hi test for poultry disease diagnosis
TRANSCRIPT
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By Dr/ Hesham Kotb
Alwadi poultry company KSA
Head of Veterinary Section
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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
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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
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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
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• 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
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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
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• 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
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• 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
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• 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
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HI TEST
• For HI test either 4HAU OR 8HAU are used
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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
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• 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
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• 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
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• 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
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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
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• 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
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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
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Thank you
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Reference
• Istituto Zooprofilattico Sperimentale delleVenezie
• Laboratorio Virologia