IGG ANTI-ATHEROX TEST KIT
Device Facts
| Record ID | K072032 |
|---|---|
| Device Name | IGG ANTI-ATHEROX TEST KIT |
| Applicant | Corgenix, Inc. |
| Product Code | MSV · Immunology |
| Decision Date | Apr 4, 2008 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 866.5660 |
| Device Class | Class 2 |
| Attributes | Real-World Evidence |
Real-World Evidence
| Submission | Device | Sponsor | RWD Sources | RWE Use Summary | Key Tags |
|---|---|---|---|---|---|
| K072032 · Apr 4, 2008 | IGG ANTI-ATHEROX TEST KIT | Corgenix, Inc. | Retrospective clinical serum samples from patient cohorts (healthy controls, rheumatoid arthritis, and systemic lupus erythematosus) | Retrospective clinical samples were used to compare the performance of the IgG Anti-AtherOx test kit against the REAADS IgG anti-cardiolipin predicate device to establish clinical agreement. | Retrospective clinical samples; Clinical agreement; Diagnostic performance |
Clinical Evidence
| Study Design | Population | Comparator | Key Endpoints |
|---|---|---|---|
| Clinical performance evaluation; Retrospective comparative study | 205 healthy controls, 99 patients with rheumatoid arthritis, and 143 patients with systemic lupus erythematosus; Sample Size: 447 total samples | REAADS IgG anti-cardiolipin test kit | Positive/negative percent agreement and overall percent agreement |
Indications for Use
An enzyme-linked immunoassay (ELISA) for the detection of IgG antibodies to complexes formed by oxidized low-density lipoprotein (oxLDL) with β2-glycoprotein I (β2GPI) in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (antiphospholipid syndrome).
Device Story
The IgG Anti-AtherOx Test Kit is an indirect ELISA used in clinical laboratories to detect IgG antibodies against oxLDL/ß2GPI complexes in human serum. The device utilizes a 96-well polystyrene microtiter plate coated with oxLDL/ß2GPI complex. Patient serum is incubated in the wells, allowing specific IgG antibodies to bind to the immobilized antigen. After washing, HRP-conjugated anti-human IgG is added, followed by a TMB/hydrogen peroxide substrate. The resulting color intensity, measured via spectrophotometer, is proportional to the concentration of IgG anti-oxLDL/ß2GPI antibodies. Results are calculated using a log-log regression analysis against provided calibrators. By identifying these autoantibodies, the test assists clinicians in assessing the risk of venous and arterial atherothrombosis in patients with autoimmune conditions like SLE and APS, potentially aiding in the management of hypercoagulability.
Clinical Evidence
Clinical evaluation compared the device against the REAADS Anti-Cardiolipin test using 448 samples (205 healthy controls, 99 rheumatoid arthritis, 143 SLE). Overall agreement was 90.2%. In SLE patients, positive percent agreement was 76.0% and negative percent agreement was 86.4%. In patients with secondary APS, positive percent agreement was 73.9% and negative percent agreement was 88.7%. Subgroup analysis for arterial thrombosis showed 90.5% overall agreement.
Technological Characteristics
Indirect ELISA; 96-well polystyrene microtiter plate coated with oxLDL/ß2GPI complex; HRP-conjugated goat polyclonal anti-human IgG detection; TMB/H2O2 chromogenic substrate; 0.36 N sulfuric acid stop solution; spectrophotometric readout; high-complexity laboratory test; manual/automated processing.
Indications for Use
Indicated for detection of IgG antibodies to oxLDL/β2GPI complexes in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (antiphospholipid syndrome). For prescription use only.
Regulatory Classification
Identification
A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).
Predicate Devices
- REAADS Anti-Cardiolipin IgG/IgM Semi-Quantitative kit (k022992)
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k072032
B. Purpose for Submission:
New Device
C. Measurand:
IgG oxidized low-density lipoprotein (oxLDL) beta2-glycoprotein 1 complex (anti-oxidized LDL/β2GPI) antibodies in human serum.
D. Type of Test:
Semiquantitative ELISA
E. Applicant:
Corgenix, Inc.
F. Proprietary and Established Names:
IgG Anti-AtherOx™ Test Kit
G. Regulatory Information:
1. Regulation section:
21 CFR 866.5660 Multiple autoantibodies immunological test system
2. Classification:
Class II
3. Product code:
MSV - system, test, antibodies, β2-glycoprotein 1 (β2-GP1)
4. Panel:
82 - Immunology
H. Intended Use:
1. Intended use(s):
An enzyme-linked immunoassay (ELISA) for the detection of IgG antibodies to complexes formed by oxidized low-density lipoprotein (oxLDL) with β2-glycoprotein I (β2GPI) in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (antiphospholipid syndrome).
2. Indication(s) for use:
Same as Intended Use
3. Special conditions for use statement(s):
For prescription use only
4. Special instrument requirements:
Microplate reader capable of measuring OD 450 nm.
I. Device Description:
Each device contains the following: stabilized oxLDL-β2GPI antigen (human) coated microwell strips, with frame, sample diluent, IgG calibrator sera (human) (1-high, 2-moderate, 3-low), IgG positive control serum (human), IgG normal control serum (human), IgG anti-human (goat) HRP-conjugated antibody solution, one-component substrate (TMB and H₂O₂), stopping solution, and wash concentrate.
J. Substantial Equivalence Information:
1. Predicate device name(s):
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REAADS Anti-Cardiolipin IgG/IgM Semi-Quantitative kit
2. Predicate K number(s):
k022992
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Indications for Use | For individuals with SLE and lupus-like disorders (anti-phospholipid syndrome) | Same |
| Specimen Type | Serum | Same |
| Technology | ELISA | Same |
| Assay format | Semi-quantitative | Same |
| Solid phase | Coated polystyrene microwell plates | |
| Enzyme-conjugate | Horseradish peroxidase conjugated to goat anti-human IgG | Same |
| Substrate | TMB chromogen | Same |
| Results determination | Multipoint calibration curve | Multipoint calibration curve (or single point calibrator) |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | An enzyme-linked immuno-assay (ELISA) for the detection of IgG antibodies to complexes formed by oxidized low-density lipoprotein (oxLDL) with β2-glycoprotein I (β2GPI) in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (anti-phospholipid syndrome). | For the detection and semi-quantitation of anti-cardiolipin antibodies in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (anti-phospholipid syndrome). |
| Analyte | IgG anti-oxidized LDL/β2GPI antibodies | IgG anti-cardiolipin antibodies |
| Capture antigen | OxLDL/β2-GPI complexes | Purified cardiolipin |
| Positive & negative controls | Dilute 1:100 | Ready to use |
| Assay time | 150 min at RT | 40 min at RT |
| Quantitation range | 10-100 G Units | 0-100 GPL Units |
| Sample type and dilution | Human serum at 1:100 | 1:50 |
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# K. Standard/Guidance Document Referenced (if applicable):
EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods
EP6-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline
EP17-A Protocols for Determination of Limits of Quantitation; Approved Guideline
# L. Test Principle:
Diluted serum samples, calibrator(s), and controls are incubated in microwells coated with the oxLDL- $\beta_{2}$ GPI complex. Incubation allows the IgG anti-oxLDL- $\beta_{2}$ GPI antibody present in the samples to react with the immobilized antigen complex. After the removal of unbound serum proteins by washing, anti-human IgG antibodies, labeled with horseradish peroxidase (HRP), are added forming complexes with the bound IgG anti-oxLDL- $\beta_{2}$ GPI antibody. Following another washing step, the bound enzyme-antibody conjugate is assayed by the addition of a solution containing tetramethylbenzidine (TMB) and hydrogen peroxide $(\mathrm{H}_2\mathrm{O}_2)$ as the chromogenic substrate. Color develops in the wells at an intensity proportional to the serum concentration of IgG anti-oxLDL- $\beta_{2}$ GPI antibody. Results are obtained by reading the OD (optical density or absorbance) of each well in a spectrophotometer. Calibrator sera are provided, with the IgG anti-oxLDL- $\beta_{2}$ GPI antibody concentration expressed in G Units. A log-log regression analysis is performed with calibrator values plotted against calibrator mean ODs. Controls and patient results are determined from the calibration curve.
# M. Performance Characteristics (if/when applicable):
# 1. Analytical performance:
# a. Precision/Reproducibility:
Precision: Serum samples with concentrations spanning the range of the assay were tested in the Corgenix laboratory by 2 operators in duplicate on each of 20 days over 30 calendar days. One reagent lot was tested, and assays were calibrated each day.
| Sample | Mean Conc. (G Units) | Inter-assay Precision (%CV) | Within-laboratory Precision (%CV) |
| --- | --- | --- | --- |
| Low | 9.6 | 3% | 13% |
| Med-Low | 20.2 | 4% | 12% |
| Med | 28.1 | 7% | 13% |
| Med-High | 38.4 | 5% | 12% |
| High | 76.8 | 4% | 10% |
Reproducibility: Three operators at one site ran three clinical samples (low, medium, and high) over four consecutive days. The medium and high samples were run in triplicate and the low was run in quadruplicate. Results were determined from 48 measurements for the low sample and 36 for the medium and high sample.
| | Samples | | |
| --- | --- | --- | --- |
| | Low | Medium | High |
| Mean (G Units) | 19 | 48 | 94 |
| Intra-assay %CV | 4% | 1% | 2% |
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b. Linearity/assay reportable range:
Linearity: Independently duplicate dilutions of a strongly-positive sample, mixed at various fixed ratios with an IgG-depleted pooled serum, were prepared to produce 11 evenly-spaced concentrations that were predicted to extend past the linear range of the assay. Acceptance criterion was set at $\leq 5\mathrm{G}$ units between predicted and recovered values. Evaluation of the data indicated that the linear range of the assay was $10 - 100\mathrm{G}$ units.
c. Traceability, Stability, Expected values (controls, calibrators, or methods): No recognized reference material for IgG anti-oxLDL- $\beta_{2}$ GPI. The results are reported in arbitrary units.
Controls: The standards are prepared in-house and values are assigned during the development process. Positive and negative controls are prepared in-house.
Stability: Calibrators, controls, a sample diluent alone were used to assess the real-time stability of four kits every 4-6 months for a 24 month period. All kits were shown to be stable up to 12 months.
d. Detection limit:
Limit of Blank (LoB): Three serum samples were mixed and depleted of IgG using a protein G column. The IgG-depleted serum, four clinical samples, and a negative sample (sample diluent) were tested multiple times on two different plate readers. Ninety-two results were generated from the negative samples and 228 for the clinical samples. The frequency distribution were tabulated and the $95^{\text{th}}$ percentile of the negative results at 6.1 G units
Limit of Detection (LoD): was defined the lowest level where $5\%$ or fewer of the observed measurements are below the LoB based on a sample set showing non-Gaussian distribution. Since none of the results for the positive data set were at 7.0 G units or below, so the LoD was set as 7 G units.
e. Analytical specificity:
Five clinical samples with the addition of increasing amounts of hemoglobin (to $500\mathrm{mg / dL}$ ), conjugated bilirubin (to $20\mathrm{mg / dL}$ ), triglycerides (to $3\mathrm{g / dL}$ ) and rheumatoid factor (to 500 units/ml) were tested and showed that these levels had no effect on IgG Anti-AtherOx test kit results. However, it is recommended in the package insert that results obtained from grossly hemolyzed or lipemic specimens be interpreted with caution and that grossly lipemic samples can be clarified by centrifugation.
f. Assay cut-off:
A cut-off value of $20\mathrm{G}$ Units was established by testing 75 serum samples from healthy volunteers in duplicate. Values ranged from 3.5-17.8 G Units.
| N | Range [G units] | Mean (Median) [G units] | SD | 95th percentile | Mean + 3 SD |
| --- | --- | --- | --- | --- | --- |
| 75 | 3.5-17.7 | 8.8 (8.9) | 3 | 13.8 | 17.7 |
2. Comparison studies:
a. Method comparison with predicate device:
Serum samples from 143 Systemic lupus erythematosus (SLE), 99 rheumatoid
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arthritis and 205 normal healthy controls were tested for elevated IgG antiphospholipid antibody levels using the Corgenix IgG Anti-AtherOx and REAADS IgG anti-cardiolipin kits. Each group is summarized below:
SLE: Serum samples from 143 SLE patients (94 SLE with secondary APS, 19 SLE only and 30 with unknown APS status) were tested on the IgG Anti-AtherOx™ Test Kit and the REAADS IgG Anti-Cardiolipin Test. The mean IgG anti-oxLDL- $\beta_{2}$ GPI antibody level of this group was determined to be 24.4 G Units. Of the SLE/APS subgroup, the mean IgG anti-oxLDL- $\beta_{2}$ GPI antibody level was 28.8 G Units. Twenty five SLE/APS samples were positive when tested with the new device and 17 of these were also positive for anticardiolipin antibodies by the predicate device. Since the new and predicate devices detect different analytes, it is likely that some patients have one and not the other autoantibodies. Comparison with the predicate device for the SLE/APS group is shown below:
| SLE/APS (N = 94) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix IgG Anti-AtherOx | Pos | 17 | 8 | 25 |
| | Neg | 6 | 63 | 69 |
| | Total | 23 | 71 | 94 |
Positive Percent Agreement $= 73.9\%$ (17/23)
Negative Percent Agreement $= 88.7\%$ (63/71)
Overall % Agreement = 85.1% (80/94)
The APS patients were then further analyzed according to their clinical history of APS manifestations: arterial thrombosis, venous thrombosis, or pregnancy morbidity. Results, including the SLE only group are summarized in the following tables:
| Pregnancy Morbidity (N = 15) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix IgG Anti-AtherOx | Pos | 0 | 1 | 1 |
| | Neg | 2 | 12 | 14 |
| | Total | 2 | 13 | 15 |
Positive Percent Agreement $= 0\%$ (0/2)
Negative Percent Agreement $= 92.3\%$ (12/13)
Overall % Agreement = 80.0% (12/15)
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| Arterial Thrombosis
(N = 42) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix
IgG Anti-AtherOx | Pos | 11 | 1 | 12 |
| | Neg | 3 | 27 | 30 |
| | Total | 14 | 28 | 42 |
Positive Percent Agreement = 78.6% (11/14)
Negative Percent Agreement = 96.4% (27/28)
Overall % Agreement = 90.5% (38/42)
| Venous Thrombosis
(N = 37) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix
IgG Anti-AtherOx | Pos | 6 | 6 | 12 |
| | Neg | 1 | 24 | 25 |
| | Total | 7 | 30 | 37 |
Positive Percent Agreement = 85.7% (6/7)
Negative Percent Agreement = 80.0% (24/30)
Overall % Agreement = 81.1% (30/37)
For the 19 SLE patients without APS, the mean IgG anti-oxLDL- $\beta_{2}$ GPI antibody level was 12.0 G Units. One sample was positive with the new device which was negative with the predicate.
| SLE only
(N = 19) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix
IgG Anti-AtherOx | Pos | 0 | 1 | 1 |
| | Neg | 0 | 18 | 18 |
| | Total | 0 | 19 | 19 |
Summary results for the SLE group are shown below:
| SLE and SLE/APS
(N = 143) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix
IgG Anti-AtherOx | Pos | 19 | 16 | 35 |
| | Neg | 6 | 102 | 108 |
| | Total | 25 | 118 | 143 |
Positive Percent Agreement = 76.0% (19/25)
Negative Percent Agreement = 86.4% (102/118)
Overall % Agreement = 84.6% (121/143)
Normal Healthy controls: Samples from 205 normal, healthy controls were tested and six samples were positive for IgG anti-AtherOx assay but negative for IgG anti-cardiolipin using the cut-off established in the initial study. The overall % agreement with the predicate device is 97.1% (199/205).
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| Healthy Controls
(N = 205) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix IgG Anti-AtherOx | Pos | 0 | 6 | 6 |
| | Neg | 0 | 199 | 199 |
| | Total | 0 | 205 | 205 |
Rheumatoid arthritis: Serum samples from 99 rheumatoid arthritis patients were tested on the IgG Anti-AtherOx™ Test Kit and the REAADS IgG Anti-Cardiolipin Test. The mean IgG value of this population was determined to be 16.4 G Units. One sample was positive for both device but there were 17 samples positive only with the new device, where 58.8% (10/17) were near the cut-off for the new device. For this group of patients, the overall % agreement is 82.8% (82/99).
| Rheumatoid Arthritis
(N = 99) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix IgG Anti-AtherOx | Pos | 1 | 17 | 18 |
| | Neg | 0 | 81 | 81 |
| | Total | 1 | 98 | 99 |
A summary of all samples tested in the method comparison study is as follows:
| All Subjects
(N = 447) | REAADS IgG Anti-Cardiolipin | | | |
| --- | --- | --- | --- | --- |
| | | Pos | Neg | Total |
| Corgenix IgG Anti-AtherOx | Pos | 20 | 39 | 59 |
| | Neg | 6 | 382 | 388 |
| | Total | 26 | 421 | 447 |
Positive Percent Agreement = 76.9% (20/26)
Negative Percent Agreement = 90.7% (382/421)
Overall % Agreement = 89.9% (404/447)
b. Matrix comparison:
Both assays use serum as the matrix.
3. Clinical studies:
a. Clinical Sensitivity and Specificity:
The tables below show the same samples from the above comparison data, but presented according to the diagnosis.
| Disease state | N | Corgenix anti-AtherOx
% positive |
| --- | --- | --- |
| SLE + Secondary APS | | |
| Preg. Morbidity | 42 | 28.6% (12/42) |
| Atrial thrombosis | 37 | 32.4% (12/37) |
| Venous thrombosis | 15 | 6.7% (1/15) |
| Total | 94 | 26.6% (25/94) |
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| Disease state | N | Corgenix anti-AtherOx % positive |
| --- | --- | --- |
| No APS | | |
| Rheumatoid Arthritis (RA) | 99 | 18.2% (18/99) |
| SLE (no secondary APS) | 19 | 5.3% (1/19) |
| SLE (APS status unknown) | 30 | 26.7% (8/30) |
| Total | 148 | 18.2% (27/148) |
| Total number of patient samples | 242 | |
| | APS | | | |
| --- | --- | --- | --- | --- |
| | | (+) | (-) | Total |
| Corgenix IgG Anti-AtherOx | (+) | 25 | 27 | 52 |
| | (-) | 69 | 121 | 190 |
| | Total | 94 | 148 | 242 |
Sensitivity: $26.6\%$ (25/94)
Specificity: $81.8\%$ (121/148)
4. Clinical cut-off:
See Assay cut-off.
5. Expected values/Reference range:
Expected value in the normal population is negative. Six (6) samples were positive by the Corgenix IgG Anti-AtherOx™ assay.
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.
O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
