The iDart™ Lyme IgG ImmunoBlot Kit is an immunoblot assay intended for the in vitro qualitative detection of IgG antibodies to Borrelia burgdorferi in human serum. The iDart Lyme IgG ImmunoBlot Kit is intended to detect antibodies to LSA and multiple other B. burgdorferi antigens following a modified two-tier test methodology. Positive results from the iDart Lyme IgG ImmunoBlot Kit are supportive evidence for the presence of antibodies and exposure to B. burgdorferi. Negative results do not preclude infection with B. burgdorferi. iDart™ Lyme IgG ImmunoBlot Kit is intended to aid in the diagnosis of Lyme disease and the test kit should only be used on samples from patients with clinical history, signs and symptoms consistent with Lyme disease. The iDart Lyme IgG Immunoblot Kit is not intended as a screen for asymptomatic patients.

Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures.

For in vitro diagnostic use only
For professional use only
For prescription use only

Device Description

The iDart™ Lyme IgG ImmunoBlot tests are line immunoblot assays. Antigenic proteins specific for Borrelia species that cause Lyme Disease are produced by recombinant DNA technology in Escherichia coli. The purified proteins are then applied as discrete lines on a nitrocellulose membrane along with two control proteins.

The iDart™ Lyme IgG ImmunoBlot Kit contains IgG ImmunoBlot strips and the proteins are applied in the following order: C1 (lgG/lgM - conjugate control), C2 (Protein L - calibrator/serum control), P93, P41, P39, P23, P31, P66, P58, P45, P34, P30, P28, P18 and LSA (a chimeric VISE peptide termed the Lyme Screen Antigen).

AI/ML Overview

Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria for the iDart™ Lyme IgG ImmunoBlot Kit are primarily demonstrated through its analytical performance (reproducibility, analytical specificity, cross-reactivity, interference) and clinical performance (method comparison with STTT, clinical sensitivity/specificity using a CDC panel).

Acceptance Criteria Category	Specific Metric / Evaluation	Acceptance Threshold (Implied/Explicit)	Reported Device Performance (as stated)
Analytical Performance
Reproducibility	Agreement across sites, operators, runs, days	100% agreement expected	100% agreement of all bands among all runs, all days and across 3 sites for negative, moderate negative, high negative, moderate positive, and high positive samples (Table 1).
Analytical Specificity (Endemic)	Specificity in healthy individuals from endemic areas	High specificity	99.36% (2 false positives out of 313 samples from CDC and Bay Area Lyme Foundation) (Table 2).
Analytical Specificity (Non-Endemic)	Specificity in healthy individuals from non-endemic areas	High specificity	100% (0 false positives out of 112 samples from CDC and CA) (Table 3).
Cross-Reactivity	False positivity with various conditions	Low/no cross-reactivity	100% specificity for LSA, 98.67% specificity for ≥2 bands, and 100% specificity for IgG Positive across 376 potentially cross-reactive samples from various disease states and infections (Table 4). Minor false positives (5 for ≥2 bands) were noted but resulted in 0% IgG positive or only a single band out of the two required for positivity.
Interference	Effect of endogenous analytes	No interference	No interference observed for bilirubin, albumin, cholesterol, triglycerides, and hemoglobin at specified low and high concentrations on positive, low positive, and negative Borrelia IgG samples (Table 5).
Clinical Performance
Method Comparison (STTT)	Positive Percent Agreement (PPA) with STTT	High PPA and NPA	Bay Area Lyme Foundation (n=290): PPA: 95.00% (95% CI: 76.39% – 99.11%), NPA: 86.67% (95% CI: 82.09% – 90.21%) (Table 7) IGeneX Inc. Cohort 2 (n=248): PPA: 95.00% (95% CI: 89.52% – 97.69%), NPA: 90.63% (95% CI: 84.33% - 94.56%) (Table 8) IGeneX Inc. Cohort 3 (n=230): PPA: 90.91% (95% CI: 62.27% – 98.38%), NPA: 96.80% (95% CI: 93.55% – 98.44%) (Table 9)
Clinical Sensitivity	Performance against CDC Reference Panel	High sensitivity for later stages	Stage I: 58.33% (higher than STTT at 30.00%) Stage II: 90.00% (equal to STTT) Stage III: 100% (equal to STTT) Overall: 71.11% (higher than STTT at 52.22%) (Table 10).
Clinical Specificity	Performance against CDC Reference Panel	High specificity	Healthy controls: 100% (equal to STTT) Disease Controls: 100% (equal to STTT) (Table 10).
Fresh and Frozen Sample Comparability	Consistent results between fresh and frozen samples	Consistent results	All IgG positive samples remained positive and all negative samples remained negative after freezing (Table 11).
Antibody Class Specificity	Specificity of anti-human IgG conjugate	Specific to IgG	All positive samples tested without treatment or with human IgM remained positive, and all negative samples remained negative. When treated with human IgG, all positive samples became negative, confirming specificity (Table 12).

2. Sample Size Used for the Test Set and Data Provenance

Reproducibility: 90 samples for each of the 6 sample types (High Positive, Moderate Positive, Negative-1, Negative-2, Negative-3, Low Positive). Total of 540 tests performed across 3 sites, 2 operators, 5 days, 2 runs/day. Data provenance is not explicitly stated beyond "blinded and coded samples."
Analytical Specificity (Healthy Individuals):
- 313 samples from endemic areas (CDC, Bay Area Lyme Foundation - NY, MA, WI).
- 112 samples from non-endemic areas (CDC, CA).
Cross-Reactivity Study: 376 samples from various disease states/infections (CDC, IGeneX (CA), New York Biological (NY), BEI, Kamineni Life Sciences Pvt. Ltd, Hydrabad (India), Warde Medical Laboratory (MI)).
Interference Study: One positive, one low positive, and one negative Borrelia IgG sample were used for each interference agent and concentration, tested in singlicate.
Clinical Studies (Method Comparison with STTT): A total of 768 serum samples.
- Site 1: 290 clinical serum samples from Bay Area Lyme Foundation.
- Site 2: 37 clinical serum samples (Cohort 2) + 230 clinical serum samples (Cohort 3) from IGeneX Inc.
- Site 3: 211 clinical serum samples (Cohort 2) from IGeneX Inc.
- Data provenance: "procured from two vendors" (Bay Area Lyme Foundation and IGeneX Inc.). Samples were "prospective banked samples" or "clinical serum samples."
Clinical Sensitivity/Specificity (CDC Serum Panel): 280 serum samples from CDC (patients with Lyme disease at different stages, look-alike conditions, healthy controls from endemic and non-endemic regions).
Fresh and Frozen Samples Comparison Study: 72 decoded left-over patient serum samples.
Antibody Class Specificity: 10 previously tested patient samples (6 negatives, 4 positives).

All clinical samples were "blinded, re-coded."

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document does not explicitly state that experts were used to establish ground truth for the test set derived from clinical samples. Instead, the ground truth for these clinical performance studies appears to be based on:

STTT (Standard Two-Tier Test Methodology): This is a laboratory-based diagnostic algorithm involving an EIA/IFA screen followed by an immunoblot. Its results serve as the comparator (ground truth) for the method comparison study.
CDC Reference Panel: For the clinical sensitivity/specificity, the CDC reference panel implicitly has established diagnoses for Lyme disease stages and other conditions. The process by which CDC established these diagnoses (e.g., expert consensus, other gold standards) is not detailed here.

For the reproducibility study, the samples were "blinded and coded" with "expected result" (e.g., High Positive, Negative). It's not specified how these initial categorizations were established.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method involving multiple experts resolving discrepancies for the test set results. The evaluations are primarily against established comparators like STTT or reference panels (CDC). For the reproducibility study, the agreement was 100%, so no adjudication would have been required for discrepancies.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and Effect Size

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not done. The performance studies focus on the device's standalone accuracy against existing diagnostic methods/reference panels, rather than how human readers' performance might improve with or without AI assistance. The device is an ImmunoBlot Kit, not an AI-assisted diagnostic imaging or interpretation system.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) Was Done

Yes, the studies reflect the standalone performance of the iDart™ Lyme IgG ImmunoBlot Kit. The "Principle of procedures" describes manual interpretation ("A strip reading guide included in each test kit shows the location of specific antigens in the test strip. ... Any band found having a visual intensity equal to or greater than the C2 control band intensity is considered as a significant (positive) band. Depending on the observed bands pattern, one can interpret the presence or absence of Lyme specific IgG antibodies in the patient serum."). The "Result Generation" for the device is listed as "Manual reading" in the comparison table. This indicates the studies assess the kit's performance as a laboratory test, interpreted by a human, but without a "human-in-the-loop" AI system.

7. The Type of Ground Truth Used

Clinical Performance (Method Comparison): The "ground truth" was established by Standard Two-Tier Test Methodology (STTT), which involves FDA-cleared EIA and immunoblot tests, performed by laboratory personnel.
Clinical Sensitivity/Specificity: The "ground truth" was established by the CDC Reference Panel, which represents diagnosed cases of Lyme disease at various stages, look-alike conditions, and healthy controls. The methods for establishing these CDC diagnoses are not detailed but likely involve a combination of clinical assessment and established laboratory criteria.
Analytical Specificity / Cross-reactivity: Ground truth for these samples was "known to contain potentially cross-reactive antibodies to Lyme infection" or "healthy individuals" or specific disease states, implying prior clinical diagnoses or sample characterization.
Reproducibility: Samples were initially characterized as "High Positive," "Negative," etc., which served as the expected result for comparison. The method for this initial characterization is not specified.
Fresh/Frozen & Antibody Class Specificity: Ground truth was based on "previously tested patient samples" with known IgG status.

8. The Sample Size for the Training Set

The document does not describe a "training set" in the context of machine learning or AI. The iDart™ Lyme IgG ImmunoBlot Kit is an immunoassay kit, not an AI-based device that requires model training. Therefore, this question is not applicable to the information provided.

9. How the Ground Truth for the Training Set Was Established

As the device is not an AI/ML product, there is no "training set" or corresponding ground truth establishment process described in the document.

Summary

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ID-FISH Technology, Inc. Jyotsna Shah VP of Research and Development 556 Gibraltar Drive Milpitas, California 95035

August 12, 2024

Re: K233367

Trade/Device Name: iDart Lyme IgG ImmunoBlot Kit Regulation Number: 21 CFR 866.3830 Regulation Name: Treponema Pallidum Treponemal Test Reagents Regulatory Class: Class II Product Code: LSR Dated: July 5, 2024 Received: July 8, 2024

Dear Jyotsna Shah:

We have reviewed your section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (the Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database available at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Additional information about changes that may require a new premarket notification are provided in the FDA guidance documents entitled "Deciding When to Submit a 510(k) for a Change to an Existing Device" (https://www.fda.gov/media/99812/download) and "Deciding When to Submit a 510(k) for a Software Change to an Existing Device" (https://www.fda.gov/media/99785/download).

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Your device is also subject to, among other requirements, the Quality System (QS) regulation (21 CFR Part 820), which includes, but is not limited to, 21 CFR 820.30, Design controls; 21 CFR 820.90, Nonconforming product; and 21 CFR 820.100, Corrective and preventive action. Please note that regardless of whether a change requires premarket review, the QS regulation requires device manufacturers to review and approve changes to device design and production (21 CFR 820.30 and 21 CFR 820.70) and document changes and approvals in the device master record (21 CFR 820.181).

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR Part 803) for devices or postmarketing safety reporting (21 CFR Part 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safetyreporting-combination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR Part 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR Parts 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Noel J. Gerald -S

Noel J. Gerald, Ph.D. Branch Chief Bacterial Respiratory and Medical Countermeasures Branch Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K233367

Device Name iDart™ Lyme IgG ImmunoBlot Kit

Indications for Use (Describe)

Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures.

For in vitro diagnostic use only For professional use only For prescription use only

Type of Use (Select one or both, as applicable)

Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)
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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION SUMMARY

BACKGROUND INFORMATION: l.

A. 510(k) Number

K233367

B. Applicant

ID-FISH Technology, Inc. 556 Gibraltar Drive Milpitas CA 95035 USA

C. Contact Person

Dr. Jyotsna Shah VP of Research and Development regulatory@idfishtechnology.com 1-650-269-8610

D. Date Prepared

July 30, 2024

E. Proprietary and Established Names

iDart™ Lyme IqG ImmunoBlot Kit

F. Regulatory Information

Product Code(s): LSR - Reagents, Borrelia Serological Reagent Classification: Class II Regulation Section: 21 CFR 866.3830 - Treponema Pallidum Treponemal Test Reagents Panel: MI - Microbiology

II. SUBMISSION/DEVICE OVERVIEW:

A. Purpose for Submission:

To obtain a substantial equivalence determination for a new device.

B. Measurand:

lgG antibodies to Borrelia burgdorferi (B. burgdorferi)

C. Type of Test:

ImmunoBlot Assay

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III. INTENDED USE/INDICATIONS FOR USE:

A. Intended Use(s):

The iDart™ Lyme IgG ImmunoBlot Kit is an immunoblot assay intended for the in vitro qualitative detection of lgG antibodies to Borrelia burgdorferi in human serum. The iDart Lyme IgG ImmunoBlot Kit is intended to detect antibodies to LSA and multiple other B. burgdorferi antigens following a modified two-tier test methodology. Positive results from the iDart Lyme IgG ImmunoBlot Kit are supportive evidence for the presence of antibodies and exposure to B. burgdorferi. Negative results do not preclude infection with B. burgdorferi. iDart™ Lyme IgG lmmunoBlot Kit is intended to aid in the disease and the test kit should only be used on samples from patients with clinical history, signs and symptoms consistent with Lyme disease. The iDart Lyme IgG Immunoblot Kit is not intended as a screen for asymptomatic patients.

Test results are to be used in conjunction with information obtained from the patient's clinical evaluation and other diagnostic procedures.

B. Indication(s) for Use:

iDart™ Lyme IgG ImmunoBlot Kit should be used for diagnosing Lyme Disease in patients with a clinical history, and signs and symptoms consistent with Lyme Disease. It is not intended for use in asymptomatic patients.

C. Special Conditions for Use Statement(s):

For in vitro diagnostic use only For professional use only For prescription use only

IV. DEVICE CHARACTERISTICS:

A. Device Description:

B. Principle of procedures:

During the test procedure, diluted human serum is added to the immunoblot strip. Antibodies to Borrelia infection, if present, will bind to antigen lines on the nitrocellulose strips. After removing serum and unbound antibodies by washing, the nitrocellulose strip is incubated with alkaline phosphatase conjugated anti-human IgG antibody for detecting IgG antibodies.

After removing the unbound alkaline phosphatase conjugated antibody by washinq, the antibody complex is visualized as bands by adding the alkaline phosphatase substrate 5-bromo, 4-chloro, 3indolylphosphate (BCIP) and nitro blue tetrazolium (NBT) to form a strong bluish-purple precipitate. The reaction is stopped by washing the nitrocellulose strip with distilled or deionized water. A strip reading guide included in each test kit shows the location of specific antigens in the test strip. Every immunoblot strip has two functional control bands. The test strip is only valid when both control bands are visible after completion of the test. Any band found having a visual intensity equal to or greater than the C2 control band intensity is considered as a significant (positive) band. Depending on the observed bands pattern, one can interpret the presence or absence of Lyme specific IgG antibodies in the patient serum.

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SUBSTANTIAL EQUIVALENCE INFORMATION: V.

A. Predicate Device Name(s):

Viramed Borrelia All-In-One ViraChip Test Kit

B. Predicate 510(k) Number(s):

K220016

C. Comparison with Predicate(s):

Item	Device:	Predicate:
	iDart Lyme IgG ImmunoBlot Kit	Viramed Borrelia All-In-One ViraChip Test Kit(K220016)
Similarities
IntendedUse	The iDartTM Lyme IgG ImmunoBlot Kit is animmunoblot assay intended for the in vitroqualitative detection of IgG antibodies to Borreliaburgdorferi in human serum. The iDart Lyme IgGImmunoBlot Kit is intended to detect antibodies toLSA and multiple other B. burgdorferi antigensfollowing a modified two-tier test methodology.Positive results from the iDart Lyme IgGImmunoBlot Kit are supportive evidence for thepresence of antibodies and exposure to B.burgdorferi. Negative results do not precludeinfection with B. burgdorferi. iDartTM Lyme IgGImmunoBlot Kit is intended to aid in the diagnosisof Lyme disease and the test kit should only beused on samples from patients with clinicalhistory, signs and symptoms consistent with Lymedisease. The iDart Lyme IgG Immunoblot Kit is notintended as a screen for asymptomatic patients.Test results are to be used in conjunction withinformation obtained from the patient's clinicalevaluation and other diagnostic procedures.Test results are to be used in conjunction withinformation obtained from the patient's clinicalevaluation and other diagnostic procedures.For in vitro diagnostic use onlyFor professional use onlyFor prescription use only	The Viramed Biotech AG Borrelia All-In-One ViraChipis an in vitro qualitative microarrayassay for the detection of IgM and IgG antibodies toBorrelia burgdorferi in human serum. The assay isintended for testing serum samples from symptomaticpatients or those suspected of Lyme Disease. It isintended to detect antibodies to VIsE and multipleother B. burgdorferi antigens following a modified two-tier test methodology. Positive results from theViramed Biotech AG Borrelia All-In-One ViraChip aresupportive evidence for the presence of antibodiesand exposure to B. burgdorferi, the causative agentfor Lyme disease. Negative results do not precludeinfection with B. burgdorferi. Test results are to beused in conjunction with information obtained from thepatient's clinical evaluation and other diagnosticprocedures as an aid in diagnosis of Lyme disease.The Viramed Biotech AG Borrelia All-In-One ViraChipTest must be used with a ViraChipReader and the ViraChip Software.
SpecimenType	Serum	Serum
AntibodiesDetected	IgG	IgM and IgG
Controls	Positive Control serum, Negative Control Serum	Positive Control serum, Negative Control Serum
Method	Qualitative	Qualitative

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Differences
ltem	Device	Predicate
AssayTechnology	ImmunoAssay	Antigen Coated wells(Microarrays)
Antigens	Recombinant antigenic proteins -LSA antigen (chimeric VIsE peptide termed theLyme Screen Antigen), 93 kD, 66 kD, 58 kD, 45kD, 41 kD, 39 kD, 34 kD, 31 kD, 30 kD, 28 kD, 23kD and 18 kD.	VlsE, 93 kD, 58 kD, 45kD, 39 kD, 30 kD, 23kD, 21 kD, 19 kD, 18kD, and 17 kD antigens of B.burgdorferi
Procedure	Line Blot Assay BorreliaIgG antibodies to specific antigen bands.Wash between sampleand conjugate incubation steps,incubate with substrate	Wash after Sample andConjugate Step
SampleVolume	10 µl neat serum in 1000 ml sample diluent	Samples diluted 1:76 and 100 µL added per well
Reagents	Sample diluent, Wash Buffer, Milk powder,Conjugate Buffer, Substrate solution	10X Wash Buffer, Sample Buffer,Chromogen/Substrate Solution
ResultGeneration	Manual reading	Automated with ViraChip Reader

STANDARDS/GUIDANCE DOCUMENTS REFERENCED: VI.

Establishing the Performance Characteristics of In Vitro Diagnostic Devices for the Detection of Antibodies to Borrelia burgdorferi - Guidance for Industry and FDA Staff - MARCH 2013

PERFORMANCE CHARACTERISTICS (IF/WHEN APPLICABLE): VII.

A. Analytical Performance:

1. Reproducibility:

The iDart™ Lyme IgG ImmunoBlot Kit was tested in a blind study to evaluate reproducibility across 3 separate sites each with 2 operators over 5 days, 2 runs a day, using a panel of blinded and coded samples of negative, moderate negative, high negative, moderate positive and high positive samples for IgG. It is concluded that there was 100% agreement of all bands among all runs, all days and across 3 sites for the iDart™ Lyme IgG ImmunoBlot Kit (See Table 1).

Sample #	Sample Type	Number ofBandsPresent/sample	lgG	# of Samples(+)	ExpectedResult	% samplesmatched toexpected result
GA	High Positive	13	P	90/90	P	100%
GB	Moderate Positive	10	P	90/90	P	100%
GC	Negative-1	5	N	0/90	N	100%
GD	Negative-2	0	N	0/90	N	100%
GE	Negative-3	1	N	0/90	N	100%
GF	Low Positive	5	P	90/90	p	100%

Table 1. Reproducibility Study Summary - Overall - All Sites - 6 operators/5 days/ 3 replicates

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2. Analytical Specificity/Interference:

Analytical Specificity

Table 2 and 3 below shows the results of testing iDart™ Lyme IgG ImmunoBlot Kit on samples collected from a population of 313 apparently healthy individuals from endemic areas, and 112 samples collected from healthy individuals in non-endemic areas in the US.

Source	N (313)	IgG Positive
CDC	62	0
BAY AREA LYME FOUNDATION(NY, MA, WI)	251	2
TOTAL	313	2
Specificity		99.36%

Table 2: iDart Lyme IgG ImmunoBlot results for samples collected from healthy individuals in endemic areas.

Table 3: iDart Lyme IgG ImmunoBlot results for samples collected from healthy individuals in non-endemic areas.

Source	N (112)	IgG Positive
CDC	62	0
CA	50	0
TOTAL	112	0
Specificity		100%

Cross-Reactivity Study

A cross reactivity study was performed on specimens known to contain potentially cross-reactive antibodies to Lyme infection. Serum samples from patients with bacterial/viral infections and sera from patients with diagnoses that can be confused with the late manifestations of Lyme disease were tested. Based on the data presented in Table 4, there was no cross-reactivity with antibodies to all non- Borrelia pathogens or autoimmune diseases tested with the iDart™ Lyme IgG ImmunoBlot Kit.

Table 4: iDart™ Lyme IgG ImmunoBlot Kit - Cross Reactivity
	Disease State		IgG			% Cross-
Source		N (376)	LSA	2+Bands	IgGPositive	reactivity
	Fibromyalgia	15	0	0	0	0%
	Mononucleosis	15	0	1	0	0%
CDC	Multiple sclerosis	15	0	0	0	0%
	Rheumatoid arthritis	15	0	0	0	0%
	Severe periodontitis	15	0	0	0	0%
	Syphilis	15	0	0	0	0%
	Babesiosis	28	0	0	0	0%
	Bartonellosis	48	0	0	0	0%
IGeneX (CA)	Ehrlichiosis	5	0	0	0	0%
	Anaplasmosis	7	0	0	0	0%
	Rickettsiosis	22	0	0	0	0%
	Tick Borne Relapsing Fever	14	0	0	0	0%
	HIV*	12	0	0	0	0%
	RPR	23	0	2	0	0%
New York Biological	HSV1	8	0	1	0	0%
(NY)	HSV2	2	0	0	0	0%
	CMV	13	0	0	0	0%
	EBV	27	0	0	0	0%
	RSV	4	0	0	0	0%
BEI	FLU	21	0	0	0	0%
Kamineni Life Sciences Pvt.	Pregnant women	12	0	0	0	0%

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Ltd, Hydrabad (India)	H. pylori	10	0	0	0	0%
Warde Medical Laboratory(MI)	Parvovirus-19	10	0	0	0	0%
CDC	Leptospira	10	0	0	0	0%
False Positive			0	5	0	0%
Specificity			100%	98.67%	100%

Interference from Endogenous Analytes

The potential interfering effect of endogenous substances in patient samples when using the iDart Lyme IgG ImmunoBlot was evaluated using one positive, one low positive and one negative Borrelia IgG samples were spiked with the endogenous substances at the final concentrations listed in the table below. All samples were tested in singlicate. No interference was observed in he tested samples.

Table 5. Effect of Interference Substances on iDart™ Lyme IgG ImmunoBlot Kit

Agent	Concentration inserum	iDart™ Lyme IgG result			Effect on ImmunoBlotKit
		High Positive	Low Positive	Negative
Bilirubin	1mg/dL (low)	Positive	Positive	Negative	No effect
Bilirubin	15mg/dL (high)	Positive	Positive	Negative	No effect
Albumin	3.5g/dL (low)	Positive	Positive	Negative	No effect
Albumin	5g/dL (high)	Positive	Positive	Negative	No effect
Cholesterol	150mg/dL (low)	Positive	Positive	Negative	No effect
Cholesterol	250mg/dL (high)	Positive	Positive	Negative	No effect
Triglycerides	150mg/dL (low)	Positive	Positive	Negative	No effect
Triglycerides	500mg/dL (high)	Positive	Positive	Negative	No effect
Hemoglobin	10g/dL (low)	Positive	Positive	Negative	No effect
Hemoglobin	20g/dL (low)	Positive	Positive	Negative	No effect

1. Assay Reportable Range: Not applicable.
1. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods): Not applicable.
1. Detection Limit: Not applicable.
1. Assay Cut-Off: Not applicable.

B. Clinical Studies:

1. Method Comparison with comparators (STTT):
  The performance of the iDart™ Lyme lgG ImmunoBlot Kit for detection of Borrelial-specific antibodies was compared to FDA-cleared ElA and immunoblot as part of the standard two-tier test methodology (STTT) .Results are summarized below. A total of 768 serum samples were procured from two vendors and tested at three clinical sites. Table 6 bellow summarizes the distribution of samples per testing site and cohort.

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Table 6: Sample distribution by clinical site and cohort.

	Number ofSamples	Sample Type	Vendors ProvidingSamples
Site 1	290	Clinical serum samples	Bay Area Lyme Foundation
Site 2	37	Clinical serum samples - Cohort 2	IGeneX Inc.
Site 2	230	Clinical serum samples - Cohort 3	IGeneX Inc.
Site 3	211	Clinical serum samples - Cohort 2	IGeneX Inc.

All samples were blinded, re-coded, and tested at the respective clinical sites as per the instructions for use for the iDart Lyme IgG ImmunoBlot Kit. Performance by cohort is summarized in tables 7 through 9.

Table 7: Performance Summary on prospective banked samples from Bay Area Lyme Foundation (n=290). iDart Lyme IgG ImmunoBlot test versus STTT.

		STTT
		Positive (+)	Negative (-)
iDart Lyme IgG ImmunoBlot	Positive (+)	19	36
	Negative (-)	1	234
	Total	20	270
	PPA (95% CI)	95.00% (76.39% – 99.11%)
	NPA (95% CI)	86.67% (82.09% – 90.21%)

Table 8: Performance Summary on samples from IGeneX Inc. Cohort 2 (n=248). iDart Lyme IgG ImmunoBlot test versus STTT.

		STTT
		Positive (+)	Negative (-)
iDart Lyme IgG ImmunoBlot	Positive (+)	114	12
	Negative (-)	6	116
	Total	120	128
	PPA (95% CI)	95.00% (89.52% – 97.69%)
	NPA (95% CI)	90.63% (84.33% - 94.56%)

Table 9: Performance Summary on samples from IGeneX Inc. Cohort 3 (n=230). iDart Lyme IgG ImmunoBlot test versus STTT.

		STTT
		Positive (+)	Negative (-)
iDart Lyme IgG ImmunoBlot	Positive (+)	10	7
	Negative (-)	1	212
	Total	11	219
	PPA (95% CI)	90.91% (62.27% – 98.38%)
	NPA (95% CI)	96.80% (93.55% – 98.44%)

2. Clinical Sensitivity/Specificity:

CDC Serum Panel:

A Panel of 280 serum samples was received from CDC. These samples were from patients diagnosed with Lyme Disease at different stages (Stages 1, 2, and 3), Lyme disease look-like infectious mononucleosis, multiple sclerosis, rheumatoid arthritis, fibromyalgia and severe periodontitis), and from healthy controls living in endemic and non-endemic regions of Lyme disease. Results are analyzed according to disease stages and compared to STTT (See Table 10).

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DiseaseStage	Stage I		Stage II		Stage III		Overall		Healthycontrols		DiseaseControls
N	60		10		20		90		100		90
Test Kit	iDart	STTT	iDart	STTT	iDart	STTT	iDart	STTT	iDart	STTT	iDart	STTT
Positive	35	18	9	9	20	20	64	47	0	0	0	0
Negative	25	42	1	1	0	0	26	43	100	100	90	90
Sensitivity	58.33%	30.00%	90.00%	90.00%	100%	100%	71.11%	52.22%
Specificity									100%	100%	100%	100%

Table 10. iDart Lyme IgG ImmunoBlot Kit performance using the CDC Reference Panel

3. Other Clinical Supportive Data:

Fresh and Frozen Samples Comparison Study:

72 decoded left-over patient serum samples were tested twice with iDart Lyme lgG ImmunmoBlot Test Kit, pre and post freezing. As shown below in Table 11, after freezing, all IgG positive samples remained positive and all negative samples remained negative. Clinical Performance of iDart™ Lyme IgG ImmunoBlot Kit for fresh and frozen samples are comparable and the test kit can be used on both fresh and frozen samples.

Table 11. : Fresh and Frozen Samples Tested with iDart™Lyme IgG ImmunoBlot Kit

Sample Type	Tested	N	IgG
			LSA (+)	≥2 bands	IgG (+)	Neg
Fresh Samples	Within 2 weeks of collection(stored refrigerated)	72	39	47	33	39
Frozen Samples	After being Frozen (9-44 days)	72	39	47	33	39

Antibody Class Specificity:

A study was conducted to demonstrate the specificity of the goat anti-human IgG Conjugate used in the iDart™ Lyme IgG ImmunoBlot Kit. 10 previously tested patient samples that includes 6 negatives and 4 positives were included study. 3 sets of anti-human lgG conjugate were prepared and tested:

1ug/ml of human IgM
. 1ug/ml of human IgG
Control no additives .

The data in Table 12 demonstrates the IgG antibody class specificity performance of iDart™ Lyme IgG ImmunoBlot Kit. All positive samples tested with goat anti-human IgG conjugate without treatment with human IgG or with treatment with human IgM remained positive; and all negative samples remained neqative.

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Sample#	None			Human IgM			Human IgG
	LSA	BandGroup	Result	LSA	BandGroup	Result	LSA	BandGroup	Result
1	P	P	P	P	P	P	N	N	N
2	N	P	N	N	P	N	N	N	N
3	P	P	P	P	P	P	N	N	N
4	P	P	P	P	P	P	N	N	N
5	P	P	P	P	P	P	N	N	N
6	N	N	N	N	N	N	N	N	N
7	N	P	N	N	P	N	N	N	N
8	N	P	N	N	P	N	N	N	N
9	N	N	N	N	N	N	N	N	N
10	N	P	N	N	P	N	N	N	N

Table 12. iDart Lyme IgG ImmunoBlot Antibody Class Specificity Study results

C. Clinical Cut-Off:

Not applicable.

D. Expected Values/Reference Range:

Not applicable.

PROPOSED LABELING: VIII.

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10

CONCLUSION: IX.

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

Regulation Number and Section

§ 866.3830

Treponema pallidum treponemal test reagents.(a)
Identification. Treponema pallidum treponemal test reagents are devices that consist of the antigens, antisera and all control reagents (standardized reagents with which test results are compared) which are derived from treponemal sources and that are used in the fluorescent treponemal antibody absorption test (FTA-ABS), theTreponema pallidum immobilization test (T.P.I.), and other treponemal tests used to identify antibodies toTreponema pallidum directly from infecting treponemal organisms in serum. The identification aids in the diagnosis of syphilis caused by bacteria belonging to the genusTreponema and provides epidemiological information on syphilis.(b)
Classification. Class II (performance standards).