The ultimate aim of this study is to invent a ready to use immunochromatographic test (ICT) test kit for double diagnosis of leptospirosis and Dengue fever by means of antigen detection. The research activities on invention of ICT test kit for leptospirosis diagnosis were carried out sequentially which included: 1) determination of suitability of the pre-existing mouse monoclonal antibodies to Leptospira antigens (clones LD5, LE1 and LF9 from Faculty of Medicine Siriraj Hospital, Mahidol University and clones 3, 81, and 82 from Faculty of Medicine, Chulalongkorn University); 2) development of an ICT test kit using suitable MAb1-MAb2, MAb-PAb, or Pab-MAb format; 3) acquisition of serum samples of patients with febrile illnesses from collection of Prof. Dr. Yupin Suputthamongkol, Siriraj Hospital; and 4) validation of the ICT test kit which was performed in blinded manner in comparison to the previous laboratory and clinical diagnoses. The activity 1 was carried out by Dr. Surasak Wongrattanacheewin, Department of Microbiology, Faculty of Medicine, Khon-Kaen University (third party). It turns out that suitable monoclonal antibodies were from clones LD5 and LE1 (specific to pathogenic Leptospira) from Siriraj Hospital. Therefore, the MAbLE1 was used to conjugate with the colloidal gold particles and impregnated into the gold conjugate pad while the MAbLD5 was immobilized at the Test (T) line. Control (C) line was immobilized with goat anti-mouse immunoglobulin. Alternatively, rabbit polyclonal immunoglobulin to pathogenic Leptospira was used to conjugate with the gold particles, MAbLD5 was immobilized at the Test (T) line, and goat anti-rabbit immunoglobulin was at the Control line. Both formats of the ICT test kit had the lowest detection limit at 0.375 g of Leptospira whole cell proteins/strip. They are specific only to Leptospira and did not detect antigens of 27 other bacterial species.
Sixty serum samples of patients with febrile illnesses (kept frozen) were obtained from Prof. Dr. Yupin for use for the ICT test kit validation. They were subsequently known after the ICT testing to be 14 sera diagnosed leptspirosis, 16 sera diagnosed scrub typhus, and 30 sera diagnosed Dengue viral infections. All 14 sera diagnosed leptospirosis gave positive results by the ICT test kit. Nevertheless, 11 of the 16 diagnosed scrub typhus and 3 of 30 diagnosed Dengue viral infections gave positive results by the ICT test kit. Thus, the ICT test kit shows 100% sensitivity, 70% specificity, 77% accuracy, 0% negative predictive value and 30% positive positive predictive value. It is noteworthy that, we have sent the bands at the test lines of three samples diagnosed scrub typhus which were positive by our leptospirosis ICT test kit for mass spectrometry. The results of the mass spectrometry revealed the presence of Leptospira peptides and the absence of scrub typhus antigen; implying that the original diagnosis was inaccurate. The second batch of 248 serum samples was obtained from Prof. Dr. Yupin. Among them, 201 samples were positive for leptospirosis by the ICT test kit. However, we have not obtained the diagnoses of these sera from Prof. Dr. Yupin as yet.
We have prepared ICT test kit for detection of Dengue virus antigen. The monoclonal antibodies to Dengue virus were from Dr. Chanya Putthikhan, Siriraj Hospital. One MAb preparation (SI4) is a pool of 4 MAbs (2 MAbs were mouse IgG1 and the other two were IgG2b) and another MAb (SI1) is IgM. We have used the MAbSI4 to conjugate with gold particles and used the MAbSI1 at the test (T) line. Goat anti-mouse immunoglobulin were immobilized at the control (C) line. Because the supply of Dengue virus antigens and the MAbs from Dr. Chanya was inadequate for optimization of the test kit, we cannot accomplish the test kit invention as yet.