Establishment And Application Of PRRSV-2 Antigen And Antibody Detection Methods Based On Monoclonal Antibodies

Porcine reproductive and respiratory syndrome virus（PRRSV）mainly causes reproductive failure in pregnant sows and respiratory diseases in piglets and fattening pigs.The latest classification criteria have divided the original two genotypes into two different species:Betaarterivirus suid 1（European type/PRRSV-1）and Betaarterivirus suid 2（North American type/PRRSV-2）,respectively,which means that the diagnosis and prevention of these two types PRRSV should be distinguished.The rapid diagnosis of PRRSV in the early stage of infection can effectively control its spread and epidemic.At present,PRRSV-2 is the main epidemic strain and PRRSV-1 is a potential epidemic strain in China.Combined with the needs of rapid detection of PRRSV vaccine and local clinical samples,and specific detection of PRRSV antibody,this study aims to establish a rapid detection method for PRRSV-2antigen and antibody by preparing monoclonal antibodies（MAbs）specific to PRRSV-2.In this study,mice were immunized with purified PRRSV-2 Hu N4-F112 strain whole virus protein and prokaryotic expressed SD53-1603 strain recombinant N protein as immunogens,respectively.Five MAbs against PRRSV N protein and one against M protein were successfully obtained by the fused hybridoma cells.Among them,1H9,1G5 and 3G5,which can specifically recognize PRRSV-2representative strains,were selected for pairing screening with MAbs 1C8,1H4 and 4E3 preserved in our laboratory.The optimal combination of 1H4/1H4-HRP antibody pairs was determined,and the reaction conditions were optimized.An antigen capture ELISA（AC-ELISA）method for the detection of PRRSV-2 N protein was successfully established.This method has good sensitivity,specificity,repeatability and broad-spectrum detection.It has no cross-reaction with PRRSV-1 representative strains and other major porcine viruses.It can specifically detect various PRRSV-2 strains in China,and the minimum detection limit of PRRSV-2 was 1.45×10^3.0 TCID₅₀/m L and that of recombinant N protein was 18.75 ng/m L.The coefficient of variation of the intra-batch repeated test was less than 10%,and the coefficient of variation of the inter-batch repeated test was less than 15%.There was an exponential relationship between vaccine titer and AC-ELISA test results at different time points.The relative coincidence rate of AC-ELISA and PCR method was 83.96%.The PRRSV N protein could be detected from the serum in fifth days after infection,which could be applied to the laboratory batch screening of PRRSV-2 in clinical samples.On this basis,an immunochromatographic strip（ICS）for the detection of PRRSV-2 N protein was established by using MAb 1H4 screened by AC-ELISA as both detection antibody and capture antibody,and red latex microspheres as labeling tracer.This method can specifically detect PRRSV-2representative strains in China,and has no cross-reaction with PRRSV-1 representative strains and other common viruses in pigs.The minimum detection limit for PRRSV-2 was 5×10^2.0 TCID₅₀/m L,and the minimum detection limit for recombinant N protein was 15 ng/m L.There was no difference in the detection results of intra-batch and inter-batch test strips.It can be stored at room temperature for at least 6 months,and the overall coincidence rate with the PCR method was 83.33%.The detection effect of porcine serum infected with PRRSV-2 strains（Highly pathogenic strain,NADC30-like strain,and Highly pathogenic-like strain）was good.And PRRSV-2 N protein could be detected from serum in fifth day after infection,which was very suitable for rapid screening of PRRSV-2 in the field.In order to establish an antibody detection method with higher specificity than indirect ELISA method,a MAb 1G5 that can specifically detect PRRSV-2 was screened in this study,which had the best blocking effect.On this basis,a blocking ELISA（B-ELISA）method that can specifically detect PRRSV-2 N protein antibody was successfully established.The optimal coating concentration of whole virus antigen（12μg/m L）,the optimal dilution of HRP-1G5（1:6000）and the optimal dilution of pig serum（1:2）were determined by optimizing the reaction conditions.By detecting 174 negative pig serum and 451 positive pig serum,the receiver operating characteristic curve analysis was performed to determine the optimal critical value PI of 40%when the diagnostic specificity and diagnostic sensitivity were 96%and 99.8%,respectively.The coefficient of variation of intra-batch and inter-batch repeatability tests was less than 10%,indicating that B-ELISA had good repeatability.There was no cross-reaction with PRRSV-1 and other major swine virus positive serum,indicating that B-ELISA had high specificity.B-ELISA can detect 32-fold and 256-fold diluted 14#and 71#positive serum,which is better than the 16-fold and 128-fold diluted 14#and 71#positive serum by IDEXX detecting,indicating that B-ELISA has higher sensitivity.215 clinical serum samples collected from Heilongjiang Province were detected by B-ELISA,of which 197（91.63%）were PRRSV-2 antibody positive,and the consistency with the commercial IDEXX kit was 99.06%（kappa value=0.989）.It can be used as a alternative to the imported IDEEX PRRSV kit,and provides a new detection method for PRRSV-2epidemiological investigation and antibody level monitoring.