Of these 1430, 1352 were from the asymptomatic high risk population and 78 reported signs or symptoms of HBV.

## Expected Values

A total of 1352 prospective asymptomatic subjects were tested with the MONOLISA™ Anti-HBc IgM EIA. All subjects (100%) were at high risk for viral hepatitis including intravenous drug users (N = 461), homosexual males (N = 143), sex workers (N = 172), prison history (N = 340), high risk sex partners (N = 165), high risk occupation/health care workers (N = 75), hemodialysis (N = 55), hemophiliacs (N = 3), and other (N = 481). Many had more than 1 high-risk behavior or risk factor. One hundred seventy-four (174, 12.9%) 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, (34.0%); 57 from Santa Ana, CA (4.2%); 72 from Miami, FL (5.3%); 344 from Cocoa, FL (25.4%); 254 from San Francisco, CA (18.8%); and 166 from Seattle, WA (12.3%). The group was Caucasian (36.6%), Black or African American (41.1%), Hispanic or Latino (13.2%), Asian (4.3%), Native Hawaiian or other Pacific Islander (0.7%), and American Indian or Alaska Native (2.3%), with the remaining 1.9% represented by multiple ethnic groups or unknown ethnicity. The subjects were male (70.2%) and female (29.8%) and ranged in age from 18 to 81 years.

The MONOLISA™ Anti-HBc IgM EIA results for the asymptomatic prospective population (N = 1352) are presented in Table 5 by gender and age range.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

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

| Age Range | Gender | MONOLISA™ Anti-HBc IgM EIA Result | | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | Reactive | | Borderline | | Non-reactive | | |
| | | N | % | N | % | N | % | N |
| 10-19 | F | 6 | 0.4 % | 1 | 1.3 % | 7 | 0.5 % | 6 |
| | M | 10 | 0.7 % | 1 | 1.3 % | 11 | 0.8 % | 10 |
| 20-29 | F | 104 | 7.7 % | 9 | 11.5 % | 113 | 7.9 % | 104 |
| | M | 122 | 9.0 % | 9 | 11.5 % | 131 | 9.2 % | 122 |
| 30-39 | F | 108 | 8.0 % | 7 | 9.0 % | 115 | 8.0 % | 108 |
| | M | 212 | 15.7 % | 13 | 16.7 % | 225 | 15.7 % | 212 |
| 40-49 | F | 107 | 7.9 % | 5 | 6.4 % | 112 | 7.8 % | 107 |
| | M | 348 | 25.7 % | 8 | 10.3 % | 356 | 24.9 % | 348 |
| 50-59 | F | 62 | 4.6 % | 5 | 6.4 % | 67 | 4.7 % | 62 |
| | M | 210 | 15.5 % | 11 | 14.1 % | 221 | 15.5 % | 210 |
| 60-69 | F | 11 | 0.8 % | 3 | 3.8 % | 14 | 1.0 % | 11 |
| | M | 37 | 2.7 % | 4 | 5.1 % | 41 | 2.9 % | 37 |
| 70-79 | F | 2 | 0.1 % | 1 | 1.3 % | 3 | 0.2 % | 2 |
| | M | 5 | 0.4 % | 0 | 0.0 % | 5 | 0.3 % | 5 |
| 80-89 | F | 0 | 0.0 % | 0 | 0.0 % | 0 | 0.0 % | 0 |
| | M | 1 | 0.1 % | 0 | 0.0 % | 1 | 0.1 % | 1 |
| Unknown | F | 3 | 0.2 % | 1 | 1.3 % | 4 | 0.3 % | 3 |
| | M | 4 | 0.3 % | 0 | 0.0 % | 4 | 0.3 % | 4 |
| Totals | | 1352 | 100 % | 78 | 100 % | 1430 | 100 % | 1352 |

## Reference Markers

The HBV disease classification for each subject in the total prospective population (N = 1430) was previously determined by a serological assessment using a hepatitis marker profile consisting of commercially available FDA approved reference assays. 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 HBeAg (Anti-HBe), and total antibody to hepatitis B virus surface antigen (anti-HBs). All reference assays were tested according to the manufacturer’s package insert instructions. Agreement of the MONOLISA™ Anti-HBc IgM EIA was assessed relative to the reference anti-HBc result and to HBV classification.

The data were analyzed following the assignment of specimen classification based upon the positive or reactive (+) / negative or non-reactive (-) / Indeterminate (I) patterns for the six HBV reference marker assays. Table 6 below summarizes how these classification patterns were derived. No other laboratory or clinical information was used in the disease classification process. There were 33 unique reference marker patterns observed in the MONOLISA™ Anti-HBc IgM EIA clinical study across the three clinical sites.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

Table 6 Characterization of Prospective Specimens

| FDA Characterization based on single point specimen | HBsAg | HBeAg | Anti-HBc IgM | Total HBc | Anti-HBe | Anti-HBs |
| --- | --- | --- | --- | --- | --- | --- |
| Acute infection | + | + | + | + | - | - |
| | + | + | I | + | - | - |
| | + | + | - | - | - | - |
| | + | - | + | - | - | + |
| | + | - | + | - | - | - |
| | + | - | - | - | - | - |
| Chronic infection | + | + | + | + | - | + |
| | + | + | - | + | - | - |
| | + | + | - | + | - | + |
| | + | + | - | + | + | + |
| | + | + | - | + | + | - |
| | + | - | - | + | + | - |
| | + | - | - | + | + | + |
| Early recovery | - | - | + | + | + | + |
| | - | - | + | + | + | - |
| | - | - | - | + | - | - |
| | - | - | - | + | + | - |
| | - | - | - | + | I | - |
| | - | - | I | + | + | + |
| HBV vaccine response | - | - | - | - | - | + |
| HBV vaccine response (?) | - | - | - | - | - | I |
| Not previously infected with HBV | - | - | - | - | - | - |
| Recovered | - | - | - | + | I | I |
| | - | - | - | + | - | I |
| Recovered or Immune due to natural | - | - | - | + | - | + |
| Recovery | - | - | - | - | + | + |
| | - | - | - | + | I | + |
| | - | - | - | + | + | I |
| | - | - | - | + | + | + |
| Uninterpretable | + | - | - | - | + | - |
| | - | + | - | - | - | + |
| | - | + | - | - | - | - |
| | - | - | - | - | + | - |

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

## Comparison of Results by Specimen Classification

Table 7 compares the MONOLISA™ Anti-HBc IgM EIA results with the previously determined anti-HBc IgM reference assay results for each specimen classification. The data in the table are representative of the number of specimens in each result category.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

Table 7
FDA HBV Classification of High Risk Prospective Samples
MONOLISA™ Anti-HBc IgM EIA versus FDA Approved Anti-HBc IgM EIA

| Reference Serology Classification | Reference Anti-HBc IgM Assay | | | | | | | | | Totals |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Reactive | | | GRZ^{1} | | | Non-Reactive | | | |
| | MONOLISA™ Anti-HBc IgM | | | MONOLISA™ Anti-HBc IgM | | | MONOLISA™ Anti-HBc IgM | | | |
| | R | BRD^{2} | NR | R | BRD^{2} | NR | R | BRD^{2} | NR | |
| Acute Infection | 7 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 6 | 14 |
| Chronic Infection | 1 | 0 | 0 | 0 | 0 | 0 | 2 | 1 | 75 | 79 |
| Early recovery | 3 | 0 | 0 | 0 | 1 | 1 | 5 | 5 | 91 | 106 |
| HBV vaccine response | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 5 | 300 | 309 |
| HBV vaccine response (?) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 31 | 31 |
| Not previously infected with HBV | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 7 | 596 | 606 |
| Recovered | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 11 | 12 |
| Recovered or Immune due to natural infection | 0 | 0 | 0 | 0 | 0 | 0 | 7 | 2 | 83 | 92 |
| Recovery | 0 | 0 | 0 | 0 | 0 | 0 | 16 | 8 | 149 | 173 |
| Uninterpretable | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 8 |
| Total | 11 | 0 | 0 | 1 | 1 | 1 | 38 | 28 | 1350 | 1430 |

¹ GRZ = Gray Zone Reactive; ² BRD = Borderline

## Percent Agreement

The percent agreement between the MONOLISA™ Anti-HBc IgM EIA and the reference anti-HBc IgM assays for each specimen classification was determined, including the upper and lower 95% Wilson confidence bounds. The percent agreement between the MONOLISA™ Anti-HBc IgM EIA and the anti-HBc IgM reference assay for the prospective population is presented in Table 8 for each HBV classification.

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

| HBV Classification | N = | Positive Percent Agreement | | 95% Confidence Interval | Negative Percent Agreement | | 95% Confidence Interval |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Acute Infection | 14 | (7/7) | 100.0% | 64.5%, 100% | (6/7) | 85.7% | 48.6%, 97.4% |
| Chronic Infection | 79 | (1/1) | 100.0% | 20.6%, 100% | (75/78) | 96.2% | 89.3%, 98.7% |
| Early recovery | 106 | (3/4) | 75.0% | 30%, 95.4% | (91/101) | 90.1% | 82.7%, 94.5% |
| HBV vaccine response | 309 | (0/0) | NA | NA | (300/309) | 97.1% | 94.6%, 98.5% |
| HBV vaccine response (?) | 31 | (0/0) | NA | NA | (31/31) | 100.0% | 89%, 100% |
| Not previously infected with HBV | 606 | (0/0) | NA | NA | (596/606) | 98.3% | 97%, 99.1% |
| Recovered | 12 | (0/0) | NA | NA | (11/12) | 91.7% | 64.6%, 98.5% |
| Recovered or Immune due to natural infection | 92 | (0/0) | NA | NA | (83/92) | 90.2% | 82.4%, 94.8% |
| Recovery | 173 | (0/0) | NA | NA | (149/173) | 86.1% | 80.2%, 90.5% |
| Uninterpretable | 8 | (0/0) | NA | NA | (8/8) | 100.0% | 67.5%, 100% |
| Total | 1430 | (11/12) | 91.7% | 64.6%, 98.5% | (1350/1417) | 95.3% | 94%, 96.3% |

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

1 N=Total number of samples; refer to Table 3 for correlation of borderline samples. The one specimen that was indeterminate by both assays was not included in the percent agreement calculations. Positive or negative results from the MONOLISA™ Anti-HBc IgM 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-HBc IgM EIA.

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-HBc IgM EIA.

Of the 1430 samples tested, 29 samples gave borderline results with MONOLISA™ Anti-HBc IgM EIA. Three (3) of the 1430 samples were equivocal with the reference assay. One (1) of the MONOLISA™ Anti-HBc IgM EIA borderline samples was also equivocal by the reference method and 28 were negative by the reference method.

Percent agreement can also be determined by evaluating equivocal results as agreement with the reference assay. Below are the calculations of percent agreement when the borderline results by MONOLISA™ Anti-HBc IgM EIA are considered as positive results and when the borderline results by MONOLISA™ Anti-HBc IgM EIA are considered as negative results.

| MONOLISA™ Anti-HBc IgM EIA | Positive Agreement | Negative Agreement |
| --- | --- | --- |
| Borderline considered positive | 100.0% (11/11) | 95.3% (1350/1460) |
| Borderline considered negative | 100.0% (11/11) | 97.3% (1378/1416) |

## Seroconversion Panels

The comparative sensitivity of the MONOLISA™ Anti-HBc IgM EIA was determined by testing 6 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 | # Members | MONOLISA™ Anti-HBc IgM | | Reference anti-HBc IgM test |
| --- | --- | --- | --- | --- | --- |
| | | | S/CO | Result | Result |
| PHM935A-11 | 35 | 18 | 0.23 | NR | NR |
| PHM935A-12 | 50 | | 0.22 | NR | NR |
| | 66 | | | | NR |
| PHM935A- | | | 1.83 | R | NR |

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

| Panel ID | Day since 1^{st} bleed | # Members | MONOLISA™ Anti-HBc IgM | | Reference anti-HBc IgM test
Result |
| --- | --- | --- | --- | --- | --- |
| | | | S/CO | Result | |
| 13 | | | | | |
| PHM935A-14 | 68 | | 3.02 | R | R |
| RP009-06 | 31 | 20 | 0.45 | NR | NR |
| RP009-07 | 36 | | 2.20 | R | NR |
| RP009-08 | 43 | | 5.46 | R | R |
| RP009-09 | 56 | | 5.76 | R | R |
| RP016-06 | 25 | 20 | 0.27 | NR | NR |
| RP016-07 | 57 | | 0.67 | NR | NR |
| RP016-08 | 60 | | 3.90 | R | R |
| RP016-09 | 74 | | 5.34 | R | R |
| RP017-13 | 65 | 30 | 0.37 | NR | NR |
| RP017-14 | 71 | | 0.54 | NR | NR |
| RP017-15 | 76 | | 1.14 | R | NR |
| RP017-16 | 78 | | 2.26 | R | R |
| RP017-27 | 179 | | 1.94 | R | R |
| RP017-28 | 181 | | 1.72 | R | NR |
| RP017-29 | 186 | | 1.76 | R | NR |
| RP017-30 | 188 | | 1.51 | R | NR |
| 6278-08 | 26 | 11 | 0.28 | NR | NR |
| 6278-09 | 33 | | 0.55 | NR | NR |
| 6278-10 | 37 | | 0.98 | BRD | NR |
| 6278-11 | 41 | | 3.97 | R | R |
| 6281-08 | 36 | 12 | 0.29 | NR | NR |
| 6281-09 | 41 | | 1.30 | R | NR |
| 6281-10 | 43 | | 3.41 | R | R |
| 6281-11 | 50 | | 5.02 | R | R |

In 4 of the 6 (66%) seroconversion panels, the MONOLISA™ Anti-HBc IgM EIA detected reactive levels of hepatitis B core IgM antibody 1 member before the reference anti-HBc IgM test. In 2 of the 6 (33%) seroconversion panels, the MONOLISA™ Anti-HBc IgM EIA detected reactive levels of hepatitis B core IgM antibody at the same member as the reference anti-HBc IgM test. MONOLISA™ Anti-HBc IgM EIA appears to detect IgM for a longer period than the reference assay for qualitative determination of IgM antibody to Hepatitis B core

## Clinical Performance with Acute HBV Samples

Retrospective acute HBV samples from 85 individuals were tested with the MONOLISA™ Anti-HBc IgM EIA and a reference anti-HBc IgM EIA. All testing was according to the protocol and the manufacturer’s package insert instructions. The results of the MONOLISA™ Anti-HBc IgM EIA are compared to results of the reference anti-HBc IgM method in Table 10.

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Summary of Safety and Effectiveness

Table 10
Acute HBV Sample Results
MONOLISA™ Anti-HBc IgM EIA versus Reference Anti-HBc IgM EIA

| MONOLISA™
Anti-HBc IgM Result | Reference Anti-HBc IgM Result | | | |
| --- | --- | --- | --- | --- |
| | Positive | Gray zone | Negative | Total |
| Reactive | 66 | 13 | 4 | 83 |
| Borderline | 0 | 0 | 0 | 0 |
| Non-Reactive | 0 | 0 | 2 | 2 |
| Total | 66 | 13 | 6 | 85 |

The positive percent agreement with the reference method is 100% (66/66) with a 95% confidence interval of 94.5 - 100%. The negative percent agreement with the reference method is 10.5% (2/19) with a 95% confidence interval of 2.9 - 31.4%. Of the 17 discrepant results, the MONOLISA™ Anti-HBc IgM EIA was reactive on 13 samples with gray zone reactive results on the reference assay and 4 samples that were negative on the reference assay.

## Clinical Evaluation of the MONOLISA™ Anti-HBc IgM EIA on Chronic HBV Samples

Retrospective chronic HBV samples (HBsAg positive for more than 6 months) from 120 individuals were tested with the MONOLISA™ Anti-HBc IgM EIA and a reference anti-HBc IgM EIA. All testing was according to the protocol and the manufacturer’s package insert instructions. The results of the MONOLISA™ Anti-HBc IgM EIA are compared to results of the reference anti-HBc IgM method in Table 11.

Table 11
Chronic HBV Sample Results
MONOLISA™ Anti-HBc IgM EIA versus Reference Anti-HBc IgM EIA

| MONOLISA™
Anti-HBc IgM Result | Reference Anti-HBc IgM Result | | | |
| --- | --- | --- | --- | --- |
| | Positive | Gray zone | Negative | Total |
| Reactive | 1 | 3 | 3 | 7 |
| Borderline | 0 | 0 | 1 | 1 |
| Non-Reactive | 0 | 6 | 106 | 112 |
| Total | 1 | 9 | 110 | 120 |

The negative percent agreement with the reference method is 93.8% (106/113) with a 95% confidence interval of 87.8 - 97%. The positive percent agreement with the reference method is 14.3% (1/7) with a 95% confidence interval of 2.6 - 51.4%. Of the 13 discrepant results, the MONOLISA™ Anti-HBc IgM EIA was reactive on 3 and non-reactive on 6 of the 9 samples with gray zone reactive results on the reference assay. The MONOLISA™ Anti-HBc IgM EIA was reactive on 3 and borderline on 1 of the 4 samples that were negative on the reference assay.

## Clinical Performance with Pre-HBV Vaccination Samples

Pre-vaccine samples from 38 individuals were tested on one lot of the MONOLISA™ Anti-HBc IgM EIA and a reference anti-HBc IgM EIA. All testing was according to the protocol and the

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Summary of Safety and Effectiveness

manufacturer’s package insert instructions. The results of the MONOLISA™ Anti-HBc IgM EIA are compared to results of the reference anti-HBc IgM method in Table 12.

Table 12
Pre-Vaccination Sample Results
MONOLISA™ Anti-HBc IgM EIA versus Reference Anti-HBc IgM EIA

| MONOLISA™
Anti-HBc IgM Result | Reference Anti-HBc IgM Result | | | |
| --- | --- | --- | --- | --- |
| | Positive | Gray zone | Negative | Total |
| Reactive | 0 | 0 | 0 | 0 |
| Borderline | 0 | 0 | 0 | 0 |
| Non-Reactive | 0 | 0 | 38 | 38 |
| Total | 0 | 0 | 38 | 38 |

The negative percent agreement with the reference method is 100% (38/38) with a 95% confidence interval of 90.8 - 100%.

## X. Conclusions Drawn from the Studies

Multi-centered clinical and non-clinical studies were conducted in the US to evaluate the MONOLISA™ Anti-HBc IgM EIA. A method comparison was performed with a commercially available FDA-approved assay to detect IgM antibodies to hepatitis B core antigen 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-HBc IgM EIA kits which are stored as indicated (2-8°C) are stable for the intended shelf-life of the kits.

Hepatitis B virus classification using the prospective population showed 33 unique reference marker patterns. The overall positive percent agreement between the MONOLISA™ Anti-HBc IgM EIA and the reference assay was 91.7% (11/12) in the high risk, signs and symptoms, and vaccinated populations. The overall negative percent agreement between the MONOLISA™ Anti-HBc IgM EIA and the reference assay was 95.3% (1350/1417) in the same population.

In a study of 85 retrospective acute HBV samples, the positive agreement was 100% (66/66) with the comparison method. In another study with 120 retrospective chronic HBV samples (HBsAg positive for more than 6 months), the negative percent agreement with the comparison method was 93.8% (106/113).

Precision and reproducibility of the MONOLISA™ Anti-HBc IgM EIA was established for within-run, between-day, between-lot, and between sites.

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

Specimen collection tube study results support the use of human serum and plasma (EDTA, sodium and lithium heparin, ACD, or sodium citrate) with the MONOLISA™ Anti-HBc IgM EIA.

The results from both the non-clinical and clinical studies indicate that the MONOLISA™ Anti-HBc IgM EIA can be used safely and effectively for the qualitative determination of anti-HBc IgM 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-HBc IgM 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.

## SAFETY

Based on the results of the preclinical and clinical laboratory studies, the MONOLISA™ Anti-HBc IgM 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-HBc IgM EIA has been demonstrated for use in determining if IgM antibodies to the hepatitis B core antigen are present in an individual’s serum or plasma. A reasonable determination of effectiveness of the MONOLISA™ Anti-HBc IgM EIA assay for aiding in the diagnosis 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

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Bio-Rad MONOLISA™ Anti-HBc IgM EIA
Summary of Safety and Effectiveness

The applicant's manufacturing facilities at Seattle and Woodinville were inspected on May 25 and May 31 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.

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