Antigen: The CF antigen is prepared by GenBio and standardized using ImmunoWELL IgM units.

1. Frequency of Mycoplasma pneumoniae Complement Fixation IgM in a Healthy, Adult Population B. Kiehl1 and S. Jeansson2 1GenBio, San Diego, 2Oslo University Hospital, Oslo, Norway Abstract Summary Table 2: Percentile Rank of U.S. healthy adult population expressed in IgM Units Methods & Materials Mycoplasma pneumoniae is the causative agent of atypical pneumonia. Serological diagnosis detecting antibodies to either an immunodominant membrane protein or the complement fixing lipid antigen is often used. These Mycoplasma antibodies are cross-reactive with other Mycoplasma pneumoniae species, so clinical interpretation is made using a clinical cutoff. The clinical cutoff of ImmunoWELL™ Mycoplasma Pneumoniae IgM Test, using a purified CF antigen, is evaluated using presumptively healthy U.S. blood donors. Materials and Methods: Fourteen hundred twenty-six (1426) sera collected from potential U.S. blood donors collected at centers located in thirteen states is tested. Tests are performed using ImmunoWELL Mycoplasma IgM Test following package insert instructions. Results: Figure 1 illustrates specific antibody distribution in a U.S. healthy, adult population. Summary: The 95th percentile (736 to 917 units/mL)is consistency with ImmunoWELL’s 950 units/mL clinical cutoff. The 75th percentile is 295-319 units/mL. Figure 1 Antigen: The CF antigen is prepared by GenBio and standardized using ImmunoWELL IgM units. Specimens: Fourteen hundred twenty-six (1426) sera collected from potential U.S. blood donors during 2010 and 2011 are tested. The specimens are collected in thirteen states by Interstate Blood Bank, Memphis, TN, USA. ImmunoWELL:GenBio tested approximately eighty specimens every one to two weeks using ImmunoWELL Mycoplasma Pneumoniae IgM Test following package insert instructions. ImmunoFLOW:ImmunoFLOW Mycoplasma, a flow-through rapid test method, uses this same CF antigen and its clinical cutoff is calibrated using the above standards. A small study conducted by an outside European laboratory tested eighteen (18) specimens using ImmunoCard (Meridian Diagnostics) and ImmunoFLOW following respective package insert instructions. Mycoplasma pneumoniae specific antibody is detected in healthy subjects, presumably because of surface antigen cross-reactivity between Mycoplasma species. Prior to commercial test kits with clinical cutoffs defined by prospective clinical study, acute and convalescent sera (“serum pair”) was required for clinical interpretation. A statistical difference between the specimens is used to aid disease diagnosis. To assure specificity, kits with a clinical cutoff (opposed to the analytic cutoff or older kits) are set at a much higher antibody threshold. This study measures specific IgM in a presumptively healthy population using the semi-quantitative test ImmunoWELL Mycoplasma Pneumoniae IgM. This test detects specific IgM units/m reporting values above 200 units/mL; however, the clinical cutoff is 950 units/mL. Although presumptively healthy U.S. blood donor adult population is not the intended test population, it is easily tested and used by the manufacturer as a means to assure product performance clinical cutoff consistency. During the initial 1996 ImmunoWELL prospective validation study a similar U.S. blood donor population was tested and reported as expected results. The current study results are more extensive and report substantially the same results. Table 2 shows the percentile rankings, corresponding to results. The 95-percentile ranking is 807 units/mL (736 to 917 units/mL) which is consistent with ImmunoWELL’s clinical cutoff. It was also shown that by using ImmunoWELL’s semi-quantitative results to establish a qualitative test method (ImmunoFLOW), good correlation to an alternative qualitative clinical test (see Table 1) is seen. Results Introduction Figure 1 illustrates anti-CF distribution measured using ImmunoWELL Mycoplasma Pneumoniae IgM Test in a presumptively healthy U.S. adult population. Mycoplasma pneumoniae is the causative agent of atypical pneumonia. Sero-diagnosis detects antibodies against either an immunodominant membrane protein or complement fixing (CF) lipid antigens. Because the membrane protein is water soluble and the CF lipid is not water soluble, only one immune reaction type is detected by most assay methods. Additionally, some older serology kits report analytic results (“epidemiological results”) while newer kits are required to report a clinical cutoff. In the 1980’s, Dr. Stig Jeansson developed an enzyme immunoassay (EIA) method detecting anti-CF. ImmunoWELL (unpublished method). Mycoplasma Pneumoniae IgM Test uses this original design and more recently ImmunoFLOW Mycoplasma Pneumoniae IgM Test also uses the antigen. Originally, the ImmunoWELL™ Mycoplasma Pneumoniae IgM Test’s clinical cutoff was established using healthy U.S. blood donors and validated in a prospective study comparing its single IgM interpretation to acute and convalescent (“paired serum”) results. This new study illustrates the incidence of Mycoplasma pneumoniae IgM in a similar U.S. healthy adult population. Conclusions • These results are substantially the same as originally observed (1996) to establish the kit’s clinical cutoff. • Using standards defined using th3 IgM semi-quantitative test, a rapid qualitative test using the same M. pneumoniae CF antigen yields correlative results to an alternative qualitative test. Figure 1: M. pneumoniae IgM Distribution in U.S. Healthy Adult Population Table 1 shows Mycoplasma IgM test results of eighteen clinical specimens using ImmunoCard and ImmunoFLOW, a flow-through test method using the same Mycoplasma pneumoniae CF antigen. Note: ImmunoFLOW is not available within the U.S.; however it is available in the European Union (CE Mark) and other international markets. Table 2: ImmunoCard and ImmunoFLOW Comparison Contact Information Bryan Kiehl (bkiehl@GenBio.com) Mobile (+1 760-855-8999)