The Mycoptasma IgG ELISA kit is an Enzyme-Linked Immunosorbent Assay (ELISA) for semi-quantitative or qualitative determination of IgG antibodies in human serum to Mycoplasma pneumoniae for the determination of immunological experience The Mycoplasma IgG ELISA kit may be used to evaluate paired sera for seroconversions and the presence of a significant increase in specific IgG as an aid in the diagnosis of Mycoplasma pneumoniae infection in the adult population.

Device Description

The Mycoplasma IgG ELISA kit is an Enzyme-Linked Immunosorbent Assay (ELISA) for semiquantitative or qualitative determination of IgG antibodies in human serum to Mycoplasma pneumoniae for the determination of immunological experience. The Mycoplasma IgG ELISA kit may be used to evaluate paired sera for the presence of seroconversions and a significant increase in specific IgG as an aid in the diagnosis of Mycoplasma pneumoniae infection in the adult population. For In Vitro Diagnostic Use Only.

The Mycoplasma IgG ELISA test is an enzyme linked immunosorbent assay to detect IgG antibodies to Mycoplasma pneumoniae. Purified Mycoplasma pneumoniae antigen is attached to a solid phase microtiter well. Diluted test sera is added to each well. If the antibodies are present that recognize the antigen, they will bind to the antigen in the well. After incubation the wells are washed to remove unbound antibody. An enzyme labeled anti-human IgG is added to each well. If antibody is present it will bind to the antibody attached to the antigen on the well. After incubation the wells are washed to remove unbound conjugate. A substrate solution is added to each well. If enzyme is present the substrate will undergo a color change. After an incubation period the reaction is stopped and the color intensity is measured photometrically, producing an indirect measurement of specific antibody in the patient specimen.

AI/ML Overview

K971393 Mycoplasma IgG ELISA Test Kit: Acceptance Criteria and Study Details

1. Acceptance Criteria and Reported Device Performance

The acceptance criteria for the Mycoplasma IgG ELISA Test Kit are primarily defined by its "relative" performance against a predicate commercial IFA kit, rather than absolute diagnostic accuracy against a confirmed disease state.

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance (Study 1)	Reported Device Performance (Study 2)
Relative Sensitivity	Not explicitly stated, but generally expected to be high.	95.1% (95% CI: 91.2% - 99.0%)	98.5% (95% CI: 96.4% - 100.0%)
Relative Specificity	Not explicitly stated, but generally expected to be reasonably high.	55.6% (95% CI: 40.7% - 70.4%)	45.9% (95% CI: 29.6% - 62.3%)
Relative Agreement	Not explicitly stated, but generally expected to be high.	84.5% (95% CI: 78.1% - 90.1%)	87.1% (95% CI: 82.0% - 92.3%)
Linearity (r-value)	r ≥ 0.974	All r-values ≥ 0.974 (for 20 positive sera)	Not applicable (linearity assessed for all 20 sera)
Paired Sera Sensitivity	100% for detecting a four-fold increase in antibody level.	100% (56/56 pairs showed >46% rise in ISR values)	100% (7/7 pairs showed >46% rise in ISR values for CF comparison)
Precision (CV)	Intra and Inter-assay CV < 15% (for appropriate technique)	Generally < 15% across varied sera (Tables 4, 5, 6)	Generally < 15% across varied sera (Tables 4, 5, 6)
Reproducibility (Inter-site correlation)	Pearson product moment correlation coefficients > 0.987	> 0.987 (between three sites)	Not applicable
Reproducibility (Percent Agreement of Expected Results)	Not explicitly stated.	99.3% (145/146 excluding equivocals)	Not applicable

Note on "Relative" Performance: The report explicitly states, "Please be advised that 'relative' refers to the comparison of this assay's results to that of a similar assay. There was not an attempt to correlate the assay's results with disease presence or absence. No judgment can be made on the comparison assay's accuracy to predict disease." This is crucial context for interpreting the results.

2. Sample Size and Data Provenance

Test Set (Comparative Studies):
- Study 1: 187 frozen retrospective sera.
- Study 2: 176 frozen retrospective sera.
- Data Provenance: From two R&D laboratories at commercial companies in Maryland and New York, affiliated with the manufacturer. Sera in Study 1 were from "normal individuals of various ages, gender, from Lyme diseases endemic and non-endemic areas." Sera in Study 2 were "randomly selected sera from normal individuals of various ages, gender, and geographical location." Crucially, none of the performance characteristics were established with specimens from patients having documented mycoplasma infections.
Test Set (Linearity): 20 positive sera (serially diluted).
Test Set (Paired Sera Evaluation): 56 pairs of sera (acute sera with ISR < 2.18).
Test Set (Complement Fixation Paired Serum Study): 11 serum pairs from patients suspected of having acute Mycoplasma pneumoniae infection (7 usable pairs).
Test Set (Reproducibility Study): 50 different sera with various levels of activity.

3. Number of Experts and Qualifications for Ground Truth

Number of Experts: Not applicable in the context of expert review for image-based diagnostics. The "ground truth" for the comparative studies was the result of a commercial IFA kit (predicate device).
Qualifications of Experts: Not specified, as the comparison was against another diagnostic test, not expert clinical judgment of individual cases solely.

4. Adjudication Method for the Test Set

No adjudication method described for establishing the primary ground truth (IFA results). The IFA results were taken as the reference.
In the comparative studies (Tables 2 and 3), "equivocals were not included in the above calculations" for sensitivity and specificity, indicating a simple exclusion rather than an adjudication process for those specific cases.
For the 20 sera found positive by IFA but negative by the Wampole ELISA in both studies, it is noted that "* All 20 sera were found to be positive by an alternate ELISA." This suggests a secondary verification, but not a formal adjudication process involving multiple independent readers to establish a definitive consensus ground truth.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, an MRMC comparative effectiveness study was not done. This report describes the performance of a diagnostic kit against a predicate device and its internal consistency, not the improvement of human readers with AI assistance.

6. Standalone Performance Study (Algorithm Only)

Yes, a standalone performance study was done. The entire submission details the performance of the "Mycoplasma IgG ELISA Test Kit" (an assay, not an algorithm in the modern AI sense) as a standalone diagnostic device. The reported sensitivity, specificity, agreement, linearity, paired sera detection, precision, and reproducibility demonstrate its performance without human-in-the-loop assistance for interpretation beyond reading the assay results.

7. Type of Ground Truth Used

Comparative Reference Standard: The primary "ground truth" for the relative sensitivity and specificity studies was the results from a commercial IFA kit (predicate device).
Internal Consistency: For linearity, precision, and reproducibility, the ground truth was derived from the expected behavior of serially diluted samples, repeated measurements, and inter-site comparisons.
Clinical Correlation (Limited): For the paired sera analysis, the "ground truth" was a four-fold increase in antibody level or comparison against Complement Fixation (CF) results for a subset. However, the report explicitly states that there was no attempt to correlate the assay's results with disease presence or absence.

8. Sample Size for the Training Set

Not Applicable. This is an ELISA test kit, not a machine learning algorithm. Therefore, there is no "training set" in the context of AI/ML model development. The development of the kit involved internal optimization and validation, but this is distinct from training a statistical or AI model.

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

Not Applicable. As noted above, there is no training set for this type of device. The development process would have involved establishing the optimal antigen coating, reagent concentrations, incubation times, and cutoff values through internal technical validation, rather than an ML-style "ground truth" establishment for training.

Summary

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K971393

Summary of Safety and Effectiveness Information Mycoplasma IgG ELISA Test Kit

JUL 14 1997

I. Immuno Probe Inc. 1306 Bailes Lane, Suite F Frederick, Maryland 21701 Contact person: William Boteler Telephone: 301-695-7920 Date of preparation: June 11, 1997

II. Description of Device

III. Predicate Device

The Mycoplasma IgG ELISA test is substantially equivalent to the IFA test. Equivalence is demonstrated by the following comparative results:

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PERFORMANCE CHARACTERISTICS

Relative Sensitivity and Specificity

Two different sites compared the Wampole Mycoplasma IgG ELISA test relative to a commercial IFA kit. The two sites were R&D laboratories at commercial companies located in Maryland and New York, and affiliated with the manufacturer of the kit. The 187 frozen retrospective sera from the first study were from normal individuals of various ages, gender, from Lyme diseases endemic and non-endemic areas. The results of the first study are summarized in Table 2. The 176 frozen retrospective sera from the second study were randomly selected sera from normal inividuals of various ages, gender, and geographical location. The results of the second study are summarized in Table 3. None of the performance characteristics were established with specimens from patients having documented mycoplasma infections.

Table 2 Comparison of Wampole Mycoplasma IgG ELISA and IFA Study 1

Wampole Mycoplasma IgG ELISA

	+	eq	-	Total
+	117	13	6	136
MycoplasmaIFA (1:32) -	20*	6	25	51
Total	137	19	31	187

Relative Sensitivity = 117/123 = 95.1%	95% Confidence interval = 91.2% - 99.0%
Relative Specificity = 25/45 = 55.6%	95% Confidence interval = 40.7% - 70.4%
Relative Agreement = 142/168 = 84.5%	95% Confidence interval = 78.19 - 90.1%

All 20 sera were found to be positive by an alternate ELISA.

Equivocals were not included in the above calculations.

The 95% Confidence Intervals were calculated using the normal method.

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Table 3 Comparison of Wampole Mycoplasma IgG ELISA and IFA Study 2

Wampole Mycoplasma IgG ELISA

		+	eq	-	Total
	+	132	5	2	139
MycoplasmaIFA (1:32)	-	20*	0	17	37
	Total	152	5	19	176

Relative Sensitivity = 132/134 = 98.5%	95% Confidence interval = 96.4% -100.0%
Relative Specificity = 17/37 = 45.9%	95% Confidence interval = 29.6% - 62.3%
Relative Agreement = 149/171 = 87.1%	95% Confidence interval = 82.0% - 92.3%

All 20 sera were found to be positive by an alternate ELISA. Equivocals were not included in the above calculations. The 95% Confidence Intervals were calculated using the normal method.

Please be advised that 'relative' refers to the comparison of this assay's results to that of a similar assay. There was not an attempt to correlate the assay's results with disease presence or absence. No judgment can be made on the comparison assay's accuracy to predict disease.

PRECISION

Seven sera were assayed ten times each on three different assays at two different sites. Both sites were affiliated with the manufacturer of the kit. The intra and inter assay precision at each site is shown in Tables 4 and 5. The intersite precision is shown in Table 6. With appropriate technique the user should obtain precision of <15% CV.

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Table 4 Mycoplasma IgG ELISA Intra and Inter Assay Precision Study 1

Assay 1 (n=10)			Assay 2 (n=10)			Assay 3 (n=10)			Inter-Assay(n=30)
Sera#	X	SD	CV	X	SD	CV	X	SD	CV	X	SD	CV
1	0.42	0.054	12.86%	0.36	0.033	9.18%	0.47	0.025	5.48%	0.42	0.054	13.02%
2	0.29	0.043	14.58%	0.23	0.026	11.38%	0.29	0.034	11.91%	0.27	0.043	15.93%
3	3.54	0.274	7.73%	3.24	0.244	7.53%	3.50	0.273	7.78%	3.43	0.274	7.98%
4	1.89	0.133	7.05%	1.76	0.142	8.09%	1.90	0.103	5.42%	1.85	0.133	7.21%
5	0.42	0.059	13.93%	0.33	0.051	15.21%	0.42	0.044	10.45%	0.39	0.059	15.02%
6	1.09	0.103	9.49%	1.03	0.088	8.56%	1.16	0.096	8.28%	1.09	0.103	9.45%
7	2.31	0.218	9.44%	2.21	0.286	12.98%	2.41	0.160	6.66%	2.31	0.218	9.46%
HPC										4.51	0.078	1.72*
Cal										2.50	0.072	2.89%**
LPC										1.31	0.135	10.33%*
NC										0.49	0.049	10.14%*

n = 3
** n = 9

Table 5 Mycoplasma IgG ELISA Intra and Inter Assay Precision Study 2

	Assay 1 (n=10)			Assay 2 (n=10)			Assay 3 (n=10)			Inter-Assay(n=30)
Sera#	X	SD	CV	X	SD	CV	X	SD	CV	X	SD	CV
1	0.23	0.013	5.74%	0.22	0.024	10.76%	0.25	0.028	11.04%	0.24	0.025	10.75%
2	0.17	0.022	12.71%	0.17	0.023	13.58%	0.19	0.018	9.45%	0.18	0.023	12.90%
3	3.58	0.199	5.56%	3.58	0.161	4.51%	3.70	0.154	4.17%	3.62	0.176	4.87%
4	1.84	0.102	5.57%	1.84	0.173	9.41%	2.07	0.135	6.55%	1.91	0.174	9.07%
5	0.35	0.016	4.54%	0.33	0.028	8.37%	0.37	0.029	7.90%	0.35	0.029	8.29%
6	1.15	0.070	6.09%	1.14	0.074	6.47%	1.27	0.078	6.15%	1.19	0.092	7.78%
7	2.22	0.147	6.64%	2.16	0.154	7.13%	2.31	0.105	4.56%	2.23	0.148	6.63%
HPC										3.48	0.191	5.48%*
Cal										2.51	0.056	2.21%**
LPC										1.43	0.140	9.78%
NC										0.13	0.03	23.08%

n = 3
** n = 9

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Table 6 Wampole Mycoplasma IgG ELISA Inter Site Precision Study

Inter-Assay
Sera#	X	SD	CV	n
1	0.33	0.101	30.89%	60
2	0.22	0.057	25.73%	60
3	3.52	0.247	7.01%	60
4	1.88	0.157	8.33%	60
5	0.37	0.050	13.43%	60
6	1.14	0.108	9.50%	60
7	2.27	0.189	8.34%	60
HPC	4.00	0.581	14.53%	6
CAL	2.51	0.063	2.50%	18
LPC	1.37	0.140	10.24%	6
NC	0.31	0.199	64.46%	6

A total of 456 determinations were made at the two sites. The only sera to change status was sera # 6 which was positive 38 times and equivocal 22 times.

X = Mean

SD = Standard Deviation

CV = Coefficient of Variation = SD/X x 100

The methods in NCCLS EP5 were utilized for precision parameters.

LINEARITY

Simulated paired sera evaluation

To evaluate the linearity of the assay 20 positive sera were serially two-fold diluted and run on the assay. The ISR values were compared to logs of dilution by standard linear regression. The r values were all ≥0.974. The data indicates that the antibody can be semi-quantitated by using a single serum dilution. The detection of a significant antibody increase may be made only by an evaluation of paired specimens, acute and convalescent. To validate the sensitivity of the paired sera procedure the percent rise in ISR values were calculated for 56 pairs that had a four-fold dilution where the acute sera had a value of less than 2.18. All 56 pairs demonstrated a >46% rise in ISR value, showing a significant rise in antibody. Therefore, the paired sera procedure demonstrated 100% sensitivity in being able to detect a four-fold increase in antibody level when the acute sera has a value of <2.18.

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Figure 1 illustrates the linearity of a representative serum. The ISR values were compared to log2 of dilution by standard linear regression

Image /page/5/Figure/1 description: The image is a graph titled "Linearity of Mycoplasma ELISA". The x-axis is labeled "Dilution Factor" and has values Neat, 1:2, 1:4, 1:8, and 1:16. The y-axis is labeled "ISR Value" and has values from 0 to 3.5. The graph shows a curve that decreases as the dilution factor increases. The text box on the right side of the graph shows that r = 0.989, slope = 0.66, and y inter = -0.24.

Complement Fixation Paired Serum Study

Eleven serum pairs tested by CF from patients suspected of having acute Mycoplasma pneumoniae infection were assayed on the Wampole Mycoplasma IgG ELISA assay. Each serum pair was evaluated to determine a significant rise in antibody. Four serum pairs could not be used due to the acute serum being too high. The remaining seven pairs all demonstrated a >46% rise in ISR values thus giving a 100% sensitivity versus CF for showing a significant rise in antibody for serum meeting the paired sera criteria.

Reproducibility Study

Fifty different sera with various levels of activity were assayed at three different sites. Two sites were R&D laboratories at commercial companies located in Maryland and New York. The third site was a large clinical laboratory located in Pennsylvania. The data from the three sites show good correlation with Pearson product moment correlation coefficients of >0.987 between the sites. Excluding equivocals (n = 4) one determination varied from its expected result (negative result for a positive specimen) giving a percent agreement of expected results between the three sites of 99.3% (145/146). The expected results were derived from previous Wampole ELISA testing of the samples. Three sera changed status in this study: one serum was equivocal at one site and negative at the other two sites; the second serum was equivocal at two sites and positive at the third serum was positive at one site, equivocal at second site, and negative at the third site.

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Image /page/6/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized eagle or bird symbol, with three human profiles incorporated into the design. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" is arranged around the bird symbol.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

William L. Boteler President -Immuno Probe, Inc. 1306F Bailes Lane Frederick, Maryland 21701

JUL 1 4 1997

K971393 Trade Name: Mycoplasma IgG ELISA Test Kit Regulatory Class: I Product Code: LJZ Dated: June 11, 1997 Received: June 11, 1997

Dear Mr. Boteler:

Re:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to 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 (Act). You may, therefore, market the device, subject to the general controls provisions of the Act. 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.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Paris 800 to 895. A substantially equivalent determination assumes compliance with the Good Manufacturing Practice for Medical Devices: General (GMP) regulation (21 CFR Part 820) and that, through periodic GMP inspections, the Food and Drug Administration (FIDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal Laws or Regulations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770)488-7655. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll free number (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Sutman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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Page 1 of 1

510(k) Number: Not known

Device Name: Mycoplasma IgG ELISA

Indications For Use: The Mycoptasma IgG ELISA kit is an Enzyme-Linked Immunosorbent Assay (ELISA) for semi-quantitative or qualitative determination of IgG antibodies in human serum to Mycoplasma pneumoniae for the determination of immunological experience The Mycoplasma IgG ELISA kit may be used to evaluate paired sera for seroconversions and the presence of a significant increase in specific IgG as an aid in the diagnosis of Mycoplasma pneumoniae infection in the adult population.

PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE)

Prescription Use v (Per 21 CFR 801.109)


OR

Over-The Counter Use (Optional Format 1-2-96)

Aila Rinke

(Division Sign-Off) Division of Clinical Laboratory Devices 510(k) Number _

Regulation Number and Section

§ 866.3375

Mycoplasma spp. serological reagents.(a)
Identification. Mycoplasma spp. serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies toMycoplasma spp. in serum. Additionally, some of these reagents consist ofMycoplasma spp. antisera conjugated with a fluorescent dye (immunofluorescent reagents) used to identifyMycoplasma spp. directly from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria belonging to the genusMycoplasma and provides epidemiological information on diseases caused by these microorganisms.Mycoplasma spp. are associated with inflammatory conditions of the urinary and respiratory tracts, the genitals, and the mouth. The effects in humans of infection withMycoplasma pneumoniae range from inapparent infection to mild or severe upper respiratory disease, ear infection, and bronchial pneumonia.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 866.9.