T.T.D TESTING

HIV Elisa test:
 Assay procedure:
  1.   Bring all the reagents to room temperature
  2.  Arrange required no. of wells and proceed
  3.  25µl of conjugate -1 to each well (ready to use
  4. 75 µl of negative control (R3) in well1A1
  5. 75µl of cut off control (R4) in wells B1, C1, at D1
  6.  75µl of HIV positive control (R5) in well E1 & F1
  7.  75 µl of test sample to other remaining wells Mix well, seal with adhesive film and incubate at 37Cfor 1 hour
  8.  Wash 3 times with working wash solution (Concentrate - R2 100 ml+Dist. Water 1000 ml)
  9. 100 µl of working conjugate-2 to each well (Working conjugate-2 = Mix conjugate-R7a Feojugate dialent-R7b) Seal and incubate at room temperature for 30 miles
  10.  Wash 5 times
  11.  80µl fresh working development solution (Chromogen-R91ml+ Substrate buffer Rs.10 ml
  12. Incubate for 30 minutes at dark place.
  13. 100 µl stopping solution (ready to use) Read at 450 & 620 after 4 minutes


Calculation

                                                 Mean absorbance O.D. of cut off control
Cut-of value = -------------------------------------------------------------------------------------
                                                                                5


Assay validation
 Negative control :  < 0.7x C.O
Cut-off control:  > 0.35

 Positive control: > 0.50

Comments

Post a Comment

Popular posts from this blog

Storage of Blood and Blood Products.

Srl No   Name of the product Temperature Shelf Life 1. Whole Blood ( CPDA ) 2 -6 *c 35 Days 2. (a) Concentrated RBC ( CPDA-1) closed system (b) Concentrated RBC ( CPDA-1) open system (c) Concentrated RBC ( SAGM) 2 -6 *c 2 -6 *c 2 -6 *c 35 Days 24 Hrs 42 Days 3. Fresh Frozen Plasma -30*c 1 year 4. Thawed 2 -6 *c 12 Hrs 5. Platelet concentrate 20 – 24 *c 3- 5 days (Depending on the bag ) Other items to be observed     Pilot tubes of issued blood                            -         ...
Read more

Unexpected antibody test

Saline method of antibodies testing Method: 1.     Put 2 drops oftest serum in a labeled tube 2.     Add 1 drop of 2-4% suspension of appropriate red cells in Saline and mix 3.     Incubate at R.T for 30-45 min. In urgent cases centrifuge the tubes at 1000 rpm for 1 min after 5-10 min. incubation at room temperature (spin method) 4.     Observe the supernatant fluid for the presence of haemolysis 5.     Gently disperse the cell button and check for agglutination against a well lighted background Albumin method of antibodies testing Method: 1.     Add 2 drops oftest serum to a labeled tube 2.     Add one drop of 2-4% suspension of appropriate RBC in Saline 3.     Add two drops of bovine albumin 22% 4.     Mix and incubate at 37°C for 30-60 minutes 5.     Centrifuge at1000 rpm for 1 minutes 6.    ...
Read more

Phlebotomy

Donor Preparation: 1.     The phlebotomist should wash his/her hands with soap and water and should wear sterile gloves. 2.     Select the required bag and write donor no., date of collection and expiry. 3.      Inspect the bag for leakage or any other defect. The anticoagulant solution must . be clear. Place the bag on a pre-programmed blood collection monitor and press the start button. 4.      Choose the site of venipuncture in the anticubital are of the arm. Apply blood pressure cuff, inflate to 50-60mm of Hg and select a prominent and firm vein in the area that is free of any skin lesions/needle marks. Asking the donor to close the fist usually helps in bringing the vein in to prominence. 5.     Release the blood pressure cuff and thoroughly clean the selected site of venipuncture with an antiseptic lotion in the following way. a)      Clean 4-5 cm area starting at...
Read more