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The ImmuSTRIP® HAMA IgG ELISA Test System is a direct enzyme-linked immunosorbent assay for the detection and semi-quantitation of human antibodies to mouse IgG1. Significant titers of human anti-mouse antibody (HAMA) have been associated with patients receiving injections of murine monoclonal antibody for diagnostic and/or therapeutic purposes2 .

The ImmuSTRIP" HAMA IgG ELISA Test System is a direct enzyme linked immunosorbent assay for the semi-quantitative detection of human antibodies to mouse IgG (HAMA).

The HAMA assay is a two-stage test carried out in plastic microwell strips which have been coated with mouse IgG, whole molecule.

In the first stage, mouse IgG conjugated to horseradish peroxidase (conjugate) is added to the microwell. Diluted test sample is then added and incubated for a specified length of time. If antibody to the mouse IgG is present in the test sample, bridging will occur with the solid phase mouse IgG, the test sample antibody, and the conjugate. If antibody is not present in the test sample, the unbound conjugate will be removed in the subsequent washing step.

In the second stage, enzyme substrate is added to the microwell. If bound conjugate is present, the substrate will be reduced; the reduced end product of the catalytic reaction oxidizes the colorless chromogen resulting in a colored end product. Acid is added to stop the reaction and fix the color.

The color intensity is proportional to the amount of bound conjugate and, therefore, to the amount of precipitable antibody present in the sample. The color intensity is measured with a microwell strip reader.

The ImmuSTRIP" HAMA ELISA Test System may be performed manually or with existing microtiter equipment. Results, which are determined by optical density at 488-492 nm, are available in less than one hour. The test has been standardized against primate anti-mouse IgG serum and has a sensitivity of approximately 40 ng/ml. Final values are reported as nanograms of precipitable antibody equivalents per ml.

The assay system is a rapid, efficient, and semi-quantitative method. Specific reagent formulation has eliminated the problem of background interference currently existing in this type of assay.

Acceptance Criteria and Device Performance Study for ImmuSTRIP® HAMA IgG ELISA Test System

1. Acceptance Criteria and Reported Device Performance

• ImmuSTRIP HAMA IgG: 71.4% positive (30/42)
• Reference Method: 64.3% positive (27/42)
• With Reference Method as GT: Sensitivity = 85.2%, Specificity = 66.7%, Accuracy = 78.6%, PPV = 82.1% |
  | Clinical Utility (Impact on Pharmacokinetics/Dosimetry) | Demonstrate relationship between HAMA levels and impact on pharmacokinetics and dosimetry. | Study demonstrated that at HAMA titers below 300 ng/ml, no effect on antibody clearance rates was apparent. Above 300 ng/ml, a rapidly increasing plasma and whole-body clearance rate was observed, reflected by decreasing red marrow and whole-body doses. |

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

• Reproducibility (Within-run and Between-run): Not explicitly stated how many individual replicates were performed, but CVs were calculated for replicates of three for low, medium, and high control samples.
• Lot-to-lot Reproducibility: 6 prepared serum controls and 15 normal serum samples.
• Specimen Dilution: 4 positive HAMA specimens.
• Recovery: A normal human serum pool spiked at 6 different HAMA concentrations (50 ng/ml to 400 ng/ml), assayed in quadruplicate for each concentration.
• Specificity (Normal Population): 464 healthy individuals (250 males, 110 non-pregnant females, 104 pregnant females). Data Provenance: Dianon Systems, Inc. (Stratford, CT), retrospective.
• Specificity (Rheumatoid Factor Samples): 57 serum samples with varying concentrations of rheumatoid factors. Data Provenance: Samples sequestered from Universal Reagents, Inc., and Scantibodies Laboratories, Inc., assayed by Dianon Systems, Inc.
• Clinical Utility (HAMA detection in patients receiving MAb):
  • Retrospective Study: 26 patients from Center for Molecular Medicine and Immunology (CMMI), Newark, NJ, and 51 patients from VA Medical Center, Buffalo, NY. A total of 305 pre- and post-injection samples from 77 patients.
  • Prospective Study: 240 patients (out of 382 entered) from CEA-Scan Phase III study who had baseline and at least one follow-up measurement.
  • Data Provenance: Clinical studies were conducted at two medical institutions (CMMI and State University of New York at Buffalo, Dept. of Nuclear Medicine) and one reference laboratory (Dianon Systems, Inc.). The original submission states retrospectively for determination of HAMA incidence in normal population and in patients, and prospectively for patients receiving CEA-Scan.
• Clinical Utility (Comparison to Reference Method): 61 samples (19 pre-injection, 42 post-injection) from 28 patients. Data Provenance: Samples provided by Dr. Robert Sharkey of CMMI, assayed by Dr. M. Khazaeli of HAMA-KINE, Inc.
• Clinical Utility (Impact on Pharmacokinetics/Dosimetry): 57 patients with CEA-expressing tumors. Data Provenance: From a published study in The Journal of Nuclear Medicine, June 1997.

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

The document does not explicitly state that experts were used to establish the ground truth for the test set. Instead, the primary "ground truth" or reference for performance characteristics relies on:

• Known concentrations: For reproducibility, specimen dilution, and recovery, purified HAMA was added to serum to create known concentrations.
• Reference Method: For comparative studies, a "double-antigen, radiometric assay method," referred to as "The Reference Method," was used as a comparator (implicitly considered a reference for comparison, not necessarily a 'ground truth' established by experts in the typical sense of diagnostic imaging reader studies). The assay was performed by Dr. M. Khazaeli, of HAMA-KINE, Inc. (no specific qualifications other than being responsible for that lab's testing).
• Clinical Outcomes/Diagnosis: For clinical utility sections, the patient's status (e.g., received murine monoclonal antibody, diagnosed with rheumatoid arthritis) serves as context for HAMA detection, rather than an expert ground truth for individual HAMA presence.
• Pathology/Outcomes Data: For the study on pharmacokinetic/dosimetry impact, clinical outcomes data (e.g., antibody clearance rates, red marrow and whole-body doses) linked to HAMA levels were used.

4. Adjudication Method for the Test Set

No explicit adjudication method is mentioned for establishing ground truth or resolving discrepancies, as the studies primarily rely on quantitative measurements against known standards or a reference assay. For the comparative study with the reference method, discrepancies between the ImmuSTRIP® ELISA and the RIA method are reported as false positives/negatives, suggesting a direct comparison rather than an adjudicated outcome being derived.

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

No, an MRMC comparative effectiveness study was not done. The device is an ELISA assay, not an imaging or diagnostic algorithm typically evaluated with MRMC studies involving human readers.

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

Yes, the studies presented are all standalone validations of the ImmuSTRIP® HAMA IgG ELISA test system. The device is an in-vitro diagnostic test, and its performance is evaluated directly through laboratory measurements, not as an AI algorithm that assists human interpretation.

7. The Type of Ground Truth Used

• Known concentrations: For reproducibility, specimen dilution, and recovery, the ground truth was established by spiking normal human serum with purified HAMA to create samples with known concentrations of HAMA.
• Reference Assay: For the comparative study, the "double-antigen, radiometric assay method" (referred to as "The Reference Method") served as the comparative baseline for evaluating agreement, implicitly acting as a form of ground truth for comparing detection rates.
• Clinical Status/Literature: For specificity in a normal population, the absence of known exposure to murine antibodies was considered "negative." For clinical utility in exposed patients, the patient's history of receiving murine monoclonal antibodies was the context, with elevated HAMA levels being the documented finding. Discussion also includes agreement with "distribution of HAMA values from similar populations cited in numerous publications."
• Pathology/Outcomes Data: For pharmacokinetics and dosimetry, the clinical parameters and physiological responses (clearance rates, doses) were the ground truth for assessing the impact of HAMA.

8. The Sample Size for the Training Set

The document does not describe a "training set" in the context of machine learning or AI. This is a traditional IVD device. However, the reference standard and controls used for calibrating and evaluating the assay were developed from baboons immunized with murine anti-CEA monoclonal antibody (IMMU-4 IgG). The process involved:

• Immunization of baboons to produce anti-mouse antibodies.
• Collection of serum samples during immunization.
• Testing by RID until significant levels of anti-mouse antibody were achieved.
• Pooling collected sera and freezing at -80°C.
• Determining HAMA activity by RID.
• Preparing a dilution of the pooled sera to 220 ng of precipitable antibody equivalents/ml to serve as the reference standard.

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

As noted above, there isn't a "training set" in the AI sense. However, the reference standard for the ImmuSTRIP® HAMA assay (which guides the assay's performance and quantification) was established by:

• Immunizing baboons with a known murine monoclonal antibody (IMMU-4 IgG) to induce the production of anti-mouse antibodies (HAMA).
• Monitoring the development of anti-mouse antibodies in the baboon sera using Radial Immunodiffusion (RID) until significant levels were reached.
• Quantifying the HAMA activity in the pooled baboon sera via RID to precisely determine the concentration of precipitable antibody equivalents. This allowed for the standardization of the reference material at 220 ng precipitable antibody equivalents/ml. Thus, the ground truth for the reference standard's concentration was established through a validated immunological method (RID) on a well-characterized biological source.

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