Manual of basic techniques for a health laboratory - libdoc.who.int

348 Manual of basic techniques for a health laboratoryFig. 11.22 RPR test3. Add one drop of RPR antigen to each well of the test plates.4. Place the test plates on the rotator and rotate for 8 minutes at 100rpm. (Therecommended speed is between 95 and 105rpm and this should be checkeddaily as part of quality control.)If a mechanical rotator is not available, tilt the plates back and forth and rotatethe plates carefully for 8 minutes at 80–85rpm.5. Examine the test plates for flocculation (Fig. 11.22) and compare the reactionsof the test sera with those of the controls.6. Prepare a twofold dilution of any positive sera and examine the dilutionsas described in steps 2–5. The highest dilution of serum to give flocculation isthe titre.11.10.2 TPHA testMaterials and reagents●●Test-tubesTest-tube rack● Commercially available TPHA test kit containing microtitre plates, micropipettes(with disposable tips), absorbing diluent, erythrocytes sensitized with T. pallidum,unsensitized erythrocytes, positive and negative control sera● Distilled water.The reagents and controls should be reconstituted before use according to themanufacturer’s instructions.Method1. Dilute the test and control sera 1:20 with absorbing diluent.2. Using a micropipette, dispense 25ml of the negative control serum into wells 1and 2 of the first horizontal row of the microtitre plate (A in Fig. 11.23).3. Dispense 25ml of the positive control serum into wells 1 and 2 of the secondhorizontal row of the microtitre plate (B in Fig. 11.23).4. Dispense 25ml of the first test serum into wells 1 and 2 of the third horizontalrow of the microtitre plate (C in Fig. 11.23). Repeat the procedure with theremaining test sera. If necessary, use the adjacent wells (e.g. 3 and 4 in Fig.11.23).5. Add 75ml of the control erythrocytes to the wells in the first vertical row (1) andevery other row (3, 5, 7, 9 and 11), as appropriate.