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Homework answers / question archive / Human Haematology and Clinical Immunology ASC_5_22/23 Laboratory 1 Manual - Covid-19 Total Antibody ELISA Covid-19 Total Antibody ELISA Introduction End of 2019, a novel respiratory disease emerged in the city of Wuhan, China and soon spread rapidly worldwide leading to Covid-19 pandemic

Human Haematology and Clinical Immunology ASC_5_22/23 Laboratory 1 Manual - Covid-19 Total Antibody ELISA Covid-19 Total Antibody ELISA Introduction End of 2019, a novel respiratory disease emerged in the city of Wuhan, China and soon spread rapidly worldwide leading to Covid-19 pandemic

Health Science

Human Haematology and Clinical Immunology ASC_5_22/23

Laboratory 1 Manual - Covid-19 Total Antibody ELISA

Covid-19 Total Antibody ELISA

Introduction

End of 2019, a novel respiratory disease emerged in the city of Wuhan, China and soon spread rapidly worldwide leading to Covid-19 pandemic. There was a high demand from the clinical laboratories to order Covid-19 kits to test and diagnose patients with Covid-19 symptoms.  Now, there are many Covid-19 tests available from different companies and for this laboratory students will be using a Covid -19 Total Antibody (IgG and IgM) ELISA kit to test controls and samples

Note: No human samples used for this assay.  Negative and positive controls have been aliquoted and used as samples

The purpose of this practical is not to get knowledge in the Covid -19 disease but to understand the principle of the indirect ELISA and practice laboratory skills (like how the procedure is carried out in clinical setting).

Principle of the assay

This is a qualitative two-step incubation indirect ELISA technique which determines both IgG and IgM Covid -19 antibodies.

Polystyrene microwell strips are coated with specific antigens (in this case recombinant SARS-CoV2 antigen).  Controls and samples are added and during the first incubation the antibodies in the sample will bind to the specific antigens coated in the microwells forming Ag/Ab complex. 

The microwells are then washed to remove unbound sample proteins and a second antibody conjugated (labelled) with the enzyme HRP (horse radish peroxidase) is added and during incubation this will bind to the Ag/Ab complex inside the microwells forming Ag/1stAb/2ndAb sandwich immunocomplex.

The microwells then are washed again to remove unbound conjugate and chromogen (TMB - 3,3', 5,5;-tetramethylbenzidine) solution is added into the microwells.  The colourless chromogens are hydrolysed by the bound HRP conjugate to a blue coloured product.  The intensity of this product is proportional to the amount of specific antibodies in the sample. A week sulphuric acid (0.2M) is added to stop the enzymatic reaction. This produces a yellow endpoint colour. Absorbance at 450nm is read using and ELISA microtiter plate reader.

 

Fig. 1 Principle of the indirect ELISA

Materials for each bench

Microwell plate 96 tests coated with recombinant antigen - two strip for each pair of students 

Negative control  (Neg) – Green tube

Positive control (Pos) – Red tube

HRP – conjugate reagent - ready to use (Red bottle)

Chromogen A ( Green bottle) and Chromogen B (Black bottle)– ready to use

Wash buffer – ready to use

Stop solution – ready to use (Yellow bottle)

Paraffin ( plate sealer)

Pipettes (100 and 1000?l) and tips for these pipettes.

96 well microtiter plate reader (at least 2 plate readers for the whole class)

Water bath 37 °C

PPE (gloves, lab coats, waste container, sharp bins, alcohol/disinfectants,)

Timer

Clean towels

Fine markers

 

Procedure

Step 1 – Preparation

•             Work in pairs

•             Each pair will use 2 strips (16 wells) of the 96-microtiter plate (Fig1).

•             Using a fine marker mark your initials on your strip e.g., ST & PT

•             Using a fine marker, mark well A1 as Blank (B), B1 as Negative Control (Neg) and C1 as Positive Control  (Pos) and D1-H2 as Patient Sample ( S1 – S13) as shown in fig.1 below.

 

Step 2 – Add controls/samples and incubate plate (1st incubation) Note: use a separate disposable pipette tip for each sample and control to avoid contamination

•             Using a 100 ?l pipette add 50?l of negative and positive control into their respective wells (excluding blank)

•             Using a 100?l pipette add 50??l sample into their respective wells

•             Mix by taping the plate gently

•             Cover the plate with paraffin and incubate in a water bath (37°C) for 25 minutes.

Note: Use this time to answer questions 1-5 at the end of this booklet

 

Step 3 – 1st Wash

•             Remove the plate from the water bath - take off the paraffin and discard it in the clinical waste bin.

•             Set a 1000?l pipette at 300?l and using the same tip add 300?l of wash buffer in each well (excluding blank) – leave it to soak for 30-60 seconds and discard wash in the sink (be careful with the wells not to come out).

•             Repeat the above step 2 more times (in total 3x washes).

•             After the final wash carefully turn down the plate onto a clean towel and tap it gently to remove any wash buffer residue (gently remove any bubbles with a tip).

 

Step 4 – Add HRP conjugate and incubate ( 2nd incubation)

•             Using a 100?l pipette and using the same tip add 100?l of HRP- conjugate into each well (excluding blank).

•             Cover the plate with paraffin and incubate at a water bath (37°C) for 25 mins.

Note: Use this time to answer questions 5-10 at the end of this booklet.

 

Step 5 – 2nd Wash 

•             Remove the plate from the water bath - take off the paraffin and discard it in the clinical waste bin.

•             Set a 1000?l pipette at 300?l and using the same tip add 300?l of wash buffer in each well (excluding blank) – leave it to soak for 30-60 seconds and discard the wash in the sink (be careful with the wells not to come out).

•             Repeat the above step 2 more times (in total 3x washes).

•             After the final wash carefully turn down the plate onto a clean towel and tap it gently to remove any wash buffer residue (gently remove any bubbles with a tip).

 

 

Step 6 – Colour development 

•             Set a 100?l pipette at 50?l and using the same tip add 50?l of Chromogen A in each well including the blank.

•             Set a 100?l pipette at 50?l and using the same tip add 50?l of Chromogen B in each well including the blank

•             Mix by tapping the plate gently

•             Cover the plate with the plate sealer and incubate it in a water bath (37°C) for 15 mins avoiding light.

Note: If the test has worked the enzymatic reaction between the HRP and chromogen (TMB) will produce a blue colour in positive control and SARS-Cov 2 antibody-positive sample wells.

Negative samples - colourless wells.

 

Step 7 – Stop the reaction

•             Remove the plate from the water bath - take off the paraffin and discard it in the clinical waste bin.

•             Set a 100?l pipette at 50?l and using the same tip add 50?l stop solution in each well and mix by tapping the plate gently.

Note: If the test has worked an intensive yellow colour will develop in the well containing the positive control and SARS-Cov 2 antibody positive sample wells. Negative samples – colourless wells

 

Step 8 – Measure the absorbance

•             Read the absorbance at 450nm using a 96-well microtiter plate reader as soon as stopping the reaction.

 

Note: An absorbance microplate reader (or spectrophotometer plate reader) is a piece of equipment capable of detecting and quantifying the light photons absorbed/transmitted by a liquid sample present in a microplate when exposed to light at a specific wavelength. Absorbance detection is an absolute measurement quantified in Optical Densities (ODs). OD is defined as the logarithmic ratio between the intensity of light hitting a sample and the intensity of the light transmitted through it. Compared to a standard spectrophotometer that measures one sample at a time in a cuvette, a microplate reader can measure the same absorbance assay with higher throughput. It can measure a full 96-well microplate in seconds.

 

Step 9 – Clean the bench! 

 

Step 10 - Assay validation (Quality control)

Each microplate well should be considered separately when calculating and interpreting the results of the assay.

1              Subtract blank absorbance (O.D) from each control and sample result (record results in table 1)

2              Calculate cut-off absorbance (O.D) using the formulae below:

Cut/off absorbance (O.D) = Negative (O.D) + 0.16

3              The assay has worked if the below Quality control criteria are fulfilled:

-              The blank absorbance (O.D) value which contains only Chromogen and Stop solution must be <0.08

-              The positive control absorbance (O.D) value must be > or = to 1.9 after subtracting the blank

-              The negative control absorbance (O.D) value must be < or = to 0.1 after subtracting the blank.  Note: if your negative control is less than 0.03 take it as 0.03.

-              If negative and positive control values do not meet the criteria the test has failed (in clinical practice the test must be repeated using fresh control aliquots).

Step 11 Interpretation of results

Example

-              Negative control = 0.02.  as the value is <0.03 so the value of 0.03 is used to calculate the cut/off

-              Positive control = 2.5

-              Cut/off absorbance = 0.03 +0.16 = 0.19

Interpretation of the results - for the example above:

•             Negative results = all samples giving absorbance (O.D) less than the Cut-off value.  Following the example above all results with O.D < 0.19 are reported as negative. This indicates no SARS Cov-2 Abs, hence no serological indications for current or past COVID-19 disease.

•             Positive results = for this test all samples giving an absorbance (O.D) equal to or greater = 1.9 are reported as Positive. This indicates that SARS-Cov2 antibodies are present, and patients might be currently infected or have a past infection.  To differentiate between present infection and past infection separate serology IgM and IgG must be performed.  If IgM is positive and IgG negative = present infection. If IgG is positive and IgM is negative  =  past infection.

•             Borderline results = all samples giving absorbance (O.D) between cut/off and positive values.  All results with O.D between 0.19 – 1.9  are reported as indeterminate or borderline.  In the clinical laboratory, a repeat test is required for these samples (usually separate IgM and IgG). 

Table 1.  Use the table below to calculate, interpret and report your results (enter results on the lab report)

                O.D result            O.D result after subtracting the Blank     Report results as: positive, negative, or borderline                 Comment e.g., no colour change after adding 2nd Ab – what might have happened? 

Blank    

 

                N/A        N/A       

Negative control             

 

                                               

Positive control

 

                                               

Cut/off

                                N/A        N/A       

Sample 1             

 

                                               

Sample 2             

 

                                               

Sample 3             

 

                                               

Sample 4             

 

                                               

Sample 5             

 

                                               

 

 

 

 

 

 

 

 

Answer the questions below (include a copy at the end of the laboratory report)

 

Question 1.  What does ELISA stand for?

 

Question 2.  What are microtiter wells coated with in the Covid-19 total Ab indirect ELISA?

 

Question 3. What happens in the 1st incubation of the Covid-19 total Ab indirect ELISA?

 

Question 4.  Why is it important to use separate pipette tips when adding controls and samples?

 

Question 5. Covid-19 total Ab indirect ELISA has high sensitivity and specificity – what does this mean?

 

Question 6.  What are the causes of the high reading background for this assay?

 

Question 7.  What can be the causes of no signal/colour development in the wells for this assay?

 

Question 8.  Indirect ELISAs are prone to cross-reactivity – what does this mean?

 

Question 9.   What is the difference between a primary Ab and a secondary Ab in indirect ELISA?

     Question 10.  Why is a substrate (TMB) and stop solution (weak acid) used in the ELISA technique?

 

 

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