By Dr/ Hesham Kotb

Alwadi poultry company KSA

Head of Veterinary Section

Collecting blood for RBCS SOLN

• Collecting blood from SPF chicken or chicken not vaccinated nor in contact with birds kept for other purposes against the diseases we are going to use RBCS for Diagnosis

• Collect the blood on anti coagulant either

• Alsever’s soln 1:1 or

• Acid Citrate dextrose 1: 3 blood

Preparation of washed RBCS

• We prepare 1 % washed RBCS soln in 0.05 bovine serum albumin phosphate buffer saline

• Use of bovine serum albumin for RBCS is to nourish and extend the shelf life

• We will need a graduated sterile glass cylinder , glass bottle , plastic tube for RBCS suitable for centrifugation 50 ml

Washing RBCS

• Centrifuge blood directly at 1500-2000 g for 10 minutes

• Discard supernatant

• Add phosphate buffer saline and mix gently by inversion

• Centrifuge at 1500-2000 for 10 minutes and discard the supernatant

• Repeat the last step for 4-5 times till get clear supernatant

• Withdraw the supernatant with pipette but take care not to disturb RBCS

• Add 1 ml of washed RBCS to 99 ml PBS

• Accuracy of of washed RBCS soln has to be controlled either by

• 1- spectrophotometer or

• 2- RBCS cell count chamber or

• 3-micro hematocrit

• As too concentrated or too diluted RBCS soln may give false positive or false negative results

• Store the RBCS soln at +4c for 5 days

Preparation of 4 HAU antigen solution

• Lyophilized antigen and antisera should be stored at -20 c

• Once antigens are reconstituted store them at -80c

• while reconstituted positive and negative control sera should be stored at – 20c

• Keep the reagents on ice while they are out of refrigerator is strongly recommended

• Determining the titer of reference antigen by means of HA test each time the antigen solution is prepared

• Reconstitute the lyophilized antigen with 1 ml distilled water

• Mark the microtiter plates with subtype of antigen in use

• Distribute 25 microliter of PBS into the first row for antigen titration(A1to A12)

• add 25 microliter of PBS into the second row for RBCS control

• Dispense 25 microliter of antigen in the first well of the first row (A1)

• Make 2 fold dilution across the plate A1 to A12

• Discard the last 25 microliter

• Add 25 microliter PBS to all wells A1-A12 B1 TO B12

• Gently shake RBCS solution and add 25 microliter of it to each well

• Mix by tapping the microtiter plate gently

• Incubate at 20 c for 30 minutes or at 4 c for 60 minutes

• Read results when RBCS of the control wells have settled forming distinct pellet in the bottom of the well

• Reading is done by tilting the plate and observing the presence or absence of tear shaped streaming of RBCS

• At last well of no hemolysis this dilution is the 1HAU

• In the following picture 1HA=1:512

HI TEST

• For HI test either 4HAU OR 8HAU are used

Preparation of 4HAU solution

• Calculate the total amount of antigen solution required to test all the sera under examination

• Calculate HA titer contain 4HAU by deviding 1HAU BY 4

• Example 1: 512/4=1:128

• Means 1 antigen to 128 PBS

• Calculate antigen solution quantity according to samples number

• For example for preparation of 25 ml 4HAU solution

• =(25ml*1000 micron)/128 =195.3 microliter

• So add 196 microliter antigen solution to 24804 microliter phosphate buffer saline

• Use A,B,C,D.E for serum samples

• Use F for positive control serum

• Use G for negative control serum

• Use first 6 wells of H for back titration of antigen solution while the last 6 for RBCS control

• Add 25 microliter PBS in all wells except the first well H1

• Dispense 25 microliter of 1st serum sample in A1

• dispense 25 microliter of 2nd ,3rd ,4th ,5th serum samples at B1,C1,D1.E1 respectively using new micro pipette tip for each sample

• Use 25 microliter from positive serum at F1

• Use 25 microliter from negative serum at G1

• It is recommended to use reference control sera for each batch of samples under examination

• Using multichannel micropipette make 2 fold serial dilution of the sera across the plate and discard the last 25 microliter with exception of the last row

• add 25 microliter of the relevant antigen suspension containing 4HAU across the plate with exception of the last row

Back titration of the 4HAU sol

• Dispense 25 micoliter of the 4HAU soln into the H1,H2

• Then 2 fold serial dilution fron H2 to H6

• Discard 25 micoliter from H6

• Dispense 25 microliter of PBS with addition of albumin if prefered in all wells of the last row to bring volume to 75 microliter

• Mix by gentle tapping and incubate plate

• Between 20-22c for30 min or 4c for 60 min

• Finally add 25 microliter of 1% RBCS suspension solution into all wells of the plate

• Mix by gentle tapping and incubate plate Between 20-22c for30 min or 4c for 60 min

• Results are read when RBCS are settled done by tilting the plate and recording the presence or absence of tear shaped streaming as control wells containing RBCS alone

Read results

• HI titer is the highest dilution of serum causing complete inhibition of the HA

• The test is valid when the negative control serum has titer of less than 1:8 against 4 HAU antigen solution

• The titer of positive control serum should coincide with that declared

• One dilution of difference is permitted

Thank you

Reference

• Istituto Zooprofilattico Sperimentale delleVenezie

• Laboratorio Virologia