Immunoglobulin G subclass switching impacts sensitivity of an immunoassay targeting Francisella tularensis lipopolysaccharide

Abstract

The CDC Tier 1 select agent Francisella tularensis is a small, Gram-negative bacterium and the causative agent of tularemia, a potentially life-threatening infection endemic in the United States, Europe and Asia. Currently, there is no licensed vaccine or rapid point-of-care diagnostic test for tularemia. The purpose of this research was to develop monoclonal antibodies (mAbs) specific to the F. tularensis surface-expressed lipopolysaccharide (LPS) for a potential use in a rapid diagnostic test. Our initial antigen capture ELISA was developed using murine IgG3 mAb 1A4. Due to the low sensitivity of the initial assay, IgG subclass switching, which is known to have an effect on the functional affinity of a mAb, was exploited for the purpose of enhancing assay sensitivity. The ELISA developed using the IgG1 or IgG2b mAbs from the subclass-switch family of 1A4 IgG3 yielded improved assay sensitivity. However, surface plasmon resonance (SPR) demonstrated that the functional affinity was decreased as a result of subclass switching. Further investigation using direct ELISA revealed the potential self-association of 1A4 IgG3, which could explain the higher functional affinity and higher assay background seen with this mAb. Additionally, the higher assay background was found to negatively affect assay sensitivity. Thus, enhancement of the assay sensitivity by subclass switching is likely due to the decrease in assay background, simply by avoiding the self-association of IgG3.

Development of Immunoassays for Detection of Francisella tularensis Lipopolysaccharide in Tularemia Patient Samples

“…holarctica (FRAN-012), F. novicida strain U112 and Francisella philomiragia , a near neighbor [ 41 ]. Representative blots showing reactivity of all ten mAbs plus previously isolated F. tularensis mAb 1A4 IgG1 [ 42 ] with purified LPS, and reactivity of the ten newly purified mAbs with pathogenic F. tularensis Schu S4 (Type A) and holarctica (FRAN-012) (Type B) are shown in Figure 1 .…”

“…To evaluate the potential of the isolated antibodies in an LFI format for development into a rapid diagnostic test, all ten mAbs were evaluated in both the capture and detection position for reactivity with purified F. tularensis LPS, along with 1A4 IgG1, an anti- F. tularensis LPS antibody isolated in a previous study [ 42 ]. Initial evaluation of LFIs involved testing with a standard concentration of LPS in buffer compared to a control of buffer alone.…”

Levels of serum IgG subclasses in patients with liver disease: A retrospective study

“…When antigens enter the body, their chemical compositions stimulate an immune reaction, resulting in differential patterns of class switching. For different antigens, such as adhesion protein desmoglein 3, interleukin (IL)-4 and IL-10, class switching tends to occur from IgG1 or IgG3 to IgG4 ( 44 , 45 ).…”

Burkholderia pseudomallei Detection among Hospitalized Patients, Sarawak

“…18 Also, secreted CPS does appear to pass from the blood into the kidneys, where it may accumulate to higher concentrations in urine. 19,20 Serum samples tend to have a low detection rate consequentially.…”