Generation and evaluation of monoclonal antibodies

None of the polyclonal antibodies detected endogenous CFAP43 convincingly in immunocy-tochemistry so far. Therefore monoclonal antibodies were generated against a purified frag-ment of CFAP43 expressed in bacteria. Three expression constructs (see figure 2.7 A) were prepared comprising fragments of CFAP43, which would based on structure predictions be expected to fold correctly independently from the rest of the protein. These fragments were MBP-tagged, because MBP was reported to increase the solubility of proteins (Kapust and Waugh, 1999) and earlier attempts of CFAP43 overexpression and purification resulted in insoluble protein (Mai, 2012). Expression of the coiled-coil domain (referred to as fragment 2, figure 2.7 A) resulted in the highest protein amount, but non of the fusion constructs was soluble under the tested conditions. Thus, monoclonal antibodies were generated using fragment 2 for immunization. The fusion protein was expressed in E. coliunder control of an IPTG-inducible promoter. The fractions resulting from protein overexpression and purifi-cation are shown in figure 2.7 B. When comparing the lysates of non-induced vs. induced

Figure 2.7: Purification of a region of the CFAP43 coiled-coil domain for monoclonal antibody generation. AThree fragments of CFAP43 were expressed as MBP-fusion proteins for generation of monoclonal antibodies. BA coomassie-stained SDS-PAGE of the purification steps showed most of the overexpressed fragment 2 to be in the insoluble part of the bacteria culture. Inclusion bodies were purified and tested for solubilization. C Concentration of the purified CFAP43 coiled-coil domain (fragment 2) was determined by comparing known amounts of albumin with varying volumes of the resuspended inclusion bodies.

bacteria the overexpressed fusion construct is clearly visible as a band with the expected apparent molecular weight of 60 kDa. Since most of the protein remained in the insoluble fraction despite different attempts for solubilization, inclusion bodies were purified. Protein concentration was determined comparing different amounts of the purified inclusion bodies with known amounts of albumin. Finally 1 mg protein was used for immunization of rats and mice and the subsequent generation of monoclonal antibodies in collaboration with Dr.

Elizabeth Kremmer (Helmholtz center Munich). By ELISA screening 62 antibodies from rat as well as 16 antibodies from mouse were identified, which interacted specifically with the CFAP43 fragment used for immunization.

All resulting antibodies were further characterized by their ability to detect overexpressed CFAP43 in western blot and immunocytochemistry. Antibodies, which detected overex-pressed CFAP43 in western blot, were also tested regarding their ability to detect endoge-nous CFAP43 in western blot (exemplary shown in figure 2.8 A). The blots contained lysates

2.2 Generation and characterization of antibodies against CFAP43

Figure 2.8: Characterization of monoclonal antibodies. ARepresentative western blots showed some monoclonal antibodies detecting overexpressed and endogenous CFAP43 (asterisks). For comparison a western blot containing the same samples was stained with the P4 antibody. B As for P4 (see figure 2.6 A), detection of CFAP43 by monoclonal antibodies in immunofluorescence was tested on L-cells expressing endogenous CFAP43 as well as after doxycyclin-induced overexpression and siRNA-mediated knock-down ofCfap43. As representative images of a antibody recognizing overexpressed CFAP43 pictures of cells stained with M41 are shown. CImmunohistochemistry was carried out to test the antibodies for their ability to detect endogenous CFAP43. Selected pictures are shown from

of CFAP43-overexpressing L-cells, L-cells expressing only the endogenous protein and testis lysate from an adult mouse. The expected size of CFAP43 is marked by the green box. Under these conditions P4, which was used as a positive control, detected only the overexpressed protein. In contrast some monoclonal antibodies showed a faint band of expected size also in the testis lysate (e.g. M41, M42, M58, M71, M73, M74). To test the ability of the anti-bodies to detect CFAP43 in cells or tissue sections, immunocytochemistry was performed on L-cells allowing for inducible CFAP43 overexpression, as already performed with antibody P4 (see section 2.2.1). The results for M41 are exemplary shown in figure 2.8 B. Antibodies, which resulted in a strong signal of overexpressed CFAP43 in L-cells were further tested in immunohistochemistry on lung and testis sections (see figure 2.8 C). Some antibodies pro-duced high background (e.g. M43, c, d), whereas others resulted in a specific pattern lining the epithelia of large airways in the lung (arrowheads, a, c, e, g) and a strong signal in seminiferous tubules of the testis during different phases of spermiogenesis (M7, M53, red asterisks; or M43, 78, black asterisks). The latter case resembles more the sectionin situ hy-bridization pattern described by Mai (2012), nevertheless staining with different antibodies did not result in the same or a very similar pattern between the different antibodies, which is most obvious in the testis sections (e.g. compare M7 with M43 and M53). The differing patterns seen in testis sections can be explained by the antibodies staining other proteins than CFAP43 or by expression of CFAP43 during different stages of spermiogenesis. Thus it is not clear, whether the tested antibodies indeed detected CFAP43 in this experiment. All experiments performed for characterization of the antibodies are summarized in table 2.1.

2.2 Generation and characterization of antibodies against CFAP43

Table 2.1:Summary of the results of the monoclonal antibodies^∗screening No host WB OE WB testis ICC OE IHC lung IHC testis

1 rat ✗ n.d. ✓ n.d. n.d.

∗Antibodies, which did not detect CFAP43 under any tested conditions are

∗excluded from this table.

Abbreviations: WB = western blot, OE = overexpression, ICC = immunocytochemistry, IHC = immunohistochemistry n.d. = not determined