Use of antibodies to analyze body fluids and determine species of origin

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Use of antibodies to analyse body fluids and determine species of origin

Introduction

In vitro interactions between antigen and antibody are widely used for diagnostic purposes. Immunoassays can detect serum antibodies to specific infectious agents and identify microorganisms in clinical specimens; they are also useful in forensic science, for example for the detection of drugs and toxins, and identification of the nature of a biological fluid.

Antigen-antibody interactions can result in a variety of consequences, including precipitation of soluble antigens, agglutination of particulate antigens, neutralization of toxins and viruses, and activation of complement. Some assays depend on these secondary events (eg precipitation).

Carry out experiments 1a and 1b in parallel, starting with experiment 1a.

The parts of the form to be completed and submitted (as a record and extension of the lab file results) are shown in red.

Background

Cross-linking of multiple antigen molecules by antibody is required for precipitation, agglutination or complement fixation, and it is possible only if the antigen is multivalent and the antibody is at least bivalent [either intact or F(ab')₂].

The ability of antibodies to precipitate antigens from solution (precipitation reaction) is one of their best known properties and is dependent on the antigen binding sites of antibodies specifically binding to a number of determinants on a single antigen molecule. A single antigen binding site is specific to one antigenic determinant, a property conferred by its variable domains, but a polyclonal antibody (derived from many B lymphocyte clones) will be directed to a number of determinants on an antigen. The precipitation reaction takes place when polyclonal antibodies and their soluble antigens are mixed in the correct proportions. The lattice theory states that precipitation is a result of the increase in size of antigen-antibody aggregates such that each molecule of antigen is linked to more than one antibody molecule and vice versa. When the aggregates exceed some critical volume, they settle out of solution spontaneously.

Precipitate formed when adding increasing amounts of antigen to an antibody.

Precipitation reactions in gels

Precipitation reactions can be performed gels.

Most proteins can readily diffuse through an agar gel but, if a precipitating antibody contacts the appropriate antigen whilst migrating through the gel, an immunoprecipitate will form at equivalence and this can be visualised.

Experiment 1a. Double immunodiffusion for determining which sample is human serum

In this technique antigen and antibody are loaded in separate wells bored in the agar; these proteins diffuse out into the agar forming gradients of concentration, with the highest concentrations closest to the wells. Somewhere between the two wells, the reacting antigen and antibodies will be present at proportions that are optimal for the formation of a precipitate; (equivalence). The precipitin line can be visualised. This assay is called the Ouchterlony or double immuno diffusion technique.

PURPOSE OF EXPERIMENT

To determine which blood sample is human, using a human albumin specific antibody.

EXPERIMENTAL

Week 1

1. Carefully pour the tube of 1% (w/v) agar solution at 55°C into plastic template. Leave to set at room temperature for 15 minutes.
2. Cut neat holes in the agar with a cutter, using the paper template as a guide to give two sets of holes as show below.
3. Remove the agar plugs using a vacuum line, leaving empty holes (GET A DEMONSTRATOR TO HELP).
4. Label the underside of the plastic template
5. Carefully introduce 10 ml of four samples to the outer wells (one sample/hole) of one set.
6. To the other set, introduce 10 ml of ‘standards’ (1mg/ml; human albumin, 1mg/ml bovine albumin, 1mg/ml horse albumin and 1mg/ml goat albumin)
7. To the central wells of both sets add 10ml of antibody A (goat anti human albumin antibody)
8. Incubate the plate in a closed humidity chamber at 4°C for 1 week.

O                                       O                                             O                                             O

Sample 1               Sample 2                                             Goat alb                      Human alb

                        O                                                                                             O

                        Antibody A                                                                             Antibody A

O                                       O                                             O                                             O

Sample 3               Sample 4                                 Bovine alb                   Horse alb

Week 2

9. Remove the plates, dry the bottom of the plate and observe the zones of precipitation.

10. Record your observations.

Insert a photo of your plate, or draw a schematic of it, in the space below. Label the wells and indicate the position of the precipitin lines.

Precitiptin lines indicate that ‘Antibody A’ travelled to the ‘Human albumin’ on both the left and right side of the plate.
ANSWER THE FOLLOWING QUESTIONS IN THE SPACE BELOW:

1. Give a definition of polyclonal and monoclonal antibodies (use between 150 and 200 words)

The terms monoclonal antibodies are used to represent an antibody population that recognises a single epitope specific to an antigen. Typically, production of monoclonal antibodies involves using a single B lymphocyte of an immunised mouse to generate a clonal antibody population. These antibodies are all identical to each other and recognise the same epitope within an antigen. As a result, they possess low cross reactivity in the presence of non-specific antigens, making them quite attractive for application in various biological assays.

On the other hand, polyclonal antibodies are a heterogeneous antibody population that recognises a variety of epitopes within an antigen and combines with the antigen, exhibiting notable variations. The response to the antigen, in general, involves the B-cell activation that targets specific epitopes on the antigen. Consequently, antibodies are produced in large numbers with varying epitope affinities and specificities. Production of polyclonal antibodies entails purification from serum derived from immunised animals, where B-lymphocytes produce a range of antibodies specific to a particular antigen in response to antigen stimulation. Ideally, polyclonal antibodies can be used in suitably in sandwich assays where they act as antigen detectors in the second stage. They are often tagged with alkaline phosphatase or horseradish peroxidase (Harlow & Lane 2008).

2. Briefly describe the technique of ”double immunodiffusion” (use between 50 and 100 words)

Double immunodiffusion allows both the antibody and antigen to diffuse into a gel. The technique has often been used when studying evolution as it can test if two antigens bear similarity (Stanley 2002).  There are 3 wells in this procedure; two of these are loaded with the antigens set for identification while the third well containing a known antibody is put between and below the antigen wells in a triangular pattern. A range of geometrical patterns can be produced in the space between the antiserum and antigen wells, and this depends on the similarity of the antigens (Crowle 2003).

3. What was the purpose of the albumin standards in experiment 1a?

The albumin standards served as controls in the experiment.

4. 1. Observe the reaction between the antibody (anti human albumin) and the “standard” albumins: has the antibody reacted with human albumin as expected? Justify your answer.

The antihuman albumin did not react with human albumin as had been expected because there lacks similarity between the antihuman albumin and the human albumin as the epitopes on the antigen cannot bind the antibody. The lack of similarity emanates from the fact that lacks evolutionary relationship between the animal from which the antihuman albumin was derived and humans (Phillips 2011).

1. Observe the reaction between the antibody and the unknown samples. Which sample is of human origin?

Sample 3

Experiment 1b. Single radial immunodiffusion for determining the amylase content of human saliva.

In this technique antigen is loaded into wells bored in an agar gel which contains a fixed concentration of antibody. The antigen diffuses into the gel and combines with antibody until equivalence is reached, ie, the antigen is precipitated and made stationary. At this point an immunoprecipitation ‘halo’ or ring is formed around the well. Mancini showed that the diameter (area) of the ring at equivalence is related to antigen concentration in the well. Therefore, preparations of a purified antigen can be used to establish a standard calibration graph so that the unknown concentration of antigen in biological fluids can be determined.

In this experiment an anti-human amylase antibody is incorporated in the gel. In order to improve the visualisation of the precipitin ring, amylase activity is monitored rather than the precipitin itself. Active amylase is detected using a modified substrate for amylase, starch azure (starch covalently linked to Ramazol Brilliant Blue R), which is blue. When the blue substrate is digested by amylase the blue colour disappears and a clear colourless disc is produced. The edge of the colourless ring signifies where the precipitin line is; the diameter of this ring defines the amount of amylase in a sample: Using amylase ‘standards’ one can estimate the concentration of amylase in saliva.

PURPOSE OF EXPERIMENT:

To compare amylase concentrations of two samples.

EXPERIMENTAL

Week 1

40ml of 1% (w/v) starch azure agar (Blue agar) has been mixed for you with 100ml of anti-human saliva antiserum.

1. Immediately pour the agar into a plastic template. Leave to set at room temperature for 15 minutes.
2. Cut neat holes in the agar with a cutter, using the paper template as a guide to give two sets of holes as show below. MAKE SURE THE CORRECT TEMPLATE IS USED.
3. Remove the agar plugs using a vacuum line, or as directed by the demonstrators, leaving empty holes, as before
4. Label the underside of the plastic template
5. Prepare standard solutions of amylase by diluting stock (20mg/ml) with distilled water to give the following six concentrations:

10mg/ml, 5mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml and 0.25mg/ml.

6. Dilute the two unknown saliva samples with distilled water; dilute 1:1(1/2) and 1:4 (1/5).
7. Carefully introduce 10 ml of each amylase solution (prepared in step 5) into the top row (wells 1-6) of the immunodiffusion plate
8. Pipette 10 ml of neat, 1:1 and 1:4 dilutions of saliva samples into the bottom row, as illustrated below.

O         O         O         O         O         O

10        5          2          1          0.5       0.25     AMYLASE (mg/ml)

                        O         O         O         O         O         O

                        S1        S1        S1        S2        S2        S2        SAMPLES

• 1:1 1:4 0          1:1       1:4       Dilutions

9. Incubate the plate in a closed humidity chamber at 4°C for 1 week.

Week 2

10. Remove the plates; dry the bottom of the plate.
11. Measure the diameter of each ring (in mm) for the Amylase standards and the Unknown samples (S1 and S2) and fill the “Diameter”-row in the tables below (table 1 and 2) with the corresponding values.
    

Table 1

Table 2

*Enter the values given by the diluted samples first, then the actual values after taking into account the dilution factor please.

12. Use data of Table 1 to construct a standard calibration graph of diameter²versus amylase concentration (using the standards) and attach it to this form.

13. Using the standard curve, calculate the concentrations of amylase in the two undiluted and diluted samplesS1 and S2 and transcribe them in Table 2. Please SHOW CLEARLY THE STEPS LEADING TO THE CALCULATION OF THECONCENTRATIONS (including the dilution factor when applicable).

14. On the basis of your data what is the most likely concentration of S1 and S2? Justify your statement.

S1 most likely has a concentration of 4.5 and S2 0.1 because the concentration for the dilutions cannot be higher than the concentration of the actual samples.

15. Answer the questions below.

QUESTIONS

1. What effect would eating have on amylase concentration in saliva? (use between 50 and 100 words)

Eating would definitely raise the concentration of salivary amylase in saliva. The biologic function of salivary amylase is the breakdown of starch into the simple sugar, maltose. Consequently, immediately after taking a meal, the salivary amylase concentration rises steadily to digest the starch being taken. The change in concentration is especially marked when the food being taken is a snack, and when the introduction of such food into the mouth is done in high frequencies. The pH of the food being eaten would cause more increase when it is high (Talwar & Srivastava 2002).

2. What control should have been used in the experiment? Justify your statement.

A well filled with distilled water only should have acted as the control. This would then have been compared with the rings formed in the other wells. Such a control would have been suitable because distilled water has no activity on starch. Therefore, while salivary amylase in the wells showed rings, there would be apparently no reaction in the well containing distilled water.

How to dispose of the waste from this class:  Immunodiffusion

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