Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease

Abstract

The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD.

FIG 1

The flow diagram for preparation of antigen I to V. B. henselae harvested from the agar medium was sonicated and treated with sarcosine. The preparation was variously processed to obtain antigen I through antigen V.

FIG 2

The reactivity of fractions from DEAE-Sepharose to anti-B. henselae IgM antibody. The sonicated B. henselae whole-cell proteins were processed by DEAE-Sepharose Fast Flow ion-exchange chromatography. The profiles of the protein concentrations (conc) (optical density [OD] at 280 nm) and reactivity to anti-B. henselae IgM antibody are depicted by dashed and solid lines, respectively.

FIG 3

SDS-PAGE and Western blot analyses of 5 proteins associated with Bartonella henselae. An SDS-PAGE analysis was performed with the use of silver staining for 5 different protein preparations. Wc, whole-cell B. henselae; Ins, 0.4% N-lauroyl sarcosine-insoluble proteins; Sol, 0.4% N-lauroyl sarcosine-soluble proteins; R-ins, sarcosine-insoluble proteins refined by DEAE-Sepharose chromatography; R-sol, sarcosine-soluble proteins refined by DEAE-Sepharose chromatography. A Western blot analysis using a chemiluminescent reaction was performed to examine the reactivity to anti-B. henselae IgM of two pools of sera, one composed of sera from 3 CSD patients and the other composed of sera from 3 healthy individuals (shown in the right two panels). Molecular size markers (kDa) are indicated at the left at each panel. Ag, antigen.

FIG 4

Determinations of contents of the sera in the reference panel by the 5 IgM-ELISAs. The reference serum panel (5 positive and 5 negative sera) was tested by the 5 IgM-ELISAs. The test results of each ELISA are expressed as optical density (OD). The 5 different antigen preparations were sonically disrupted B. henselae (Whole cell [antigen I]); 0.4% N-lauroyl sarcosine-insoluble and -soluble proteins of B. henselae (Insoluble [antigen II] and Processed soluble [antigen III], respectively); and sarcosine-insoluble and -soluble proteins refined by DEAE-Sepharose chromatography (Refined insoluble [antigen IV] and Refined soluble [antigen V], respectively).