1. The document discusses several immunological methods and applications including ELISA, latex agglutination assay, immunochromatography assay, haemagglutination inhibition assay, immunofluorescence antibody technique, and Western blot. 2. Key principles involve antigen-antibody reactions to detect the presence of various antibodies or antigens through techniques like enzyme reactions, agglutination, fluorescent labeling, or protein separation/detection. 3. The methods can be used to diagnose conditions like hepatitis, rheumatoid arthritis, malaria, and HIV.

1. Microbiology practical
(Immunological Method & Application)
By :
Saeed Saleh Al-Ghamdi
Osama Al-Zahrani
@OSAMA_Z96

2. ‫تعرف‬ ‫شيء‬ ‫أهم‬ ‫يقول‬ ‫الدكتور‬:
1-‫ايجاب‬ ‫تحدد‬ ‫وانت‬ ‫اختبار‬ ‫نتيجة‬ ‫يعطيك‬ ‫يعني‬ ‫االختبار‬ ‫تقرا‬‫سلبي‬ ‫واال‬ ‫ي‬
2-‫االختبار‬ ‫اسم‬ ‫تعرف‬
3-‫تعرف‬‫ايش‬‫االختبار‬ ‫استخدام‬
4-‫مثال‬ ‫يعني‬ ‫االختبار‬ ‫مبدأ‬ ‫تعرف‬:
(antigen-antibody reaction)‫عمله‬ ‫مبدا‬ ‫تعرف‬ ‫يعني‬

3. ELISA
( enzyme-linked immunosorbent assay )

4. ELISA ( enzyme-linked immunosorbent assay )
• Principle of test : Antigen-Antibody reaction
• Marker : ELISA for ( Anti-HBs Ig M )
• Uses : To diagnose Hepatitis viruses by detect the surface Antigen
• Sensitivity for this test is 95-98 %
• Before first step Dilute the sample ( the control didn't dilute )

5. Micro plat
Micro plate well
One of this called (micro plate well ) , all of this called (micro plate )

6. Procedure
We used only
two rows
1- put 10 µL Anti-HBclgM Control Serum
negative into each of the first 4 wells(A1-
D1), 10 µL Anti-HBclgM Control Serum
positive into the next well(E1), and 10
µL diluted sample into each of
subsequent wells .
2- Incubate sample : Incubate for at least 15 minutes ( ≤ 30 min. )
at 18 to 25 C ° .

7. - ve control
+ ve control
Sample 1
Sample 2
Sample 3

8. 3- put conjugate: Remove the foil. Without washing
the wells, add 100 µL Conjugate Working Solution to
each well. Then seal the test plate with fresh foil and
place immediately into the incubator .
• Conjugate : is anti-antibody binding with enzyme .
• Dilute the conjugate before this step .

9. 4- Incubate conjugate: Incubate for 60 ±2 minutes at 37 ±1 °C,
then proceed immediately to the wash step .
5- Wash : Remove foil and aspirate all wells. Fill each well with
approx. 300 µL diluted washing solution POD, aspirate the plate,
and repeat the wash cycle three times. After completing the wash
cycles, control the effectiveness of the aspiration and proceed
immediately to the next reagent dispensing step(otherwise the wells
may try out).
• we will wash in the machine ( microplate washer )
• washing solution : is solution have 25 times concentration we will
Dilute to become 20 concentration , and we will put it in washing
solution box ( distal water and buffer solution )

10. In this box : We
will put washing
solution for wash
the well .
In this box : We
will put distal
water for wash
the machine after
wash .
In this box : the
contaminated
water will go out
the machine and
collect in this box .

11. 2 Device (machine ) of microplate of ELISA :
1- Microplate washer
for wash the well
2- Microplate reader
for read & print the result

12. 6- put substrate: Pipette 100 µl Chromogen working
solution into each well, then seal the microtitration plate
with fresh foil.
7- Incubate substrate: Immediately after the substrate
dispensing step incubate at 18 to 25 C° for 30 ± 2 minutes,
protected from light.
• Substrate is Chromogen it is will react with Enzyme and
give a color ( Enzyme it is bind with conjugate – back to
slide 6 ) .. If didn’t show color that mean didn’t react and
that mean the conjugate remove with washing that mean
the conjugate didn’t bind with antibody that mean
antibody didn’t bind with antigen that mean the result is
( -ve normal ) ..
• But if show color that mean ( +ve HBc patient )

13. 8- stop reaction: Remove foil, and add 100 µL Stopping
Solution POD to each well, keeping to the same timing as
during the substrate dispensing step.
• Stopping Solution : is sulfuric acid
9- Read : Read the test plate by naked eye and by the
machine at 450 nm within one hour. The recommended
reference wavelength is 650 nm, or where appropriate
between 615 and 690 nm.
10 - you can print the result by Microplate printer .

14. - ve Sample
+ ve Sample
Read the result >>?
The result
Control Sample
- ve Sample

15. Latex agglutination assay

16. RF latex test kit (latex agglutination assay)
• Principle of test : Antigen-Antibody reaction
• Marker : Rheumatoid factor latex for Rheumatoid arthritis
• Uses : To diagnose Rheumatoid arthritis.
• Sensitivity for this test is 75 %
• RF = Rheumatoid factor
Latex agglutination assay

17. 1- put one drop from control +ve >> in 1
2- put one drop from control -ve >> in 2
3- put ( Rf latex ) >> in the all 1 & 2 and mix .
+ve control
and ( RF
latex )
- ve control
and ( RF
latex )
31 2
64 5
Procedure

18. Latex agglutination with +ve control
( because antibody binding with latex )
Control +ve
Latex agglutination
antibody binding with latex
Control -ve
The result

19. Immunochromatography assay

20. • Principle of test : Anti-Antigen-Antibody reaction
• Uses : For diagnose different type of plasmodium ( malaria )
Immunochromatography assay

21. Region of sample (S)
Region of Buffer (B)
1- put sample in region of sample .
2- put Buffer in region of Buffer .
wait to see the band ( result )
Procedure
The Buffer

22. How to read the result
• - If presence one band with C ( control ) that mean ( -ve result ..
Normal )
• - If presence tow band with C ( control ) and with pan ( P. vivax or P.
Ovale P. Malarie ) that mean ( +ve result .. ,malaria patient )
• If presence three band with C ( control ) and with pan and with P.F>
that mean ( +ve result .. ,malaria patient of P. Falciparum )

23. 2 Band >> +ve result >> Sick 1 Band >> -ve result >> Normal
The result

24. Haemagglutination inhibition asssy

25. • This test determine ratio of antibody in blood.
• Principle of test : Antigen-Antibody reaction
• Uses : For detecting the Antibodies
Haemagglutination inhibition assy

26. The result
+ve Sample-ve Sample

27. Immunofluorescence antibody technique

28. • Principle of test : Antigen-Antibody reaction
• Uses : to detect the Antibodies.
• Use Ultraviolet to see antibody under microscope :
- If the fluorescence show up under microscope that mean
presence of antibody (+ve test).
• Like ELISA but conjugate bond within fluorescence .
Immunofluorescence antibody technique

29. Fluorescence microscope

30. The result
+ve Sample
In -ve test will be no florescence light

31. Western blot

32. Principle of test : Detect Anti-HIV antibody
Uses : To conform the HIV
Western blot