Question
Diagnosis of HIV
Case: One of a clinic sent a patient to our laboratory to make diagnosis of HIV.
Step 1: Collection of blood.
1 - In our phlebotomy room, we have collected two blood samples one to get a serum and another to get plasm.
Step 2: ELISA (serum for screening).
1 - We used in our laboratory an instrument called Architct PLUS, and it gave us a positive result of HIV.
2- - The principle of instrument:
ELISA tests:
- Direct solid phase antiglobulin ELISA is the method most commonly used. The antigen is obtained from HIV grown in continuous T- lymphocyte cell line or by recombinant techniques and should represent all groups and subtypes of HIV-1 and HIV-2. The antigen is coated on the microtitre wells or other suitable solid surface.
The test serum is added. And if the antibody is present, it binds to the antigen.
- After washing away the unbound serum, antihuman immunoglobulin linked to a suitable enzyme is added. If test serum contains anti-HIV antibody, a photometrically detectable colour is formed which can be read by special ELISA readers.
3- - do another confirmation test which is Western Blot"
Step 3: Western Blot (plasma for conformation).
1- - Western Blot test gave us also a positive result of HIV.
2- The principle of Western Blot test:
- In this test HIV proteins separeated according to their electrophoretic mobility(and molecular weight) by polyacrylamide gel electrophoresis are blotted onto strips of nitrocellulose paper. These strips are reacted with test sera and then with enzyme conjugated antihuman globulin.
- A suitable substrate is then added, which produces a prominent colour band where the specific antibody has reacted with the separated viral protein.
- A positive reaction with proteins representing the three genes gag,pol,env is considered positive even if it shows bands against at least two of the following gene
Final report is:
The patient has infected with HIV, and we used two different test and which all of them gave us a positive result.
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Diagnosis of HIV
Aim: To diagnose a HIV patient
Introduction: HIV (Human Immunodeficiency Virus) is a retrovirus which suppresses the immune system and causes Acquired Immuno Deficiency Syndrome (AIDS). The HIV test is used for the presence of HIV in blood (serum), saliva or urine of the suspected patients. The HIV diagnosis detects the antigens of HIV such as gp120, gp41, gp160, antibodies against HIV antigens, or the genetic material of HIV (RNA).
Tests:
(A) Antibody test:
(a) ELISA test: The enzyme linked immunosorbent assay (ELISA) is the first most screening test for HIV and very sensitive also.
Step 1: Blood collected in phlebotomy room and serum isolated from that.
Step 2: Serum diluted to 400- fold and transferred to a HIV antigen coated ELISA Plates. If the serum sample has antibodies against HIV antigens it will bind to the antigen which is coated on the multi well plates.
Step 3: Then the plate will be washed to remove unbound antibodies present in the serum and other components also of the serum.
Step 4: Secondary antibodies (anti human antibodies), which is already attached with enzymes, applied and washed again. Secondary antibodies will bind to the primary antibodies. Amount of enzyme bound along with secondary antibodies is directly proportional to the amount of primary antibodies used.
Step 5: The substrate of the enzymes will be applied. The enzyme bound to the secondary antibodies catalyses the reaction of substrate and color produces.
Step 6: The intensity of the color measured photometrically by using an ELISA plate reader.
(b) Western Blotting: This is next confirmatory test for the detection of antibodies against HIV. The principle and procedure is slightly different from the ELISA test.
Step 1: The HIV antigens will be separated by polyacrylamide gel electrophoresis on the basis of their electrophoretic mobility which depends on mass of the proteins (charge on the proteins will be leveled by treating them with sodium dodecyl sulphate (SDS)).
Step 2: Once the proteins get separated, will be transferred to a nitrocellulose membrane strip.
Step 3: The test serum diluted 400 folds and applied on the nitrocellulose strip. The antibodies present in the serum may match some of the HIV antigen and rest may be unbound.
Step 4: The plate is then washed to remove unbound antibodies present in the serum and other components also of the serum
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        Backtrace
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        • 15. vendor/laravel/framework/src/Illuminate/Routing/ControllerDispatcher.php:43
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        • 17. vendor/laravel/framework/src/Illuminate/Routing/Controller.php:54
        • 18. vendor/laravel/framework/src/Illuminate/Routing/ControllerDispatcher.php:43
        • 19. vendor/laravel/framework/src/Illuminate/Routing/Route.php:260
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        1.12mstwenty4_siteHomeworkLibraryController.php#97
        Backtrace
        • 21. app/Http/Controllers/HomeworkLibraryController.php:97
        • 22. vendor/laravel/framework/src/Illuminate/Routing/Controller.php:54
        • 23. vendor/laravel/framework/src/Illuminate/Routing/ControllerDispatcher.php:43
        • 24. vendor/laravel/framework/src/Illuminate/Routing/Route.php:260
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        1.29mstwenty4_siteHomeworkLibraryController.php#97
        Backtrace
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        • 22. vendor/laravel/framework/src/Illuminate/Routing/Controller.php:54
        • 23. vendor/laravel/framework/src/Illuminate/Routing/ControllerDispatcher.php:43
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        • 25. vendor/laravel/framework/src/Illuminate/Routing/Route.php:205
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        980μstwenty4_siteHomeworkLibrary.php#201
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        Backtrace
        • 20. app/Models/HomeworkLibrary/HomeworkLibrary.php:201
        • 26. app/Http/Controllers/HomeworkLibraryController.php:105
        • 27. vendor/laravel/framework/src/Illuminate/Routing/Controller.php:54
        • 28. vendor/laravel/framework/src/Illuminate/Routing/ControllerDispatcher.php:43
        • 29. vendor/laravel/framework/src/Illuminate/Routing/Route.php:260
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        Backtrace
        • 14. app/Repositories/HomeworkLibraryRepository.php:30
        • 15. vendor/laravel/framework/src/Illuminate/Cache/Repository.php:397
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        • 18. app/Repositories/HomeworkLibraryRepository.php:39
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      • select * from `subjects` where `subjects`.`id` in (427)
        910μstwenty4_siteHomeworkLibraryRepository.php#30
        Backtrace
        • 19. app/Repositories/HomeworkLibraryRepository.php:30
        • 20. vendor/laravel/framework/src/Illuminate/Cache/Repository.php:397
        • 21. vendor/laravel/framework/src/Illuminate/Cache/CacheManager.php:419
        • 23. app/Repositories/HomeworkLibraryRepository.php:39
        • 24. app/Http/Controllers/HomeworkLibraryController.php:139
      • select * from `solutionslibrary_files` where `solutionslibrary_files`.`solutionlib_id` = 24170 and `solutionslibrary_files`.`solutionlib_id` is not null and `publish` = 'question' order by `order` asc, `id` asc
        890μstwenty4_siteHomeworkLibrary.php#260
        Bindings
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        Backtrace
        • 15. app/Models/HomeworkLibrary/HomeworkLibrary.php:260
        • 16. app/Transformers/HomeworkLibrary/HomeworkLibraryTransformer.php:58
        • 19. vendor/league/fractal/src/TransformerAbstract.php:128
        • 20. vendor/league/fractal/src/TransformerAbstract.php:107
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        • 19. vendor/league/fractal/src/TransformerAbstract.php:128
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        1.07mstwenty4_siteHomeworkLibrary.php#260
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        Backtrace
        • 15. app/Models/HomeworkLibrary/HomeworkLibrary.php:260
        • 16. app/Transformers/HomeworkLibrary/HomeworkLibraryTransformer.php:80
        • 19. vendor/league/fractal/src/TransformerAbstract.php:128
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