Serodiagnostic comparison of enzyme-linked immunosorbent assay and surface plasmon resonance for the detection of antibody to porcine circovirus type 2.

This paper describes the cloning and expression of the capsid protein of Porcine circovirus type 2 (PCV2) in an Escherichia coli expression system that was used to produce a fusion protein for subsequent immunologic studies: enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). Polymerase chain reaction was used to amplify the gene encoding the capsid protein from the DNA of PCV2. The protein was then cloned into a pRSET prokaryotic expression vector. Western blot analysis revealed that the recombinant protein gave strong signals on a polyvinylidene difluoride membrane when exposed to the serum from a pig infected with PCV2. The expressed protein was purified and used as an antigen for the ELISA and SPR study. A protein chip based on SPR was developed, and the diagnostic potential of SPR was compared with that of ELISA with the use of 70 serum samples obtained from 6 pig farms. There was a strong positive correlation between the ELISA and SPR titers (r = 0.877, P < 0.01). Therefore, this recombinant capsid protein can be used as an antigen for serologic studies, and the SPR, a label-free method, appears to be a valuable and reproducible tool in the serodiagnosis of a PCV2 infection.

Serodiagnostic comparison between two methods, ELISA and surface plasmon resonance for the detection of antibody titres of Mycoplasma hyopneumoniae.

A protein chip based on surface plasmon resonance (SPR) was developed for measuring the Mycoplasma hyopneumoniae antibody titres using a recombinant 30-kDa fragment of P97 adhesin as an antigen. The diagnostic potential of this SPR assay, for detecting the antibody titres to the M. hyopneumoniae 30-kDa protein, was compared with that of conventional ELISA using 70 pig serum samples taken from six pig farms. The SPR assay was found to be highly specific and sensitive. Moreover, there was a strong positive correlation between the SPR and ELISA titres (n = 70, r = 0.898, P < 0.01). Therefore, this recombinant 30-kDa protein can be used as an antigen for serological studies, and the SPR, which is a label-free method, is expected to be a valuable and reproducible tool in the serodiagnosis of M. hyopneumoniae infection.

Serodiagnostic comparison between two methods, ELISA and surface plasmon resonance for the detection of antibodies of classical swine fever.

A protein chip based on surface plasmon resonance (SPR) was developed to measure the antibody (Ab) titers of classical swine fever virus (CSFV) using the recombinant gp55 protein as an antigen. The diagnostic potential of this SPR assay for detecting the Ab titers to CSFV gp55 was compared that of the enzyme-linked immunosorbent assay (ELISA) using 170 serum samples from 14 pig farms. The SPR assay was highly specific and sensitive, and there were no cross-reactions detected. There was a strong positive correlation between the SPR and ELISA titers (n=170, r=0.869, p<0.01). Therefore, the SPR label-free method is a valuable tool in the serodiagnosis of CSFV infection and determining Ab titers after vaccination.

[Detection of the level of antibodies against bovine leucosis virus in the cow milk by immune sensor].

An immune sensor based on the surface plasmon resonance (SPR) was developed for express diagnostics of bovine leucosis. Sensor used for detection of the level of antibodies against bovine leukaemia virus (BLV) in the milk serum. It was shown that immune sensor analysis is more sensitive, rapid and simple in comparison with the traditional AGID test. It was stated that the developed immune sensor may be used for performance of screening of bovine leucosis at the farms and the minimal dilution of the milk serum should be 1:20.

[Application of lectins for determination of antibodies to glycoproteins of leukemia virus in bovine blood serum]. / Vykorystannia lektyniv dlia vyiavlennia antytil do hlikoproteïniv virusu leikozu v syrovattsi krovi velykoï rohatoï khudoby.

The possibility of using different lectins for the determination of level of specific antibodies to leukosis virus virus in bovine blood serum whit the help of the surface plasmon resonance was investigated. The modification of transducer surface with lectins provides for immobilisation of glycosilated proteins of leukaemia virus. It allows to make selective determination of antibodies to glicosilated protein, the quantity of which more high in blood serum of ill animals in the comparison with the immunised ones. Moreover, the treatment of surface with FGA-P and PLA lectins increases the sensitivity of immune sensor to the specific antibodies in the comparison with the non-treated one.

[Express diagnostics of bovine leucosis by immune sensor based on surface plasmon resonance]. / Ekspresna diahnostyka leikozu velykoï rohatoï khudoby za dopomohoiu imunnoho sensora na osnovi poverkhnevoho plazmonnoho rezonansu.

An immune sensor based on the surface plasmon resonance (SPR) was developed for express diagnostics of bovine leucosis. The sensor was used for detection of the level of antibodies against bovine leukaemia virus (BLV) in the blood serum. The industrially manufactured BLV antigen for screening test in the agar gel immunodiffusion (AGID) required the additional purification in order to be used in immune sensor analysis. It was shown that immune sensor analysis was more sensitive, rapid and simple in comparison with the traditional AGID test. It was stated that the developed immune sensor was capable to be used for performance of bovine leucosis screening at the farms and the minimal dilution of the serum should be 1:500.

Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae.

A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera.