MATERIALS AND METHODOLOGY FOR THE PRODUCTION OF AHG
MATERIALS AND REAGENTS USED
- Test tubes
- Test tubes racks
- Nose masks
- Potash alum
- Some syringes
- Pasture pipette
- Antisera D
- Human serum of group O+
- Phosphate buffered saline.
The phosphate buffered saline is an isotonic saline solution which is prepared as an Iso-osmotic phosphate buffer and then add Nacl making it an isotonic saline solution. The phosphate buffered saline contains 20g/L bounie serum albumin, 1g/L sodium azide, 0.1g/L Tween 80 and 9g/L sodium chloride. The sodium azide acts as the preservative.
PREPARATION OF PHOSPHATE BUFFERED SALINE (HENDRY 1961)
Prepare the phosphate buffer, Iso-osmotic first by the following proportions of solution A and B.
(A) NaH2P04.2H20 (150mmol/L) 23.4g/L
(B) NaH2P04 (150mmol/L) 21.3g/L
|PH||Solution A||Solution B|
|5.8||87 ml||13 ml.|
|6.0||83 ml||17 ml|
|6.2||75 ml||25 ml|
|6.4||66 ml||34 ml|
|6.6||56 ml||44 ml|
|6.8||46 ml||54 ml|
|7.0||32 ml||68 ml|
|7.2||24 ml||76 ml|
|7.4||18 ml||82 ml|
|7.6||13 ml||87 ml|
|7.7||9.5 ml||90.5 ml|
Normal human serum has an Osmolality of 289± 4 mmol, thus there is slight differents in concentrations of the stock solutions namely, 25.05g/L NaH2P04.2H20 and 17.92gk NaH2P04 for an iso-osmotic buffer, then add 9g/L Nacl for phosphate buffered saline.
This study was carried out in an animal farm of Ebonyi State University. The laboratory tests was carried out in the heamatology unit of the laboratory department of Federal Teaching Hospital Abakaliki, Ebonyi state with authorization coming from head of the laboratory department.
The study was carried out on a total of 2 rabbits about 10 weeks. Rabbit is conventionally used for the course of this project. Goat can also be used for this project because of the following advantages over rabbit;
- Large volume of serum can be obtained from goat while small volume is gotten from rabbit.
- The goat is not prone to infection by myxomatosis while rabbit is prone to myxomatosis.
- The immunizing and bleeding operations are simpler and quicker in goats than in rabbit.
- For the laboratory using or distributing large quantities of the reagent feeding and housing a goat is less costly than keeping 20 or 30 rabbits.
The blood sample that was used for this study was collected through Vene-puncture from the ear lobe of the rabbit. The ear is sterilized with cotton wool in 70% alcohol. 10-20ml of blood were collected from the subjects and put into a plain test tube to extract the serum.
PROCEDURE FOR LOCAL PRODUCTION OF ANTI-HUMAN GLOBULIN (DUNSFORDS I AND BOWLEY C.C 1955)
- Get human blood; check the blood group to know if it is blood group 0+.
- Then screen the blood for hepatitis C antigen, hepatitis B antigen, syphilis and HIV virus, to ensure that the blood does not have any infection.
- Pour the blood into a plain test tube to extract the serum.
- Get 25ml of serum add to 75ml of redistilled water.
- 90ml of 10% potash alum is added to the above solution adjust pH to 6.5.
- Centrifuge at 1000 rpm for two minutes discard the supernatant and milky white precipitate is collected.
- Wash the precipitate three times with large volume of saline containing 1:10,000 phosphate.
- Collect the washed precipitate and resuspend in 100ml of phosphate buffered saline.
- Collect 5ml of the precipitated mixture and inject intramuscularly (gluteal muscles) of the rabbit as a single dose.
- Inject the animals with the 5ml of this precipitate for a total of 10 injections with an interval of three days.
- Booster dose of 1ml of whole serum is administered intravenously.
- Ten days after the last injection, the rabbit are bled from the vein of the ear lobule.
- Pour the blood into a sterile bottle to get the serum.
- Then standardize the serum by heating the serum at 56oc for 30 minutes to inactive complement
- The inactivated serum is then treated with well washed A, B and O cells and absorption is effective at 37C.
- The serum is then titrated in parallel against red cells sensitized with an incomplete anti-D and against unsensitized cells.
- Then add the colorant.