Student Investigation on the Immune System and Hemeagglutination

Nancy Ridenour
Ithaca High School
Ithaca, New York
nancyvr@clarityconnect.com

The purpose of this experiment is to demonstrate several of the concepts developed in your test and during lectures and classroom discussions about the immune system. Since the system is currently under attack by the AIDS virus, it is a timely topic to handle thoroughly in the classroom. Thorough understanding of the immune system also involves discussions of genetics, DNA, biotechnology, evolution, use of animals in the classroom and in research, the transport system itself, disease causing organisms, and bioethics. This lab is an excellent focal point for collating concepts developed during the year.

This lab will demonstrate the principles of antibody-antigen binding, the secondary immune response, cross reactivity and complement fixation.


ABSTRACT

The immune system is critical to the survival of most animals. Through evolution, the system developed complexity and specificity. It enables organisms to survive the incredible number of pathogens in the environment and inside the organism. With the immune system, death by infection quickly ensues.

Since human body fluids should not be used in the biology classroom because of the potential transmission of AIDS or hepatitis, the use of animal serum and antibodies is a viable alternative for understanding the mechanisms of the immune system. This use also emphasizes the importance of the responsible use of animals in biological research. Cross reactivity of antibodies with more than one animal strengthens discussions about evolution and speciation. Rather than ignore blood studies because of recent hazards involving the use of human serum, this lab was designed to stimulate the your interest in and understanding of immunology. Since the immune system is such a key factor in survival, sufficient time should be spent in its study.

This lab will demonstrate the principles of antibody-antigen binding, the secondary immune response, cross reactivity and complement fixation.

The materials to be used include antibodies from a rabbit that was injected once with red cells from a sheep and antibodies from a rabbit that was injected three times with the red cells from a sheep. In part, this reaction mimics the reactions which occur in a mother with an Rh incompatible baby. Sera containing red cells from sheep, goat, dog and rabbit will be used to detect the specificity of antibody-antigen agglutination and possible cross reactivity between species.


MATERIALS

IMMUNOLOGY LAB A: MACRO AGGLUTINATION OF SERA

  • 4 microscope slides per person
  • 4 toothpicks per person
  • 4 Pasteur pipettes with bulb (or eyedroppers)per person
  • 1 pipette per person for antibodies
  • Microscope for viewing slidesvial of anti-sheep antibodies(1:10 high titer antibodies)
  • sera containing red cells from: sheep, goat, dog, and rabbit (50% red cells)
  • IMMUNOLOGY LAB B: HEMAGGLUTINATION AND HEMOLYSIS OF RED CELLS

  • 1 round bottom 96 Corning Cells Wells plate per team
  • 1 Eppendorf automatic pipetter (P 200) or Pasteur pipettes and bulbs
  • 20 Eppendorf 100 ul tips for above pipetter
  • 1 vial containing 200 ul of low titer (1:250) rabbit a SRBC antibody diluted at 1:40
  • l vial containing 900 ul of high titer (1:2000) rabbit anti-sheep red cell (aSRBC) antibody diluted at 1:40.
  • 1 vial containing 500 ul of fresh frozen guinea pig or dog serum for a source of complement
  • 1 vial each sera from sheep, goat, dog and rabbit in 0.3% concentration.
  • l vial buffered saline

  • PROCEDURE FOR MACROSCOPIC EXAMINATION OF AGGLUTINATION

    1. Obtain 4 microscope slides. Label them with a marking pen: sheep, goat, dog, and rabbit. >

    2. Obtain a tube of anti-sheep antibodies. With a sterile pipette, place a drop of the antibodies in the center of the slide marked sheep.

    3. Obtain a tube of sheep red blood cells and a sterile pipette and clean toothpick. Place a drop of sheep blood on top of the drop of antibodies and stir gently with the sterile toothpick for three circular motions.

    4. Allow the slide to stand for up to five minutes. If there is going to be agglutination, you should see a granulation of the red cells. Check the slide under the microscope on low power and without the use of a coverslip to observe agglutination. Record in the chart below (+) for positive results and (-) for negative results.

    5. Repeat this procedure for all the blood sources and fill in the chart. Be sure to use a sterile pipette for each blood source. The pipette used for the antibody can be used for each test, as long as you do not accidentally contaminate the pipette with blood sera. Clean toothpicks should be used for each procedure.

    6. Clean up all materials and put them away.

    RESULTS FOR PROCEDURE A:

    ANIMAL         AGGLUTINATION +/-
    SHEEP
    GOAT
    DOG
    RABBIT

    PROCEDURE B: HEMAGGLUTINATION AND HEMOLYSIS

    1. Obtain one 96 well plate, place it on a piece of white paper, and label the paper with the following:
      Label row A: Sheep + low titer
      Label row B: Sheep + high titer
      Label row C: Sheep + high titer + complement
      Label row D: Goat + high titer
      Label row E: Goat + high titer + complement
      Label row F: Dog + high titer
      Label row G: Rabbit + high titer
           1      2       3      4     5      6     7      8       9     10
      
      A
      
         
      B
      
       
      C
      
       
      D
      
       
      E
      
      
      F
      
       
      G
      
    2. You will be using wells 1 through 10 of rows A through G in this study. To help you focus on this part of the plate, remove the cover and use the marker to draw a border around this group of wells.

    3. Add 100 µl (2 drops) of phosphate buffered saline (PBS) to wells 2 through 10 of rows A through G. DO NOT PUT SALINE IN WELLS #1! Proceed one row at a time. When you are finished, hold the plate up to the light to ascertain that all wells except row 1 have saline in them.

    4. ROW A
      Use a new pipette to add 200 µl (4 drops) of 1:40 once-injected rabbit antiserum (low titer) to well 1 of row A. Follow the procedure for performing serial dilutions shown below to dilute and distribute the antiserum in well 1 to wells 2 through 9.

    5. The following serial dilution procedure should be done very carefully to insure the correct dilutions.

      • Using the original pipette for row A, suck up the contents of well 1.
      • Add 100 µl (2 drops) into well 2. Return the remainder to well 1. Very gently mix the
      • contents of the well 2 by stirring the contents with a toothpick. This is to avoid making bubbles.
      • Repeat this procedure for wells 3 through 9. Discard 2 drops removed from well 9. Note: Well 10 should NOT receive any antisera. This is your negative control for the row.

    6. ROWS B through G
      Use a new pipette to add 200 µl (4 drops) of 1:40 thrice-injected rabbit antiserum (high titer) to well 1 of row B. Use the same pipette to repeat this procedure for well 1 of rows C through G. Follow the procedure for performing serial dilutions shown above to dilute and distribute the antiserum in well 1 to wells 2 through 9 for each row B through G.

    7. ROWS C and E
      Use a new pipette to add 50 µl (1 drop) of complement proteins (Cp) to wells 1 through 10 of rows C and E. Mix the contents of each well with a clean toothpick.

    8. ROWS A, B, and C
      As you perform the following operation, periodically gently tap the tube with the 0.3% sheep RBCs (S) to mix the suspension of sheep red cells. Use a new pipette to add 100 µl (2 drops) of the RBC suspension to wells 1 through 10 or rows A, B, and C. Mix the contents of each with a clean toothpick for each row.

    9. ROWS D and E
      As you perform the following operation, periodically gently tap the container with the 0.3% goat RBCs (G) to mix the suspension of cells. Use a new pipette to add 100 µl (2 drops) of the RBC suspension to wells 1 through 10 of rows D and E. Mix the contents with a clean toothpick for each row.

    10. ROW F
      As you perform the following operation, periodically gently tap the container with the 0.3% dog RBCs (D) to mix the suspension of cells. Use a new pipette to add 100 µl (2 drops) of the RBC suspension to wells 1 through 10 of row F. Mix the contents of each well with a clean toothpick.

    11. ROW G
      As you perform the following operation, periodically gently tap the container with the 0.3% rabbit RBCs (R) to mix the suspension of cells. Use a new pipette to add 100 µl (2 drops) of the RBC suspension to wells 1 through 10 of row G. Mix the contents of each well by using a clean toothpick.

    12. When your plate has been properly set up, place the cover on it and store it in the designated area (a refrigerator). Hemagglutination requires at least 2 hours; alternatively the plate may be refrigerated for the following week.

    13. Discard all Pasteur pipettes.

    14. After 2 hours, the test may be read. Hemagglutination is scored as +4 when the red cells form a mat on the bottom of the well which is turning over around the edges. A score of +3 reactions contains a mat only. When the red cells begin to slide down and settle on the bottom in a pellet, the antibody concentration is decreasing. A solid button or pellet is scored as negative. A small ring with a center mat is a +1 and a larger ring with a center mat is a + 2.

    15. In any given row, the test is read as the last dilution before a negative score. This means that if you have a negative result with a bullet sedimentation of the red cells, the last dilution before that bullet where there is agglutination is read as the lowest dilution at which there is still hemagglutination.

    16. When complement is added to the well with antibody and red cells, hemolysis occurs if the antibody is coating the antigens on the surface of the red cells. Compare the hemolytic titers with the hemagglutination titers. Basically, more antibody concentration is needed for hemagglutination reactions than is required for hemolysis reactions.


    RESULTS

    1. Compare the HA serum titers seen against low vs. high titer sheep cells, and compare the reaction of high titer antibody to the red cells of sheep, goat, dog, and rabbit. Use the key above to indicate your findings on the following chart, drawing in the results as shown in the key. Score each circle.
       
        
                 1      2      3      4      5      6      7      8      9     10
      
          A
      
          B
      
          C
      
          D
      
          E
      
          F
      
          G
      
    2. Compare the serum antibody reaction of low titer antibody toward sheep cells with the serum antibody reaction of high titer antibody toward sheep cells.

    3. At what concentration did you last find hemolysis in row C toward the sheep cells?____________ How does this figure compare with the last concentration of hemagglutination of red cells in sheep in row B? Explain.

    4. Compare the serum from the rabbit injected three times for its HA titer against sheep cells, to the red cells of goat, to the red cells of dog, and to the red cells of rabbit.

    5. At what serum antibody concentration did you last find hemolysis of goat red cells in row E?_________How does this compare with the last antibody concentration at which you found hemagglutination in the goat? Explain.

    6. When you added antibody and complement, what were the results and why?

    7. What was the purpose of having the final wells of saline and red cells in each row for each animal.? Explain thoroughly.

    8. If patients with Type B blood receive blood from a patient with Type A blood which also has anti B antibodies in it, why don't the anti- B antibodies of the donor agglutinate the B cells of the recipient?



    Find out more about Nancy Ridenour


    Return to the Share the Wealth Directory
    Jump to the Access Excellence Homepage