STEROID 21-HYDROXYLASE ANTIBODY (21-OHAB) RIA ASSAY KIT

{0} 1 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k121046 B. Purpose for Submission: New Device C. Measurand: Steroid 21-Hydroxylase Antibody (21-OHAb) D. Type of Test: Semi-quantitative, radioimmunoassay (RIA) E. Applicant: KRONUS Market Development Associates, INC. F. Proprietary and Established Names: KRONUS Steroid 21-Hydroxylase Antibody (21-OHAb) RIA Assay Kit G. Regulatory Information: 1. Regulation section: 21 CFR § 866.5660, Multiple autoantibody immunological test system 2. Classification: Class II 3. Product code: PCG, 21-Hydroxylase Antibody (21-OHAb) antibody assay 4. Panel: Immunology (82) {1} H. Intended Use: 1. Intended use(s): The KRONUS Steroid 21-Hydroxylase Antibody (21-0HAb) RIA Assay Kit is for the semi-quantitative determination of antibodies to steroid 21-hydroxylase (21-0H) in human serum. The KRONUS Steroid 21-0HAb RIA Assay may be useful as an aid in the diagnosis of autoimmune adrenal disease, whether expressed as autoimmune Addison's disease (isolated) or Addison's disease as part of the more complex autoimmune polyglandular syndrome (APS), type I or II. The assay result is to be used in conjunction with other clinical and laboratory findings and is not a substitute for functional testing required to diagnose adrenal insufficiency. 2. Indication(s) for use: Same as Intended use. 3. Special conditions for use statement(s): For prescription only. 4. Special instrument requirements: RIA Gamma Counter set for $^{125}\mathrm{I}$ I. Device Description: The KRONUS Steroid 21-OHAb RIA Assay kit is available in two formats: 50-tube or 100-tube kits. The 50-tube kit includes one lyophilized vial each of $^{125}\mathrm{I}-21$ OH Tracer and Protein A(2.6 mL); ready-to-use 6 levels of Calibrators (0, 1, 5, 50, 500, 5000 U/mL, 0.15 mL each); one each ready-to-use positive and negative control serum (0.15mL); and one ready-to-use assay buffer (120 mL). The 100 tube kit includes 2 vials each of kit components. J. Substantial Equivalence Information: 1. Predicate device name(s): KRONUS GADAb RIA Assay Kit 2. Predicate 510(k) number(s): k051061 3. Comparison with predicate: {2} | Similarities | | | | --- | --- | --- | | Item | Device | Predicate | | Trade name | KRONUS 21-OHAb RIA Assay Kit | KRONUS GADAb RIA Assay Kit | | Method/Principle | Radioimmunoassay (RIA) | Same | | Sample Matrix | Human serum | Same | | Controls | Positive and negative | Same | | Assay Format | Semi-quantitative | Same | | Solid phase | Tubes | Same | | Precipitation Reagent | Anti-human IgG | Same | | Shaker | Vortex mixer | Same | | Signal | Radioactivity | Same | | Detection equipment | Gamma counter | Same | | Units of measure | U/mL | Same | | Cut-off | Positive: >1 U/mL Negative: ≤ 1 U/mL | Same | | Differences | | | | --- | --- | --- | | Item | Device | Predicate | | Indication for Use | As an aid in diagnosis of autoimmune adrenal disease whether expressed as autoimmune Addison's disease (isolated) or Addison's disease as part of the more complex autoimmune polyglandular syndrome (APS), type I or II. | As an aid in diagnosis of Type I Diabetes mellitus (autoimmune mediated diabetes) | | Intended Use | Measurement of 21-OH Ab antibody | Measurement of GADAb antibody | | Analyte | Steroid 21-hydroxylase (21-OH) Autoantibodies | Glutamic Acid Decarboxylase Autoantibodies | | Analytical sensitivity | 0.2 U/mL | 0.11 U/mL | # K. Standard/Guidance Document Referenced (if applicable): None # L. Test Principle: Calibrators, controls and patient samples are incubated overnight with $\mathrm{I}^{125}$ 21-0H. During this incubation, antibody binds to tracer. Protein A is added and the tubes are incubated for one hour during which time the antibodies present are bound by Protein A and removed {3} from solution. Assay buffer is then added and the tubes are centrifuged. After centrifugation, the supernatants are decanted or aspirated. The amount of radioactivity in the pellets is directly proportional to the amount of antibody contained in the samples. M. Performance Characteristics (if/when applicable): 1. Analytical performance: a. Precision/Reproducibility: **Intra-assay** The intra-assay precision was determined by testing six serum specimens which included one low (5.69 U/mL), one high (17.42 U/mL), two very high (1269 and 3244 U/L) and four samples close to the assay cut-off (0.9-1.7 U/mL). The low, high and very high serum specimens were tested 25 times and the samples around the cut-off, 14 times. Results showed %CV ranged from 4.59-13.5% (see table below). **Intra-assay Performance of 21-OH Ab RIA Assay** | Specimen | 1 | 2 | 3 | 4 | 5 | 6 | A | B | | --- | --- | --- | --- | --- | --- | --- | --- | --- | | n | 25 | 25 | 14 | 14 | 14 | 14 | 25 | 25 | | Mean (U/mL) | 17.42 | 5.69 | 0.9 | 1.2 | 1.5 | 1.7 | 3244 | 1269 | | SD | 0.83 | 0.33 | 0.1 | 0.2 | 0.2 | 0.1 | 220 | 58 | | CV % | 4.74 | 5.78 | 9.0 | 12.4 | 13.5 | 8.3 | 6.77 | 4.59 | **Inter-assay** The inter-assay precision was determined by testing thirteen samples: two serum specimens once a day for twenty days; eleven serum specimens once a day for 9-10 days and two additional serum samples once a day for 15 days. The serum specimens consisted of three specimens with anti-21-OH concentrations close to the assay 1 U/mL cut-off (0.6, 0.7 and 1.1 U/mL); two low levels (3.7 and 7.5 U/mL); six moderate levels (10.1, 12.9, 19.9, 22.4, 27.1, 38.6 U/mL) and two very high levels (1141 and 2861 U/mL). The %CVs ranged from 5.85-22.4% (see table below). **Inter-assay Performance of 21-OH Ab RIA Assay** | Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | A | B | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | N | 20 | 20 | 9 | 9 | 9 | 9 | 9 | 9 | 10 | 9 | 10 | 15 | 15 | | Mean | 7.5 | 22.4 | 27.1 | 19.9 | 10.1 | 3.7 | 12.9 | 38.6 | 1.1 | 0.7 | 0.6 | 2861 | 1141 | | SD | 0.49 | 1.31 | 3.7 | 2.7 | 1.4 | 0.5 | 1.2 | 5.0 | 0.1 | 0.1 | 0.1 | 174.8 | 89.1 | | CV % | 6.58 | 5.85 | 13.8 | 13.8 | 13.7 | 13.9 | 9.2 | 12.9 | 9.7 | 10.4 | 22.4* | 6.11 | 7.81 | *It is common for samples with low signals with a mean of 0.6 U/mL which is below the assay 0.1 U/mL cut-off to exhibit higher variability than higher signals. {4} Lot to lot reproducibility: Twenty nine kit lots were tested using seven specimens (two controls and five serum samples) over a period of 116 weeks. The values of the seven specimens were comparable between the twenty nine lots and demonstrated acceptable reproducibility with %CV ranging from 8.4-17.44% CV with the low concentration samples having the high %CV. b. Linearity/assay reportable range: Measuring range Three serum specimens with an initial concentration of >5000 U/mL, 3011 U/mL and 2597 U/mL, were diluted two-folds with kit negative control. The highest dilution for samples 1 and 2 was 1:256 and 1:96 for sample 3. Since these three samples did not cover the lower end of the measuring range of 0.4 U/mL, three additional samples (sample 4 with 2225 U/mL; sample 5 with 2622 U/mL and sample 6 with 5513 U/mL) were tested. Sample 4 was diluted serially from 1:2 through 1:1024 with the lowest result of 0.088 U/mL; sample 5 was diluted from 1:2 through 1:2048 with the lowest result of 0.17 U/mL and sample 6 was diluted from 1:2 through 1:1024. The neat and diluted specimens and the kit negative and positive controls were assayed using the 21-OH Ab RIA kit protocol. The measuring range was determined to be 0.4-5000 U/mL. High Dose Hook Effect No hook effect was observed for 21-OH Ab concentration up to 7826 U/mL. c. Traceability, Stability, Expected values (controls, calibrators, or methods): There is no recognized reference standard available. The 21-OH control and calibrator production are done according to approved standard operating procedures (purchased from approved supplier). Shelf life Stability was established on three lots of 21-OH Ab RIA kit. Samples used included 2 controls (4.8-8.0 U/mL and 52-86 U/mL) and 3 patient samples (from 11-54, 7-29 and 4-15 U/mL). Three additional lots were tested on two patient samples covering the lower ranges (from 0.2-1.5 U/mL). Data from 6 lots support a shelf life of 8 weeks. Real-time testings were performed at 0, 4, 8 and 9 week intervals. 5 {5} d. Detection limit: The limit of blank was computed to be 0.2 U/mL from sequentially testing the zero calibrator 25 times. The limit of detection of 0.344 U/mL was calculated from 25 replicate determinations each from four healthy blood donor specimens. e. Analytical specificity: Interference by endogenous substances: No interference was observed in 10 specimens spiked with 1000 and 3000 mg/dL Intralipids. Interferences with hemoglobin (at 500 mg/dL) and bilirubin (at 20 mg/dL) were observed in eight out of ten samples. The Package Insert states that hemolytic and icteric samples should not be used in this assay. Crossreactivity with other autoimmune diseases and other conditions for autoimmune primary adrenal insufficiency differential diagnosis: The KRONUS 21-OH Ab RIA Assay Kit was tested with 369 sera from other autoimmune diseases and conditions for autoimmune primary adrenal insufficiency differential diagnosis. These samples comprised of 2 premature ovarian failure (POF) (no Addison’s Disease [AD] or Autoimmune Polygrandular Syndrome II [APS II]), 77 Graves Disease, 67 Hashimoto’s Thyroiditis, 10 Rheumatoid arthritis (RA), 150 Type 1 Diabetes Mellitus, 35 Myasthenia gravis, 3 post-tuberculosis Addison disease, 3 hypoparathyroidism, 2 hypopituitarism, 10 celiac disease, 2 vitiligo, 2 alopecia areata, 2 Sjogrens syndrome, 2 sarcoidosis and 2 pernicious anemia. Three hundred sixty-one (361) of 369 specimens were negative with the KRONUS 21-OH Ab RIA Assay Kit. Five positive sera were as follows: 1 Graves Disease serum at 2.2 U/mL; 1 Hashimoto’s Thyroiditis serum at 1.8 U/mL; 4 Type 1 Diabetes mellitus sera at 1.03, 1.06. 1.7 and 18 U/mL; and 2 2 POF (no AD or APS II) at 11.2 and 446.3 U/mL. f. Assay cut-off: The assay cut-off (greater than or equal to 1.0 U/mL is positive) for the KRONUS 21-OH Ab Assay Kit was determined by testing specimens from 243 healthy blood donors. Ninety-eight percent (237) were negative and 6 were positive for 21-OH Ab. For the six positive samples, two had high concentrations (3.7 U/mL and 3.4 U/mL) and the remaining four samples had concentrations no greater than 1.5 U/mL. The mean result was 0.08 U/mL with a standard deviation of 0.36 U/mL. Given these results and taking into account the analytical sensitivity of the assay, values ≤ 1.0 U/mL are considered negative and values > 1.0 U/mL are {6} considered positive for 21-OH autoantibodies. # 2. Comparison studies: a. Method comparison with predicate device: Refer to Clinical studies. b. Matrix comparison: Not applicable. # 3. Clinical studies: a. Clinical Sensitivity and Specificity: The clinical performance was evaluated on 500 clinically defined patient samples. These samples had the following diagnosis: 60 patients with AD; 50 Type II APS, 6 patients with type II APS and POF; 3 patients with AD and POF; 12 Type I APS (very rare); 369 other autoimmune diseases and conditions for autoimmune primary adrenal insufficiency differential diagnosis (same samples as described in the crossreactivity study). The 21-OH Ab RIA Assay Kit sensitivity and specificity were $85\%$ (111/131) and $98\%$ (361/369) respectively (refer to table below). | | Diagnosis | | | | | --- | --- | --- | --- | --- | | | | Positive(Addison disease; Type IIAPS and Type I APS*) | Negative(Non-TargetDiseases) | Totals | | 21-OHAb RIAAssay Kit | Positive | 111 | 8 | 119 | | | Negative | 20 | 361 | 381 | | | Total | 131 | 369 | 500 | Sensitivity: $85\%$ (111/131) $(95\% C.I. = 78 - 90\%)$ Specificity: $98\%$ (361/369) $(95\% C.I. = 96\% - 99\%)$ The following table summarizes sensitivities for the various autoimmune adrenal diseases: | Target Disease | # Evaluated | # Positive | Sensitivity | | --- | --- | --- | --- | | Addison (isolated) | 60 | 44 | 73% | | Addison PAS Type II | 50 | 47 | 94% | | Addison Type I | 12 | 11 | 92% | | Addison PAS Type II with POF | 6 | 6 | 100% | | Addison with POF | 3 | 3 | 100% | {7} b. Other clinical supportive data (when a. is not applicable): Not applicable. 4. Clinical cut-off: Refer to assay cut-off. 5. Expected values/Reference range: The expected value from 237 healthy individual blood donors was 1U/mL. 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.