1. Spot test on Stored Serum/Plasma samples (Figure 4).
1. Plate out 50 µL of 1:20 serum/plasma in alternate columns 1,3,5,7,9,11 (add 2.5 µL sample to 47.5 µL PBS).
2. Add 50 µL 1:20 O-ve blood collected in (so that now sample is diluted to 1:40 and red cells at ~1% v/v)
3. Mix and transfer 50/100 µL to neighbouring columns 2,4,6,8,10,12 for -ve controls. The negative control is important because in rare cases, particularly in donors who have received blood transfusions, the sample in principle may contain antibodies to non-ABO or Rhesus D antigens.
4. Add 50 µL IH4-RBD reagent (2 µg/mL in PBS = 100 ng/well) to Columns 1,3,5,7,9,11
5. Add 50 µL PBS to columns 2,4,6,8,10,12.
6. Inc 1 hr RT
7. Tilt for 30 seconds
8. Photograph: with mobile phone use the zoom function to obtain a complete field
9. Read as Positive = No teardrop, Negative <1:40 = partial teardrop, Neg = complete teardrop.
10. Two readers should read the plates independently, and disagreements resolved by taking the lesser reading.
11. For each batch of samples set up positive control wells containing 20-100 ng monoclonal antibody CR3022 (as in Finger-Prick test below). This establishes that all of the reagents are working.
2. Titration of Stored Serum/Plasma Samples.
1. Dilute samples to 1:20 in 50 µL PBS (2.5 µL to 47.5 µL) in V-bottomed plate in Rows A-H, column 1. Prepare WHO standard serum 20/130 as above at 1:20, or CR3022 50 µL at 20 µg/mL, for calibration for each batch of titrations. WHO standard 20/130 should titrate to ~ 1:1280, and 20 µg/mL CR3022 to ~ 1:512.
2. Prepare doubling dilutions with PBS across the plate columns 1-11 (1:40 to 1:40,960), PBS control in column 12. Eight samples can be titrated per 96-well plate.
3. Add 50 µL 1:40 O-ve red cells (1% v/v or 1:40 fresh EDTA O-ve blood sample) to all wells
4. Add 50 µL IH4-RBD (2 µg/mL, = 100 ng/well). [Note: the red cells and IH4-RBD can be pre-mixed and added together in either 50 µL or 100 µL volume, to save a step. This variation in technique does not alter the measured titres.]
5. Allow red cells to settle for 1 hr
6. Tilt plate for at least 30 s and photograph. The titre is defined by the last well in which the tear drop fails to form. Partial teardrop regarded as negative.
3. Finger-prick test on capillary blood as a Point of Care Test
Preparation: Clean Hands, warm digit. Prepare a plate (96-well V-bottomed) labelled with Date and Time.
Prick skin on outer finger pulp with disposable, single use BD or another Lancet.
Wipe away first drop of blood with sterile towel/swab
Massage second drop
Take a minimum of 5 µL blood with 20 µL pipette, mix immediately into 20 µL K2EDTA (3.6 mg/mL/PBS) in Eppendorf. If possible, take 25 µL of blood and mix into 100 µL K2EDTA solution. Another approach is collection of blood drops into a BD Microtainer K2E EDTA lavender vials REF 365975 that take 250-500 µL.
For 5 µL sample dilute to 200 µL with PBS (add 175 µL PBS), for 25 µL sample dilute to 1ml (add 975 µL PBS). Sample is now at 1:40, and the red cells are at the correct density (~1% v/v assuming a haematocrit of 40%) to give a clear tear drop.
Plate 50 µL x 3 in V bottomed microtitre wells labelled T (Test), + (PC, positive Control), - (NC, negative control).
Add 10 µL of control anti RBD Mab CR3022 (2 µg/mL stock in PBS, 20 ng/well) to “+“ well
Add 50 µL IH4-RBD (2 µg/mL in PBS) to “T” (Test) and “+ve“ wells, 50 µL PBS to “-ve“ well.
Incubate 1 hour at RT for Red Cells to form a pellet in the “-ve“ well
Tilt plate against a well-lit white background for ~30 seconds to allow Tear drop to form in “-ve“ well.
The presence of antibodies to RBD is shown by loss of Tear Drop formation in the “T“ and “+ve“ wells. Occasionally a partial tear drop forms – these wells are counted as Negative.
Photograph the plate to record the results with the date and time. Results can be reviewed and tabulated later. Taking picture from a distance and using the zoom function helps to take a clear picture of all wells in a 96-well plate.
The negative (PBS) control should be done on every sample for comparison. The Positive control induced by CR3022 is used to check that all the reagents are working, and that the glycophorin epitope recognised by VHH(IH4) is present on the red cells. Absence of the IH4 epitope should be very rare (Habib et al., 2013). For setting up cohorts a positive control on every sample is therefore not necessary but should be included in every batch of samples.
15. If a 25 µL sample of blood was taken from the finger prick there should be 850 µL of the 1:40 diluted blood left. The red cells can be removed and a preparation of 1:40 O-ve red cells used as above to titrate the sample. In principle the autologous red cells could be washed x3, resuspended in the same volume of PBS, and used as indicators for the titration, however we have not attempted to do this. The supernatant is 1:40 plasma that can be used in confirmatory ELISA or other tests.