Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

The overall goal of this procedure is to quantitatively determine the influenza viral hemagglutinin and neuraminidase content in vaccines using antibodies targeting conserved epitopes. This is accomplished by first identifying conserved epitopes. The second step of the procedure is to dilute the reference and vaccine samples to improve the detection sensitivity.

The third step is to assemble the slot blot apparatus and load the samples into the slots. The fourth step of the procedure is to detect the HA or NA on the membrane using their respective universal antibodies. The final step of the procedure is to quantify the amounts of HA or NA by densitometry.

Ultimately, results can be obtained that show HA or NA quantified against their respective reference antigens. Hi, my name is Anwar Hashem. I’m a PhD student in Dr.Sean Lee’s lab.

Hi, my name is Caroline Grab and I’m a technician in Dr.Sean Lee’s lab at the Center for Vaccine Evaluation at Health Canada. Today we will be demonstrating a slot bot procedure used to quantify the HA and NA antigens of influenza viruses. The main advantage of this technique over existing methods like single radial immuno diffusion SRID, or physical chemical methods, is that it does not require strain or subtype specific antibodies contrary to SRID or sophisticated instruments like physical chemical methods.

In addition, because these antibodies target the only universally conserved epitopes of a RA, they are useful as basic research tools for interested investigators. Generally, individuals new to this method will struggle because they may encounter technical issues such as difficulty to block precipitated vaccine samples, leakage of samples between slots and loss of vacuum during the blotting step, as well as high background in diffuse bands observed following immuno detection. However, attention at some key steps can help resolve these issues.

We will be demonstrating the procedure for you today. So let’s get started. To identify conserved regions, we used cluster W alignments to analyze a total of over 6, 000 influenza A HA sequences from NCBI and found the N terminus of the HA two subunit as the highly conserved region.

The Shannon entropy and conservation rate for each position of amino acid of the identified consensus sequences was calculated. The UNI one epitope shown in red was chosen to develop a universal antibody against HA for the slot blot assay. Using a similar approach, two universally conserved sequences were identified in all influenza, A NA one close to the enzymatically active site and the other at the end terminus peptides with these amino acid sequences were used as antigens to generate universal antibodies against NA for the slot blot assay.

Before starting the slot blot procedure, prepare 20 milliliters of four MO urea solution in TBS for the hemagglutinin or HA slot blot, or 20 milliliters of a 0.01%ergen solution in TBS for the neuraminidase or NA slot blot then prepare two liters of washing buffer by adding between 20 to A TBS solution to a final concentration of 0.1%Next, prepare 80 milliliters of blocking buffer by dissolving skim milk powder to a final concentration of 5%weight to volume in washing buffer. Activate the PDF membrane and methanol for 15 seconds, followed by a two minutes rinse in double distilled water. Soak the membrane in TBS until use pre-wet three sheets of T blot SF filter paper in TBS until used influenza vaccine reference standards with predetermined HA content corresponded to the vaccine strains to be tested.

Were obtained from the Center for Biologics Evaluation and Research in the USA or the National Institute for Biological Standard and Control in the uk and were used for the HA or NA quantification by slot blot for HA reference antigens and test vaccine samples. Prepare 650 microliters of diluted HA reference antigen stock in four molo urea TBS at the listed concentrations. Then prepare 650 microliters of test vaccine samples in four mo urea TBS at three different dilutions such as 10 x, 20 x, and 40 x to achieve a twofold difference and allow the sample concentrations to fall within the standard curve for NA reference antigens and test vaccine samples.

Prepare 650 microliters each of NA antigen standards stock in 0.01%sugen TBS at the listed concentrations and a antigen standards were made in-house by using SDS page in conjunction with dense cytometry analyses. Then prepare 650 microliters of test vaccine samples in 0.01%sugen TBS at three different dilution such as 10 x, 20 x, and 40 x to achieve a twofold difference and allow the sample concentration to fall within the standard curve. Assemble a previously cleaned and dried bio SF microfiltration apparatus by placing the gasket support plate into the vacuum manifold and the ceiling gasket on top.

Place the three moist and sheets of BIO SF filter paper over the gasket. Then the pre soaked PVDF membrane. Then place the sample template on top of the membrane and finger tighten the four screws using a diagonal crossing pattern to ensure uniform application of pressure on the membrane surface Before connecting the apparatus, verify that the three-way valve is set to expose the vacuum manifold to the vacuum source.

Turn on the pump and connect the bio dot apparatus. Make sure that the flow valve is positioned at a level below the sample wells for rapid and proper drainage of the samples in the following steps. Repeat the screw tightening step using the diagonal crossing pattern.

Tightening the screws while the vacuum is applied ensures a tighter seal and prevents cross contamination between wells. Then change the flow valve to expose the vacuum manifold to air and turn off the pump. Next, apply 100 microliters of TBS to all wells using a multi-channel pipetter to rehydrate the membrane.

Apply the solution to the middle of the well as close to the bottom as possible in order to cover the slot evenly. Use a clean pipetter tip to remove any air bubbles introduced. Change the flow valve to expose the manifold to both air and the vacuum.

Turn on the pump and gently drain the buffer from the wells by putting a finger over the pore exposed to air to regulate the amount of vacuum. Then change the flow valve to expose the manifold to air. As soon as the buffer is completely drained from all wells.

Turn off the pump or disconnect the vacuum. Apply 200 microliters of each standard or vaccine sample dilution per well. Pipette samples, one at a time in the center of each well as close to the bottom as possible.

Taken care to avoid and remove airable. Put 200 microliters of sample dilution buffer in unused wells to ensure proper vacuum to the wells in use. Adjust the flow valve to expose the manifold to both air and the vacuum.

Turn on the vacuum pump and gently allow the samples to completely drain from the wells by controlling the amount of vacuum with one finger over the pore exposed to air. Remove the finger from the port to release the vacuum. As soon as all the samples have drained immediately.

Add 200 microliters of TBS to each well. Using a multichannel pipetter, apply a gentle vacuum to drain and wash the wells by controlling the amount of vacuum with one finger over the port exposed to air. Repeat the wash step once as soon as the wells have completely drained, loosen the screws of the sample template and turn off the vacuum to avoid over drying.

The wells immediately proceed to remove the membrane and place into blocking buffer. Using enough buffer to completely cover the membrane. Incubate overnight at four degrees Celsius with gentle rocking.

Following the overnight incubation and blocking buffer. Wash the membrane twice as listed, followed by rocking for all washings and incubations. Ensure the membrane is completely covered with solution.

Next, incubate the membrane with the uni one universal rabbit antibody against HA or the universal antibodies HCA dash two or HCA dash three for NA diluted in 5%weight to volume BSA in washing buffer for one hour at room temperature, dilutions of one to 4, 000 of rabbit antiserum gave optimal results for these antibodies. Following this, wash the membrane three times at room temperature with gentle rocking for 15 minutes each time. Then incubate the membrane with a one to 50, 000 dilution of goat anti IgG, IgG peroxidase conjugated antibody and blocking buffer and incubated room temperature for 30 minutes with gentle rocking after incubation, wash the membrane and washing buffer three times at room temperature rocking for 15 minutes each.

Then wash the membrane with TBS for five minutes with gentle rocking to remove residual tween 20 detergent from the membrane surface. Next, prepare the developing substrate using three milliliters of each reagent from the super signal West Dura extended duration substrate kit and mix. Well place the membrane on a dry tissue paper to gently remove any excess TBS buffer before transferring to a dry container and applying the substrate incubate for five minutes at room temperature with gentle rocking and remove excess substrate by blotting.

When finished, expose the membrane to a chemiluminescence film. The length of the exposure will need to be optimized to avoid signal saturation, but will typically range from 10 seconds to 10 minutes. Dense cytometry analysis on the developed film was done using the flora chem gel documentation system following the manufacturer’s instructions.

Average the density values for each set of reference antigen replicates and test vaccine samples. Triple kits are needed for significance testing and to evaluate experimental error. Plot the averaged density values versus the amount of HA or NA antigen in each slot.

Taken from the reference standard. Here is shown a four parameter logistic model for immunoassays that uses a reference vaccine concentration and density to extrapolate sample concentration from the density of test sample vaccines using a variable slope, nonlinear regression curve fitting approach. Graph PRISM is used to plot the standard curves and obtain concentrations from the density data obtained as shown here.

The HA universal antibody UNI one demonstrated remarkable specificity for viral sequences and is capable of binding to 13 different subtypes of influenza. A HA extracted from ENT toic fluids of virus infected embryonated chicken eggs. NP protein is the loading control.

The negative control is ENT toic fluid from uninfected eggs, and the positive control is a reference standard of H one from N-I-B-S-C. The differences observed in the mobility of the HA proteins are likely due to variations in their size and or processing stage. As shown here, the antibodies HCA dash two and HCA dash three against both conserved epitopes of NA were capable of binding to all nine subtypes of NA and showed very little cross reactivity to Alan Toic or cellular proteins and P protein control is shown in Panel C.The positive control is Alan Toic fluid spiked with R NA one of a slash new caledonia slash 20 slash 99 and probed with the corresponding antisera NP protein control is shown at the bottom.

This image shows representative results similar to those of HA of the quantification, the NA antigen in the influenza vaccine reference standard and in vaccine samples. Density analysis showed that the intensity of the signal obtained by detecting the NA antigen in various dilutions of a vaccine reference standard by slot blot is proportional to the concentration of na. While attempting this procedure, it’s important to remember to pretreat the vaccine samples to dissolve sedimented materials and ensure proper drainage from the slot.

Lot care must also be taken to prevent leakage of samples between wells. Optimization of antibody dilutions and incubation times may be necessary for any new antibody used for the detection to get sharp signals of ideal intensities while minimizing background after its development. This technique paved the way for scientists in the field of influenza vaccine research.

Further future quantitative analysis of HA and NA antigens. After watching this video, you should have a good understanding of how to quantify the important surface proteins of influenza viruses in simple assays without the use of strain specific antibodies.