Laboratory Practices Inmunology & Pathology, Gent

Clarification:
This practices where done in the Gent University together with my lab-mate Christian Martin in the Master of Biology as Erasmus.

Practices:

  • Agglutination Assay
  • Effect of the adjuvant type on vaccination
  • Identification of B-cell in tissue
  • FACS Analysis

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PRACTICE 11 — AGGLUTINATION ASSAY

Aim and principle of the experiment:

The main aim of this experiment is to know which are the concentrations of the different anti-cRBC-antibodies. We used cRBC as an antigen and observed if agglutination took place at different dilutions and concentrations. We wanted also to know which titer (maximal dilution, for which agglutination visually occurs) corresponded to each concentrations combination of cRBC and anti-cRBC.

Agglutination is the creation of a large complex which can be observed in our wells by naked eye. This complex is formed because antibodies bind multiple cRBC surface antigens, creating a network which prevents precipitation of cRBC.

Interpretation of the results:

Our plate, in which we used R2 (0.5%) cRBC and both A1 and A2 (rows D and H are — controls):

Another group´s plate, in which they used R1 (2%) and both A1 and A2 (rows D and H are — controls):

Table

As we see on the table, when antibodies are diluted, A1 antibody still produces agglutination even after being reduced its concentration several times, while A2 antibody stops producing agglutination on the first dilution or even doesn´t produce it. The cause is that the concentration of antibodies in A1 is higher than in A2, as shown on the table. As we know, the ratio between antibodies and antigens is crucial for agglutination to occur. This means that A1 has more antibodies to join with the cRBC and form the agglutination clump, and so, A1 is more plausible to still produce this process even if it is diluted. As a consequence, titers in A1 in general are higher than in A2.

On the other hand, we also see that titers on R1 cRBC sample are lower than on R2. This may seem contradictory because the concentration of cells (and so, of antigen) is higher in R1. We may conclude that having too high levels of cell concentration is not advantageous for agglutination to take place. Another explanation can be that there were pippeting mistakes when preparing those rows.

Hemagglutination is used to type blood groups (O, A, B and AB) and match compatible donors and recipients for blood transfusion. Explain:

Blood type can be determined by using antibodies that bind to the A or B blood group antigens in a sample of blood. If antibodies that bind the A blood group are added and agglutination occurs, the blood is either type A or type AB. To determine between type A or type AB, antibodies that bind the B group are added and if agglutination does not occur, the blood is type A. On the other hand, if agglutination also occurs, the blood type is AB. If agglutination does not occur with either antibodies that bind to type A or type B antigens, then neither antigen is present on the blood cells, which means the blood is type O.

Additional question for report:

a. Tube a contained a preparation cleaved by pepsin: F(ab´)2 fragments allowed agglutination because both Fab fragments were bound, and they crosslinked cRBCs forming a network that resulted in agglutination. On the other hand Fc fragment was separated of the rest of the antibody, so it couldn´t recognize complement and cause lysis.

b. Tube b contained a preparation cleaved by papain: Fab fragments recognized antigens but they couldn’t crosslink cRBCs because they were not coupled. However they were bound to cRBCs (antigens), so when the whole anti-cRBC antiserum was added, these binding places were already occupied by Fab fragments, preventing agglutination. Fc fragment was separated as in tube a, preventing complement activation.

c. Tube c contained a preparation in which antibodies weren´t cleaved. So, the whole antibodies worked properly causing agglutination thanks to Fab regions and recognizing complement through Fc region. This tube is probably a positive control.

d. Tube d contained a preparation without antibodies. That is why there is no agglutination or lysis, but when adding whole anti-cRBC serum there is agglutination. This tube is probably a negative control.

PRACTICE 12 — Effect of the adjuvant type on vaccination:

Principle of the experiment:

Vaccination of mice with OVA-antigen without any bacterial component will rather lead to a Th2 response from the CD4 T helper cells, which includes the production of OVA-specific IgG1 antibodies, but when OVA + Bacterial component (CpG) is used as antigen, the T-Helper response will shift to Th1, producing IgG2c antibodies

Relying on the highly specific antibody-antigen interaction, we can quantify analytes such as antigens or antibodies in solutions.

In this experiment we use the indirect Enzyme-Linked Immunosorbent Assay (ELISE):

First, the antigen (ovalbumin, OVA) is immobilized to the solid surface of a 96 well plate mimicking the cell surface. Secondly, serum from OVA- vaccinated mice will be added (PBS/IgG1/IgG2c). Thereby OVA-specific antibodies, present in the serum, will bind specifically to an epitope of the to the immobilized OVA on the surface. These OVA-specific antibodies can be detected by adding secondary antibodies which will bind specifically to the Fc-chain of the primary antibody.

Since the secondary antibodies are coupled to the enzyme HRP, a color shift will be visual by adding substrate to the enzyme HRP of the antigen-antibody complexes.

This enzyme is able to convert a substrate (DAB) into a non-soluble coloured reaction product which can be visualized and measured by Optical Density

Aim of the experiment:

Quantify the relative ratios of IgG1 to IgG2c anti-OVA antibodies to identify the T helper immune response in different samples of serum (N, A, B). We must to find out from the results which one contain the PBS, the OVA ag, or the OVA + CpG

Interpretation of the results:

The different values in the chart shows the Optical Density (O.D.) of the solutions during the next dilutions, from 1 to 11.

This O.D. is related with the amount of specific Antibodies of each student bound to the Antigen (OVA)

Thus, Student 1’s different samples (N,A,B) shows the correlative level of IgG1 and Student 2’s samples the correlative level of IgG2 for those same samples.

So, the sample N has a higher amount of IgG1 than IgG2, à high ratio IgG1/IgG2. That means the sample N comes from a mice vaccinated with OVA.

The sample A has low levels for IgG1 and IgG2, but higher for IgG1. The expected results for this sample was a low rate IgG1/IgG2 and thus, it would have come from a mice vaccinated with OVA+CpG, this low levels obtained could be due to the OVA antigen, non-specific for IgG2 used in the experiment. Sticking to the results, it suggests the sample A comes from a mice vaccinated with a low level of OVA.

The sample B has low levels that don’t decrease during the next dilutions, this suggest that the mice was vaccinated with PBS so it had a lack of response or non significant.

Why are IgG isotypes used as detection antibodies instead of IgM isotypes?

IgM antibody is a multivalent response of the immunology system, which arise as unspecific primary immune-response, in the other hand, IgG is a more specific antibody, that belongs to the secondary immune-response.

In order to study an specific antigen, if we use IgM the unspecific junction will produce confusing results, meanwhile using specific IgG, it will binds (or discriminate) only the antigen in which we are interested.

PRACTICE 13 — Identification of B-cell in tissue

Aim of the experiment:

The identification of b- cells in tissue(spleen) by Immunohistochemistry (IHC) using Antibodies to stain the b-cells, observing the difference between the use of a primary antibody (+) or not (-), in different samples.

Principle of the experiment:

The identification of b-cells in a specific tissue is possible by binding an antibody complex formed by a primary antibody, and a secondary antibody coupled with HRP, the enzyme HRP will convert substrate DAP into a insoluble brown product, visible by light microscope.

By washing the samples with PBS after adding each antibody we make sure that only the antibody connected with the proper b-cell epitope remains in the tissue, so only in those tissue samples where b-cells exists, (thus, with the bound antibody complex) the insoluble brown product will be shown.

To make easier the visualization of cells by microscope, an extra dye, Hematoxylin is added after the HRP-DAP staining, The Hematoxylin stains nuclei of the cells purple and make contrast with the insoluble Brown product from the HRP.

Give an example of how IHC can be used in a clinical setting:

The absence of B-Cell in tissues constitute a symptom of the deficiency of the adaptive immune system, associated with the HIV disease or the lymphoid leukemia.

The study of levels of B-cells by IHC can be used to measure the progression of the disease in an individual , or to study the growth differences of the disease between different tissues.

Interpretation of the results:

In the (+) samples we can see clearly 2 colours:

- Brown: from the presence of the B-cells in the tissue

- Purple: from the stained nuclei of the cells, by Hematixylin

In the ( — ) samples we can see mostly only 1 colour, Purple.

The lack of Brown colour is due to the different treatment of this sample, which lack the incubation of the primary antibody, this has been done to see the importance of the binding of the high specific marker(in this case, the primary antibody) to carry out the correct staining of the B-cells by the enzyme HRP of the secondary antibody, the substrate of the enzyme has been added, but the secondary antibody is not has bound to the primary antibody so it has been washed and eliminated mostly by the PBS. It gives rise to the lack of brown in the negative sample.

PRACTICE 14 — FACS ANALYSIS

Aim and principle of the experiment:

The main aim of this practice is to differentiate the different states of T-cells during their maturation in the thymus, and to know the ratio of different cells on the spleen (T-cells, NK cells, NKT cells, T-helper cells and cytotoxic T cells).

We used antibodies labelled with different fluorochromes, which bind to specific markers for each cell status or cell type. The markers were CD3 (T lymphocytes), CD4 (T-helper cells and thymocytes), CD8 (cytotoxic T cells and thymocytes), NK1.1 (NK cells and NKT cells) and TCRβ (T cells and NKT cells). The fluorochromes used were PE and FITC (red and green colour respectively when exposed under fluorescent laser).

So, depending on the type of cell present on the spleen or thymus, the corresponding antibody bound with a specific fluorochrome will give us a specific fluorescent response when analysing each cell through flow cytometry technique. The information of which levels of red or green light are related to determined markers is facilitated in the graphs by our professor Martin Guilliams, after his flow cytometry test.

Interpretation of the results :

TASK: Please calculate the percentage of CD3+CD4+ T cells among the immune cells that are not autofluorescent:

The percentage of CD3+ CD4+ T cells is a 59.0% of 295 cells (which is the total amount of CD3+ T cells). So the total amount of CD3+ CD4+ T cells among the immune cells that are not autofluorescent is 174 cells. As there are 762 immune cells which are not autofluorescent, the percentage of CD3+ CD4+ T cells is: (174/762)*100 = 22.83%.

As I said on the introduction, each marker corresponds to a type of cell: CD3 (usually expressed by T cells in general), CD4 (T-helper cells and thymocytes), CD8 (cytotoxic T cells and thymocytes). This is important for knowing which cells we are talking about.

TASK 2 = What is the percentage of CD4+CD8+ “Double Positive” T cells among: All immune cells, CD3 positive T cells, CD3 negative T cell.

Percentage among:

All immune cells: 89.9% ; CD3 positive T cells: 61.3% ; CD3 negative T cells: 95.6%

We see that the percentage of double positive cells is higher among T cells which don´t have CD3 marker. As double positive T cells are T cells which have not differentiated into single positive CD4+ cells (usually T-helper cells) or CD8+ cells (usually cytotoxic T cells), we can conclude then that CD3+ T cells are more likely to be differentiated than CD3- T cells.

Additional question for report

First of all I would differentiate the whole group of monocytes, eosinophils and macrophages from the rest of the cells of the blood sample by analysing which cells have the marker F4/80, and thus belong to this group, and which cells don´t have it. So, I would obtain the percentage of cells belonging to this group. Then I would analyse cells carrying CCR3 marker compared with cells carrying Ly-6G marker within the previous group, in order to know the relative percentage of eosinophils and neutrophils, respectively associated to each marker. The rest of the cells within the group are supposed to be monocytes.

Each marker must be associated to the correspondent antibody which carries a determined fluorochrome, for further cytometry test.

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