HumaTex Febrile Antigens

Transcription

1 Design Verification HumaTex Febrile Antigens Contents 1 Function Product Line Sensitivity... 2 Description of Control Materials... 2 Kit controls Positive and Negative Interferences Cross-reactivity Stability... 7 QM-Element: ISO9001:2000 No /7 valid of

2 1 Function The tests of the HumaTex FEBRILE ANTIGENS line has been designed as a screening and confirmatory test for the qualitative and semi-quantitative detection of antibodies associated with febrile infections caused by typical flagellar or somatic bacteria (Salmonella typhi, paratyphi, Brucella, Proteus). The test can be performed as a rapid slide agglutination test and also as a tube agglutination test (Widal test). Bacterial suspensions, vitally stained, will agglutinate in the presence of corresponding antibodies. The tests of the HumaTex FEBRILE ANTIGENS line consist of 10 different bacterial suspensions, allowing discrimination of the respective infection. Agglutination patterns may be interpreted in direct comparison with a negative and a positive control. Any agglutination stronger than that observed with the negative control should be interpreted as a positive result. The clinical diagnosis, however, should always consider the patient s anamnesis and exclude possible cross-reactions caused by infections or vaccination with related bacterial strains. 2 Product Line [REF] x 100 Tests MULTISCREENING KIT consists of 6 different febrile antigens (Proteus OX19, and antigens marked below *) and controls (PC, NC) x 100 Tests SALMONELLA KIT consists of 8 different febrile antigens ( typhi H and O, paratyphi AH, AO, BH, BO, CH and CO) Single parameter kits: x 100 Tests SALMONELLA TYPHI H * x 100 Tests SALMONELLA TYPHI O * x 100 Tests SALMONELLA PARATYPHI AH * x 100 Tests SALMONELLA PARATYPHI AO x 100 Tests SALMONELLA PARATYPHI BH * x 100 Tests SALMONELLA PARATYPHI BO x 100 Tests SALMONELLA PARATYPHI CH x 100 Tests SALMONELLA PARATYPHI CO x 100 Tests BRUCELLA ABORTUS * x 40 Tests CONTROL PC/NC 3 Sensitivity Description of Control Materials For sensitivity check an internal control is employed which is traceable to an internal master standard preparation (goat serum, reactive with the respective antigen). Due to the absence of international reference materials, the internal master standard preparation has been established against several lots of earlier released bacterial suspensions and against competitor tests. Dilutions of the internal control are performed according to the following schemes. For slide test: Starting material Vol. of start. Vol. of diluent Titer # of Standard material, µl (GBS 1) ), µl Internal control Standard # /2 2 Standard # /4 3 Standard # /8 4 Standard # /16 5 Standard # /32 6 Standard # /64 7 Standard # /128 8 Standard # /256 9 Design Verification and Product Data for HumaTex FEBRILE ANTIGENS 2/7

3 For tube test: Starting material Vol. of start. Vol. of diluent Titer No. of standard material, µl (GBS1)), µl Internal control Standard # /20 2 Standard # /40 3 Standard # /80 4 Standard # /160 5 Standard # /320 6 Standard # /640 7 Standard # / Standard # / ) GBS: glycine buffered saline Test Performance (Slide Test) 1 drop (50 µl) of the above standard dilutions are pipetted onto separate cells of the slide. 1 drop (50 µl) of the respective antigen solution (stained bacterial suspension) is applied to each of the cells and mixed with the standard dilutions. After approx. 1 minute of incubation, while the slide is slowly rotated (ca. 100 r.p.m.), the agglutionation pattern is observed under bright artificial light. Test Performance (Tube Test) 1000 µl of the above standard solutions are pipetted into separate tubes. 1 drop (50 µl) of the respective antigen solution is applied to each tube. The tubes are closed with stoppers, mixed and incubated for 24 hours at 37 C. At the end of the incubation period the agglutination pattern is observed under bright artificial light. Results Agglutinations are rated according to: 4+ grade (very strong agglutination), 3+ (strong agglutination), 2+ (distinct agglutination), 1+ (visible agglutination), ± (weak agglutination, doubtful), 0 (no agglutination). The following sensitivity results have been obtained: Parameter Titer Slide Test (Agglutination grade) Titer Tube Test (Agglutination grade) SALMONELLA TYPHI H 1/64 (1+) 1/640 (1+) SALMONELLA TYPHI O 1/32 (1+ to 2+) 1/320 (± to 1+) SALMONELLA PARATYPHI A(H) 1/256 (1+) 1/2560 (± to 1+) SALMONELLA PARATYPHI A(O) 1/16 (1+) 1/160 (1+) SALMONELLA PARATYPHI B(H) 1/128 (1+) 1/1280 (± to 1+) SALMONELLA PARATYPHI B(O) 1/16 (± to 1+) 1/160 (± to 1+) SALMONELLA PARATYPHI C(H) 1/32 (1+ to 2+) 1/320 (1+ to 2+) SALMONELLA PARATYPHI C(O) 1/16 (1+) 1/160 (1+) BRUCELLA ABORTUS 1/64 (1+ to 2+) 1/640 (± to 1+) PROTEUS OX19 1/16 (± to 1+) 1/80 (1+ to 2+) Kit controls Positive and Negative The positive and negative kit controls are employed for function testing of all 6 stained bacterial suspensions. The positive control is diluted 1/2, 1/4 and 1/8 with GB The positive control dilutions are tested with the slide test method. Design Verification and Product Data for HumaTex FEBRILE ANTIGENS 3/7

4 Results The results are summarized in the following table (agglutination degree according to ): Pos. control dil. Agglutination TYPHI H TYPHI O B. ABORTUS PROTEUS OX19 1/1 3+ to to to to 4+ 1/2 2+ to to to to 3+ 1/4 1+ to to to to 2+ 1/8 0 to 1+ 0 to 1+ 0 to 1+ 0 to 1+ Neg. control Pos. control Agglutination dil. PARATYPHI AH PARATYPHI AO PARATYPHI BH PARATYPHI BO PARATYPHI CH PARATYPHI CO 1/1 3+ to to to to to to 4+ 1/2 2+ to to to to to to 3+ 1/4 1+ to to to to to to 2+ 1/8 0 to 1+ 0 to 1+ 0 to 1+ 0 to 1+ 0 to 1+ 0 to 1+ Neg. control Interferences Hemoglobin, bilirubin, lipids (triglycerides) and rheumatoid factors have been studied for a potential interference with the HumaTex FEBRILE ANTIGENS test kits. Normal human sera have been spiked with the supposed interfering substance to the following concentrations: hemoglobin up to 1000 mg/dl, bilirubin up to 20 mg/dl, lipids up to 1000 mg/dl, rheumatoid factors up to 300 IU/ml. The spiked sera have been tested by both the slide and the tube test. Even with the highest concentrations tested no interference could have been observed in the slide and tube test. 5 Cross-reactivity Cross-reactivities have been studied for each of the respective stained bacterial suspensions, employing antisera with known specificities against a variety of bacterial antigens. In all cases the direct slide test method was used. The results are summarized in the following tables. Stained bacterial suspension: SALMONELLA TYPHI H Salmonella H:d 3+ Salmonella O f: 9 3+ Design Verification and Product Data for HumaTex FEBRILE ANTIGENS 4/7

5 Stained bacterial suspension: SALMONELLA TYPHI O Salmonella O f: 1,2 12 ± Salmonella O f: 9 3+ Stained bacterial suspension: SALMONELLA PARATYPHI AH Salmonella H:a 3+ Salmonella O f: 1, Salmonella O f: 2 ± Stained bacterial suspension: SALMONELLA PARATYPHI AO Salmonella O f: 1, Salmonella O f: 2 ± Design Verification and Product Data for HumaTex FEBRILE ANTIGENS 5/7

6 Stained bacterial suspension: SALMONELLA PARATYPHI BH Salmonella H:b 3+ Salmonella O f: 4,5 3+ Stained bacterial suspension: SALMONELLA PARATYPHI BO Salmonella O f: 5 2+ Salmonella O f: 4,5 3+ Stained bacterial suspension: SALMONELLA PARATYPHI CH Salmonella H:c 3+ Salmonella O f: 7 3+ Salmonella O f: 6,7 3+ Design Verification and Product Data for HumaTex FEBRILE ANTIGENS 6/7

7 Stained bacterial suspension: SALMONELLA PARATYPHI CO Stained bacterial suspension: BRUCELLA ABORTUS Stained bacterial suspension: PROTEUS OX19 Salmonella H:c 2+ Salmonella O f: 7 3+ Salmonella O f: 6,7 3+ Br. abortus 3+ Br. melitensis 3+ Br. suis 3+ Vibrio cholerae 3+ Francisella tulerensis 1+ Yersinia 2+ Proteus OX2 0 Proteus OXK 0 Proteus OX Stability The stability of the HumaTex FEBRILE ANTIGENS test has been confirmed by real time studies on 2 independent production lots. The lots have been tested (slide test) for correct function, employing fresh controls and fresh bacterial suspensions, respectively. Agglutination results obtained with kits fresh from production and stored over the assigned shelf life of 30 months (at C) have been compared. The results are summarized in the following. Pos. control titer (slide test) Lot 1 Lot 2 Fresh after 30 months Fresh after 28 months TYPHI H 1/64 1/64 1/64 1/64 TYPHI O 1/32 1/32 1/32 1/32 PARATYPHI A(H) 1/512 1/256 1/256 1/256 PARATYPHI A(O) 1/32 1/16 1/16 1/16 PARATYPHI B(H) 1/128 1/128 1/256 1/128 PARATYPHI B(O) 1/16 1/8 1/16 1/16 PARATYPHI C(H) 1/32 1/32 1/32 1/64 PARATYPHI C(O) 1/32 1/16 1/16 1/16 B. ABORTUS 1/128 1/64 1/128 1/64 PROTEUS OX19 1/16 1/8 1/16 1/16 With the accepted tolerance of +/- 1 titer step the above results confirm the shelf life claim of 30 months at C. Design Verification and Product Data for HumaTex FEBRILE ANTIGENS 7/7