Medical Microbiolgy - Virology Serology Laboratory Techniques

Low Wei Qi Kenneth - TG02



Hello everyone,

SO sorry for a week delay of the blog sharing. For the few weeks of attachment, i'm attached to virology serology lab which performs tests on human serum mainly detecting Ag and Ab of specific virus. The test i'm going to share is ELISA ( Enzyme linked immunosorbent assay) or EIA (enzyme immunoassay).



Test - Human Varicella Zoster Virus (VZV) IgG Enzyme immunoassay ( EIA/ELISA)



The purpose of this test is to detect Immunoglobulin G antibodies to VZV in patients' serum where VZV is a virus that commonly causes chickenpox in humans.



In my lab, they use commercial test kits which provide all the reagents and pre-coated microtiter strip wells. The base of the wells serve as the solid phase are pre-coated with VZV antigens and by adding the patients serum and incubate it at room temperature, if the IgG antibodies are present it will bind to the antigens at the solid phase. After incubation, the wells are washed using the washing solution provided in the kit. Also, the washing step time is reduced using automated microtiter plate washer which is programmed to dispense a certain amount of wash solution into each wells and remove them. So after washing, conjugates are added. The conjugates are anti-human IgG antibodies with peroxidase conjugate. The anti-IgG is added and it will bind specifically to IgG antibodies that are bound to the solid phase. Again, the wells are washed to remove unbound substances. Later, substrate and stop solution are added the wells with VZV-IgG-Antibodies will produce yellow colour. The intensity of the colour is directly proportionalo to the amount of secondary antibody present in the well. And is determine by using automated ELISA microplate reader. After the absorbance of each well is being read, the result is printed out and is compared against cut-off values.

SOoooo heres the STEP BY STEP:

1. Reagents and samples are brought to room temperature.
2. 10 ul of Patients specimens are diluted in 1ml of dillution buffer. ( 100x dilution )
3. 100ul of Positive and Negative Controls and diluted specimens are added into each wells.
4. The plate is then incubated at room temperature for 30 minutes.
5. During incubation, the wash solution is being prepared by diluting the concentrate into distilled water. ( 20x dilution)
6. After incubation, the wells are washed using the microplate washer.
7. 100 ul of Conjugate is then added into each wells except the blank well which act as a control.
8. The plate is then incubated at room temperature for 30 minutes.
9. After incubation, the wells are washed using the microplate washer.
10. 100 ul of Substrate is added into each wells
11. The plate is then incubated at room temperature for 15 minutes.
12. 100 ul of stop solution is added to each wells to stop the reaction.
13. The microplate is then place into the microtiter plate reader for results.

Results interpretation :

Quality control criteria -

• Negative Control values should be less than 0.250
• Positive Control values should be more than 0.750
• +ve control / -ve control should be more than 5
• Blank ( empty well ) should be less than 0.150

If either of the values do not meet the criteria, the whole run is rejected and rerun has to be conducted.

Interpretation -

• Cutoff value (COV) = -ve control + 0.1 x +ve control
• Positive > 1.15 x COV
• Negative < 0.85 x COV
  

To interpret the results, calculations are carried out and these formulas are provided by the supplier of the kit.

Low positive will mean that the immunity is low and the patient is susceptible to re-infection

High/ mid positive will mean that the person is immune to the virus.

Negative will mean that is patient is not reactive or not immune to the virus.

So this is the end of my sharing, to summarise, mainly in virology lab, ELISA is one of the most common test that is conducted to detect viral infection as well as the immunity of the patient to specific virus.

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