Development and validation of NLRP3 immunochromatography assay as an indicator for inflammation and bad seminal quality

IntroductionEstimation of seminal inflammation is highly necessary for management of Infertile patients, unfortunately peroxidase staining and leukocyte count is not indicative for true inflammation. This study aimed to develop a rapid NLRP3 immunochromotography (ICT) strip as an alternative tool for indication of inflammation rather than confusing Endtz test.MethodsImmunochromotography cassette developed in the Department of Microbiology/ College of Medicine/ AL-Nahrain University. Polyclonal capture rabbit anti-NLRP3 (test line) and goat anti-rabbit IgG antibodies (control line) were applied on each nitrocellulose membrane. Gold-in-a-BoxTM conjugation kit (40nm. 15OD) conjugated anti-NLRP3 were adsorbed in conjugate pad, then assembled in the test cassette. In vitro testing using serial concentration of standard NLRP3 protein using ELISA and developed ICT. Fifty infertile patients included in this study, seminal fluid analysis and Endtz test were done, seminal fluid analysis was re-evaluated after in vitro sperm activation.Resultsthe developed NLRP3 ICT was sensitive at the level of 2.5 ng/ml. NLRP3 was detected in seminal fluid of 30 (60%) of infertile patients. ICT assay showed 78.95 sensitivity and 51.61 specificity in comparison with Endtz test. Furthermore, detection of NLRP3 ICT showed 14.5 odds ratio for none improved seminal fluid analysis after in vitro sperm activation, while ELISA (> 4.5ng/ml) showed 8.33 and Endtz test (1 million/ml) showed 4.68. Positive NLRP3 cases were 14.53 greater than negative cases for their un-improvement after in vitro sperm activation 56.67% versus 95% respectively.ConclusionAn evidence of NLRP3 protein in seminal plasma may be associated with the increased leukocytospermia, bad sperm quality among infertile male with higher sensitivity than Endtz test. NLRP3 positivity may reflects bad outcome and unresponsiveness to in vitro sperm activation.

کلیدواژه ها:

NLRP3 ، Immunochromotography ، Infertility