Rapid Diagnosis of Cryptococcus neoformans Infection by Using Specific Monoclonal Antibody 18B7

Abstract

Cryptococcus neoformans is an opportunistic fungus that causes cryptococcosis in immunocompromised patients, especially for human immunodeficiency virus (HIV). The aim of this study was to develop the latex agglutination ( LAT) and lateral flow immunoassay ( LFIA) for detecting capsular antigens of Cryptococcus by using monoclonal antibody ( MAb) 18B7. In addition, MAb 18B7 is able to react with all 4 serotypes of C. neoformans. To develop the rapid tests of cryptococcosis, MAb 18B7 and capsular antigens of C. neoformans were prepared. For production of antibody, MAb 18B7 was concentrated and purified by protein G affinity chromatography, then the purity and characteristic of MAb were confirmed by SDS-PAGE and immunoblotting, respectively. The immunoreactivities of MAb 18B7 against capsular antigens of other serotypes of Cryptococcus spp. were shown as follow; serotype A ( H99) = B ( 14407) > B ( 6956) > A ( 8710) > D ( 10513) , respectively. No crossreactivity was found against other pathogenic fungi, except antigen from Trichosporon spp. The limit of detection (LOD) of LAT-18B7 against capsular antigens was ranging between 50-100 ng/ ml depended on the serotypes of Cryptococcus spp. The evaluation of LAT-18B7 performance and its comparison with the Cryptococcus Antigen Latex Agglutination Test System (CALAS) were conducted in cerebrospinal fluid (CSF) and serum of cryptococcosis as well as negative controls of healthy individual and other infectious patients. The sensitivity and specificity of LAT-18B7 against 25 positive CSF was exhibited 92% and 76%, respectively compared to CALAS. In addition, the performance against 63 serum samples was shown 69.70 sensitivity and 66.67% specificity. No false positive of LAT-18B7 was detected in both the other infectious patients and healthy volunteers. In addition to the development of LFIA-18B7, the LOD of capsular antigens of all serotypes of Cryptococcus was 0.63 ng/ml. No cross reaction was found against other pathogenic fungi, except antigen of Trichosporon spp. which the LOD was 500 ng/ml. To examine the performance of the test, LFIA-18B7 was conducted in CSF and serum compared to commercial kit of CrAg from IMMY. The sensitivity and specificity of LFIA-18B7 exhibited 92.86% and 100% tested against 28 CSF of cryptococcosis and no positive result in patients with other infections was detected. The 27 positive and 24 negative sera detected with CrAg were tested against LFIA-18B7, the sensitivity and specificity were 92.59% (24/27) and 95.83% (23/24), respectively. From the stability test both LAT-18B7 and LFIA-18B7 were still stable for 3 months. As a result, all data indicated that both diagnostic assays can detect capsular antigens of Cryptococcus, although LAT-18B7 is needed to improve more sensitivity and specificity. On the other hand, LFIA-18B7 can be applied to be a candidate to diagnose cryptococcosis in serodiagnosis with reliable, reproducible and cost-effective reagent, especially useful in countries where the commercial kit is not generally available and must be obtained at a high cost.