Immunological characterization of a recombinant 27-kilodalton antigenic protein from Paracoccidioides brasiliensis

Abstract

We report the expression in Escherichia coli of a 27-kDa antigenic protein from Paracoccidioides brasiliensis. When analyzed by immunoblotting, this recombinant antigenic protein was recognized by antibodies present in the sera of 40 of the 44 paracoccidioidomycosis patients studied. No cross-reactions were observed with sera from patients with other mycoses (histoplasmosis, aspergillosis, cryptococcosis, sporotrichosis, and chromoblastomycosis) or with tuberculosis.

Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein

“…The p27 recombinant protein was previously cloned in pBluescript IISK and expressed in E. coli DH5a cells, produced by fermentation techniques and purified by preparative electrophoresis. 10,11,13 Although, protein obtained in this system showed to be reactive when evaluated with sera from PCM patients, an important cross-reactivity was detected with sera from patients with aspergillosis and histoplasmosis; in addition, this process was expensive and time consuming.…”

“…Expression and purification of recombinant antigens have appeared as a valuable alternative to the use of crude antigenic extracts. 9,10,11 In our search for more reproducible results, we subcloned the gene coding for the p27 immunodominant antigen of P. brasiliensis and evaluated this recombinant protein in a dot blot assay, to explore its usefulness in the diagnosis of this mycosis. The use of a well-characterised antigen in the form of a recombinant protein and the appropriate test format to be used in PCM diagnosis would allow more reproducible results, making possible the standardisation of those diagnostic techniques in different laboratories.…”

“…It is known that PCM diagnosis has been based mostly on microbiological and immunological methods, 1,5,6,16,17 but both these alternatives present problems and, although, immunological tests have proved useful, one of the difficulties encountered has been the high level of cross reactivity found among the patients with other mycosis, especially histoplasmosis, mostly because of the use of crude antigens and their high associated variability. 2,8,10,11 Being aware of the need for more sensitive and specific methods and also the need of more cost-efficient and less time-consuming tests, we used the newly produced p27 protein on a dot blot format. The availability of an appropriate antigen used in combination with a standardised test, would allow its evaluation with a greater number of sera from patients with PCM in different stages of the disease to validate its use in diagnosis.…”

“…12 McEwen et al [13] cloned the protein of 27 kDa (p27) on the plasmid pBluescript; immunological characterisation of this recombinant protein showed that it was recognised by a high proportion of sera of patients with PCM. 10 When this p27 recombinant protein was evaluated on an ELISA format, the values of sensitivity and specificity obtained were of 73.4% and 87.5% respectively. 11 One of the main advantages of using recombinant antigens is the reduction of the cross-reactions that occur with other mycoses when crude antigens are used, as previously indicated.…”

“…8 An alternative to obtaining suitable antigens is the cloning, expression and characterisation of inmunodominant antigens derived from the fungus. [9][10][11] Cisalpino et al [9] cloned a P. brasiliensis immunodominant antigen, the glycoprotein of 43 kDa (gp43); this recombinant antigen showed high reactivity when evaluated with sera from patients with PCM. 12 McEwen et al [13] cloned the protein of 27 kDa (p27) on the plasmid pBluescript; immunological characterisation of this recombinant protein showed that it was recognised by a high proportion of sera of patients with PCM.…”