Development of luciferase-linked antibody capture assay based on luciferase immunoprecipitation systems for antibody detection of porcine reproductive and respiratory syndrome virus

Abstract

BackgroundEarly detection of porcine reproductive and respiratory syndrome virus (PRRSV) infection of swine is necessary to control this devastating disease. By monitoring host serum antibodies to viral antigens, early virus detection within herds is feasible. In this study, recombinant antigens were generated using recombinant DNA techniques to fuse PRRSV structural protein (N) or nonstructural protein 1α (nsp1α) with the Rellina luciferase gene. Next, fused genes were cloned into plasmids and transfected into HEK-293 T cells for transient expression. Upon co-incubation of lysates with pig sera, antigen-antibody complexes formed that bound to Protein-G coated onto microplates. By further measurement of luminance value, a modified form of Luciferase Immunoprecipitation Systems, namely luciferase-linked antibody capture assay (LACA) was developed for detection of PRRSV-specific antibodies.ResultsKnown anti-PRRSV antibody-positive or -negative serum samples (125 and 122 samples, respectively) were used to validate the LACA and compared it with IDEXX PRRS ×3 ELISA. Based on the result, N-Rluc and nsp1α-Rluc LACA results were 95.3 and 94.4% in agreement with IDEXX ELISA, suggested a similar specificity of LACA to IDEXX ELISA. Moreover, when both LACA and IDEXX ELISA were used to evaluate sequential serum samples obtained from PRRSV experimentally infected pigs, the PRRSV-specific antibody response was detectable as early as 3 days post-inoculation (dpi) using N-Rluc LACA, but undetectable until 7 dpi using IDEXX ELISA, suggesting an improved sensitivity of LACA. Meanwhile, antibodies specific for nsp1α were detected at higher levels overall, but were undetectable until 10 dpi. Furthermore,. Notably, one IDEXX ELISA positive result was not confirmed by LACA or IFA and was thus considered a false-positive result.ConclusionThe LACA exhibited similar specificity but improved sensitivity to that of the commercial IDEXX PRRS ×3 ELISA kit for detection of PRRSV-specific antibodies in pig serum. Importantly, LACA could be adapted for detecting antibodies against other PRRSV targets, such as nsp1α, to achieve earlier detection of PRRSV infection.

Porcine Reproductive and Respiratory Syndrome Virus Promotes SLA-DR-Mediated Antigen Presentation of Nonstructural Proteins To Evoke a Nonneutralizing Antibody Response In Vivo

“…It appeared that N-specific antibodies were developed within 7 dpi (Fig. 2C), which is consistent with the time point of serum conversion in infected pigs, as determined by Idexx PRRSV ELISA in our previous report (41). Meanwhile, antibodies recognizing NSPs could be detected within 14 dpi as determined by the visualization of 130-kDa bands by Western blotting; it appeared that anti-NSP antibodies in pig serum persisted at a much higher level until 28 dpi, as the relative signal strength observed in the Western blot for the 15-kDa band (potential N protein) became much weaker than the signal strength for the 130-kDa band (Fig.…”

“…All cDNA sequences for NSP immunopeptides were synthesized and cloned to the 3= end of the cDNA of NanoLuc and expressed in HEK-293T cells (Fig. 9A) according to the LACA protocol described previously (41). LACA analysis revealed that for immunopeptides originating from NSP1␤, NSP2, and NSP10, the S/N ratios of serum samples obtained from PRRSV-infected piglets increased from 5-to 10-fold and demonstrated statistical significance compared to the results for negative controls (Fig.…”

“…In our previous report, we developed a modified assay based on luciferase immunoprecipitation (IP) systems (LIPS) to quantify PRRSV-specific antibodies from serum and referred to it as the luciferase-linked antibody capture assay (LACA) (41). Based on the analysis of early antibody profiles in PRRSV-infected hosts, it was suggested that antibodies against PRRSV NSP1␣ could reach an even higher level than antibodies to the PRRSV N protein, as determined by the sample-to-negative ratio (S/N ratio) (41).…”

“…In our previous report, we developed a modified assay based on luciferase immunoprecipitation (IP) systems (LIPS) to quantify PRRSV-specific antibodies from serum and referred to it as the luciferase-linked antibody capture assay (LACA) (41). Based on the analysis of early antibody profiles in PRRSV-infected hosts, it was suggested that antibodies against PRRSV NSP1␣ could reach an even higher level than antibodies to the PRRSV N protein, as determined by the sample-to-negative ratio (S/N ratio) (41). Meanwhile, by employing a homemade monoclonal antibody (MAb) against the swine leukocyte antigen II DR ␣ chain (SLA-DR␣), we noticed that PRRSV infection of BMDCs induces upregulation of SLA-DR at the protein level and promotes surface expression of SLA-DR in PRRSV-infected BMDCs (42).…”

Evaluation of Antibody Response Directed against Porcine Reproductive and Respiratory Syndrome Virus Structural Proteins

“…This is unlikely due to the false positive results because all of the seronegative samples (e.g., serum samples collected from experimental pigs before they were infected with a PRRSV strain and ELISA-negative serum samples collected from the field) tested negative by LIPS-N, demonstrating the high specificity of this assay. Recently, a modified form of the LIPS, namely luciferase-linked antibody capture assay (LACA), was developed for detection of PRRSV-specific antibodies [ 42 ]. It was reported in this later study that the LACA was able to detect antibody specific to N protein as early as 3 dpi while the IDEXX ELISA was not able to detect PRRSV-specific antibodies until 7 dpi.…”