IMMULISA INTRINSIC FACTOR ANTIBODY ELISA
Device Facts
| Record ID | K071346 |
|---|---|
| Device Name | IMMULISA INTRINSIC FACTOR ANTIBODY ELISA |
| Applicant | Immco Diagnostics, Inc. |
| Product Code | LIG · Clinical Chemistry |
| Decision Date | Sep 24, 2007 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 862.1810 |
| Device Class | Class 2 |
Intended Use
The ImmuLisa™ Anti-Intrinsic Factor (IF) Antibody ELISA is an enzyme-linked immunosorbent assay (ELISA) for the qualitative and semi-quantitative detection of antibodies to intrinsic factor in human serum to aid in the diagnosis of pernicious anemia.
Device Story
ImmuLisa Intrinsic Factor Antibody ELISA is an in vitro diagnostic test used to detect and semi-quantitate intrinsic factor antibodies in human serum samples. The device utilizes enzyme-linked immunosorbent assay (ELISA) technology to identify these antibodies, which are associated with pernicious anemia. The test is intended for use by clinical laboratory professionals to assist physicians in diagnosing patients suspected of having pernicious anemia. Results are provided to the clinician to support clinical decision-making regarding patient diagnosis and management.
Clinical Evidence
No clinical data provided in the document; bench testing only.
Technological Characteristics
Solid-phase ELISA. Components: polystyrene microwell plates coated with recombinant glycosylated human intrinsic factor, alkaline phosphatase-labeled goat anti-human IgG conjugate, pNPP substrate. Requires spectrophotometer (405nm/620nm). Qualitative/semi-quantitative output. Manual/automated plate reader workflow. No specific ASTM materials cited.
Indications for Use
Indicated for the qualitative and semi-quantitative detection of anti-intrinsic factor antibodies in human serum to aid in the diagnosis of pernicious anemia.
Regulatory Classification
Identification
A vitamin B12 test system is a device intended to measure vitamin B12 in serum, plasma, and urine. Measurements obtained by this device are used in the diagnosis and treatment of anemias of gastrointestinal malabsorption.
Related Devices
- K061841 — QUANTA LITE INTRINSIC FACTOR ELISA · Inova Diagnostics, Inc. · Dec 22, 2006
- K240800 — Access Intrinsic Factor Ab · Beckman Coulter, Inc. · May 3, 2024
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY
A. 510(k) Number:
k071346
B. Purpose for Submission:
New device
C. Measurand:
Intrinsic factor autoantibodies
D. Type of Test:
Qualitative or semi-quantitative enzyme-linked immunosorbent assay (ELISA)
E. Applicant:
IMMCO Diagnostics
F. Proprietary and Established Names:
ImmuLisa™ Anti-Intrinsic Factor (IF) Antibody ELISA
G. Regulatory Information:
1. Regulation section:
CFR 862.1810 Vitamin B₁₂ test system
2. Classification:
Class II
3. Product code:
LIG Radioassay, intrinsic factor blocking antibody
4. Panel:
Chemistry 75
H. Intended Use:
1. Intended use:
The ImmuLisa™ Anti-Intrinsic Factor (IF) Antibody ELISA is an enzyme-linked immunosorbent assay (ELISA) for the qualitative and semi-quantitative detection of antibodies to intrinsic factor in human serum to aid in the diagnosis of pernicious anemia.
2. Indication(s) for use:
Same as intended use
3. Special conditions for use statement(s):
For prescription use only
4. Special instrument requirements:
Microwell plate reader capable of measuring OD at 405nm (and 620nm for dual wavelength readings)
I. Device Description:
The ImmuLisa Anti-Intrinsic Factor Antibody ELISA consists of microplate well strips coated with recombinant glycosylated human intrinsic factor antigen; positive and negative controls; 4 calibrators; alkaline phosphatase goat anti-human IgG conjugate; serum diluent; pNPP enzyme substrate; stop solution; powdered wash buffer; and a frame holder.
J. Substantial Equivalence Information:
1. Predicate device name(s):
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DPC Intrinsic Factor Blocking Antibody RIA Kit
2. Predicate $510(\mathbf{k})$ number(s):
k832726
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| | ImmuLisa Anti-Intrinsic Factor Antibody ELISA | DPC Intrinsic Factor Blocking Antibody RIA |
| Indications for Use | To aid in the diagnosis of pernicious anemia | To aid in the diagnosis of pernicious anemia |
| Analyte measured | Anti-intrinsic factor antibodies (Type I and II) | Anti-intrinsic blocking factor antibodies (Type I) |
| Test matrix | Serum (diluted 1:101) | Serum (undiluted) |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Method | Enzyme-linked immunosorbent assay | Radioimmunoassay |
| Calculation of results | Compared to a cut-off control | Ratio: negative reference average CPM/average CPM |
| Solid phase | Antigen coated polystyrene microwell plates | Antigen immobilized on microcrystalline cellulose particles |
| Calibrators | 4 levels | Not applicable |
| Units | Arbitrary ELISA units | Not applicable |
| Controls | Negative and positive | Negative, positive and high positive |
| Label/detector | Alkaline phosphatase labeled goat anti-human IgG | 57Cobalt-labeled vitamin B12 |
| Substrate | pNPP | Not applicable |
| Capture antigen | Recombinant human intrinsic factor | Purified intrinsic factor |
| Result interpretation | negative = <20 U/mL indeterminent = 20-25 positive = >25 | Ratios: negative = <negative cut-off + 0.10 indeterminent = >1.10 and <positive ratio - 0.10 positive = ≥ positive cut-off |
| Signal detector | Spectrophotometer (absorbance) | Gamma counter (57Cobalt) |
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K. Standard/Guidance Document Referenced (if applicable):
None referenced
L. Test Principle:
The test is performed as a solid phase immunoassay. Controls, calibrator(s) and patient sera are incubated in the antigen coated microwells to allow specific antibodies present in the serum to bind to the intrinsic factor antigen. Unbound antibodies and other serum proteins are removed by washing the microwells. Bound antibodies are detected by adding an enzyme labeled anti-human IgG conjugate to the microwells. Unbound conjugate is removed by washing. Specific enzyme substrate (pNPP) is then added to the wells and the presence of antibodies is detected by a color change produced by the conversion of pNPP substrate to a colored reaction product. The reaction is stopped and the intensity of the color change, which is proportional to the concentration of antibody, is read by a spectrophotometer at 405 nm. Specimen optical density is compared with optical density of a calibrator (qualitative result reported as positive or negative) or a four point standard curve (semi-quantitative expressed in ELISA units).
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
Intra-assay performance was based on 16 replicates of 5 specimens ranging from 22 to 150 EU/mL with %CV ranging from 1.4 to 12.3%.
| | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- |
| Mean EU/mL | 22 | 29 | 49 | 87 | 150 |
| %CV | 12.3 | 4.8 | 3.2 | 2.6 | 1.4 |
Inter-assay performance for the assay was evaluated by testing 5 specimens, by 5 operators for 3 days. The samples tested ranged from 22 to 150 Units with %CVs ranging from 5.6 to 13.9%.
| | 1 | 2 | 3 | 4 | 5 |
| --- | --- | --- | --- | --- | --- |
| Mean EU/mL | 22 | 29 | 49 | 87 | 150 |
| %CV | 13.9 | 9.9 | 9.6 | 7.9 | 5.6 |
b. Linearity/assay reportable range:
To determine acceptable linearity, plates were assayed with a positive serum diluted in doubling dilutions from 1:800 to 1:6400. The r-squared values of the resulting standard curves were determined for multiple assays (3 operators, 3 days, 3 samples). An r-squared value greater than 0.95 was considered acceptable. The average r-squared value for the assays was 0.988 and the values ranged from 0.935 – 1.0.
Dilution recovery:
For each of 5 tests, two samples of equal volume with known intrinsic factor antibody levels were mixed. Antibody concentration for the mixture was expected to be the average of the antibody concentration of the discrete samples. The antibody levels of the mixed samples were determined and used
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to calculate percent recovery. The following results were obtained:
| | Mixture | Expected (EU/mL) | Obtained (EU/mL) | Recovery |
| --- | --- | --- | --- | --- |
| 1 | 23 EU+28 EU/mL | 25.5 | 23.2 | 91% |
| 2 | 23 EU+54 EU/mL | 38.5 | 33.8 | 88% |
| 3 | 28 EU+60 EU/mL | 44 | 46.3 | 105% |
| 4 | 54 EU+81 EU/mL | 67.5 | 60.4 | 89% |
| 5 | 81 EU+137 EU/mL | 109 | 101.5 | 94% |
c. Traceability, Stability, Expected values (controls, calibrators, or methods): There is no recognized standard or reference material for anti-intrinsic factor antibodies.
Accelerated stability studies were conducted on 3 lots of assay components. Data supported a shelf life of 15 months.
d. Detection limit:
The determination of detection limit/analytical sensitivity was not performed or relevant for this assay.
e. Analytical specificity:
Grossly hemolyzed, lipemic or microbially contaminated specimens may interfere with the performance of the test and should not be used.
f. Assay cut-off:
The cut-off value was originally determined by testing 40 adult normal blood donors. In this study, the mean of the normal subjects plus 3SD was 6.7 EU and an arbitrary value of $< 20$ EU/mL was set for negative, 20-25 EU/mL indeterminant and $>25$ EU/mL as positive. A population of 30 patients in the age range at risk for pernicious anemia was tested to validate the established cut-off. All 30 samples were negative.
2. Comparison studies:
a. Method comparison with predicate device:
Testing was performed on 47 specimens that included 14 from healthy individuals, 24 from suspected pernicious anemia (PA) patients, and 9 specimens from patients with confirmed PA.
| | DPC Intrinsic Factor Antibody RIA | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| ImmuLisa Anti-Intrinsic Factor Antibody ELISA | Positive | 16 | 0 | 16 |
| | Negative | 17 | 14 | 31 |
| | Total | 33 | 14 | 47 |
Positive percent agreement: $16 / 33 = 48.5\%$
Negative percent agreement: $14 / 14 = 100\%$
Overall agreement: $30 / 47 = 63.8\%$
Twenty of the suspected PA patients had $\mathrm{B}_{12}$ that exceeded $2000\mathrm{pg / mL}$ and all were positive with the RIA assay. The 17 samples positive by RIA and negative by ELISA were from patients with $\mathrm{B}_{12}$ levels $>2000$ . To evaluate a possible cause
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for discrepant results between the IMMCO and DPC method, IMMCO spiked the normal serum samples negative for intrinsic factor antibodies on both methods with vitamin $\mathrm{B}_{12}$ . The spiked specimens were tested for vitamin $\mathrm{B}_{12}$ levels and for intrinsic factor antibodies on the RIA and ELISA systems.
| # | B12 levels unspiked (pg/mL) | B12 levels spiked (pg/mL) | RIA unspiked (ratio) | RIA spiked (ratio) | ELISA unspiked (EU/mL) | ELISA spiked (EU/mL) |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 463 | >2000 | 0.92 negative | 2.84 positive | 17 negative | 17 negative |
| 2 | 374 | >2000 | 0.97 negative | 2.11 positive | <13 negative | <13 negative |
| 3 | 257 | >2000 | 1.04 negative | 1.63 positive | <13 negative | <13 negative |
This study suggests that the IMMCO ELISA is specific for intrinsic factor antibodies and that a vitamin $\mathrm{B}_{12}$ inhibition assay such as the DPC system provide false positive results when free vitamin $\mathrm{B}_{12}$ levels in the serum are high. The DPC labeling carries a limitation to this effect.
The 17 discrepant samples in the method comparison study were most likely indicative of patients receiving $\mathrm{B}_{12}$ therapy. Literature indicates that $\mathrm{B}_{12}$ therapy is known to cause false positive results on RIA test systems.
b. Matrix comparison:
Both assays use serum as the test matrix.
# 3. Clinical studies:
a. Clinical Sensitivity and Specificity:
A total of 110 samples were tested including 20 PA, 20 healthy controls and 70 disease controls.
| Pernicious anemia, other diseases and normal subjects | N = | IF ELISA positive | IF ELISA negative |
| --- | --- | --- | --- |
| Pernicious anemia | 20 | 20 | 0 |
| Rheumatoid arthritis, Celiac disease, Hashimoto's thyroiditis, Graves' disease H. pylori, Hepatitis C | 10 | 0 | 10 |
| | 10 | 0 | 10 |
| | 10 | 0 | 10 |
| | 10 | 0 | 10 |
| | 11 | 4 | 7 |
| | 19 | 0 | 19 |
| Normal | 20 | 0 | 20 |
| Total | 110 | 24 | 86 |
Calculations
| | Pernicious Anemia disease status | | | |
| --- | --- | --- | --- | --- |
| | | Positive | Negative | Total |
| ImmuLisa Anti-Intrinsic Factor Antibody test result | Positive | 20 | 4 | 24 |
| | Negative | 0 | 86 | 86 |
| | Total | 20 | 90 | 110 |
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Clinical sensitivity: 20/20 = 100%
Clinical specificity: 86/90 = 95.6%
Overall agreement: 106/110 = 96.4%
b. Other clinical supportive data (when a. and b. are not applicable):
Not applicable
4. Clinical cut-off:
See assay cut-off.
5. Expected values/Reference range:
The expected result in the normal population is negative (≥ 20 Units). However, 0.1 – 0.2% of apparently healthy, asymptomatic individuals may test positive for anti-intrinsic factor autoantibodies.
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.
