Development of Vasoinhibin-Specific Monoclonal Antibodies

Abstract

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Keywords: 16K PRL; ELISA - enzyme-linked immunosorbent assay; monoclonal antibodies; prolactin (PRL); vasoinhibin.

Figure 1

Overview of the antibody development process. (A) A suitable, vasoinhibin-specific epitope at the loop 1 region (L1) was identified on the basis of a three-dimensional model of vasoinhibin as reported by Robles et al. (B) Antibodies obtained after immunization with two immunizing peptides comprising neighboring L1 regions were selected by their affinity to PRL, to the immunizing peptides, as well as to coated vasoinhibin antigen. (C) A sandwich ELISA consisting of two monoclonal, vasoinhibin-specific antibodies was set up.

Figure 2

ELISA of coated vasoinhibin and PRL with vasoinhibin-specific monoclonal antibodies. Four FDK-antibodies and seven HTS-antibodies were tested in a semi-quantitative indirect ELISA for their specificity to detect coated vasoinhibin in comparison to coated full-length PRL. The affinity of each antibody with vasoinhibin represented by its O.D. is plotted on the x-axis, and the affinity of each antibody with PRL by its O.D. on the y-axis, each antibody is indicated as a cross with mean and standard deviation, derived from triplicate measurements. The center point of each cross is the mean O.D. value of the triplicate measurement, and the lines show the range of the standard deviation. The green line represents the signal localization of the cross centers in case of equal affinity against vasoinhibin and PRL. The selected antibodies are shown in red. All tested antibodies are right of the green line toward the x-axis, which demonstrates a preference of vasoinhibin over PRL.

Figure 3

Sandwich ELISA of vasoinhibin and PRL with vasoinhibin-specific monoclonal antibodies. Plots of sandwich ELISA with anti-vasoinhibin monoclonal antibodies showing the affinity with vasoinhibin (1.0 µg/ml) (x-axis) and PRL (1.0 µg/ml) (y-axis). The center point of each cross is the mean O.D. value of the triplicate measurement, and the lines show the range of the standard deviation. The green line represents the signal localization of the cross centers in case of equal affinity against vasoinhibin and PRL. (A) shows sets of antibody pairs tested on Microplate A, each pair is indicated as a cross with mean and standard deviation. (B) shows the affinity of another set of antibody pairs on Microplate B with suitable affinities that do not recognize PRL. Affinities in the negative scale of the PRL y-axis are a result of wells containing PRL standard being even less reactive than blank wells. Sandwiches C10F-C50H, C1F-C50H, and C50H-C10F show high affinity to vasoinhibin and no affinity to PRL.

Figure 4

Vasoinhibin ELISA standard curves. Sandwich ELISA standard curves of antibody pairs C1-C50 (A), C50H-C10F (B) and C10F-C50H (C) against recombinant human vasoinhibin standard with the interpolated Rodbard curve with BLK-subtracted values. The O.D. at the bottom and the top of each curve is indicated. IC50 values are the analyte concentrations at the O.D. halfway between the top and the bottom.

Figure 5

Vasoinhibin ELISA in presence of PRL. Sandwich ELISA of recombinant human vasoinhibin standard in the presence of various concentrations of PRL standard using sandwich C50H-C10F (A) and C10F-C50H (B).