2 - SERIAL DILUTION

Dilutions

Necessary to make a less concentrated solution from a reagent such as an acid or a buffer in order to use the reagent in a particular procedure

Dilutions

very important in performing serologic techniques which are used to detect the interaction of antigen with antibody, quantitation of antibody against infectious agents, and non-microbial antigen

Water or saline

added to the concentrate to make the reagent the proper strength for testing

Solute

The material being diluted

Solvent/Diluent

The medium making up the rest of the solution

fraction

The relationship between solute and solvent is a ratio that can also be expressed as a _

Titer

Indicator of an antibody’s strength

Titer

Reciprocal of the highest dilution of the patient’s serum in which the antibody is detectable

antibody

Titer

The high titer indicates that a clinically significant amount of _ is present in the serum

Diluent = total volume - solute

Diluent formula

Compound Dilution

Used for very large dilutions

Preparing a serial dilution of the antibody containing solution (serum)

Adding an equal volume of antigen suspension to each dilution

Determination of the Concentration of Antibody (Titer) for a Specific Antigen

Involves 2 steps:

Serial Dilution

Simply a series of simple dilutions which amplifies the dilution factor quickly beginning with a small initial quantity of material

Two-fold Dilution

most common form of serial dilution

Doubling Dilution

Amount of serum is cut in half with each dilution

500

500

500

Procedure

1. Label the 5 tubes using numbers 1 to 5

2. Using the automatic pipette place _ uL of saline to all tubes

3. Using a new tip for the pipette, add _ uL of antibody A to tube number 1. Place the tube in a vortex

4. Draw up to _ uL from tube number 1 and transfer to tube number 2. Place again the tube in a vortex

500

500, 3

Procedure

1. Label the 5 tubes using numbers 1 to 5

2. Using the automatic pipette place 500 uL of saline to all tubes

3. Using a new tip for the pipette, add 500 uL of antibody A to tube number 1. Place the tube in a vortex

4. Draw up to 500 uL from tube number 1 and transfer to tube number 2. Place again the tube in a vortex

5. Repeat step number 4 up to the last tube

6. Discard _ uL from the last tube

7. Using new tip for the pipette, add _ uL of _% Red Blood Cell suspension to each tube

8. Mix well by using a vortex mixer or by simply tapping the tubes

30

20

agglutination

negative

Procedure

1. Label the 5 tubes using numbers 1 to 5

2. Using the automatic pipette place 500 uL of saline to all tubes

3. Using a new tip for the pipette, add 500 uL of antibody A to tube number 1. Place the tube in a vortex

4. Draw up to 500 uL from tube number 1 and transfer to tube number 2. Place again the tube in a vortex

5. Repeat step number 4 up to the last tube

6. Discard 500 uL from the last tube

7. Using new tip for the pipette, add 500 uL of 3% Red Blood Cell suspension to each tube

8. Mix well by using a vortex mixer or by simply tapping the tubes

9. Place your sets of tubes in the refrigerator _ minutes

10. Remove from refrigerator, centrifuge for _ seconds

11. Read immediately for _ by gently shaking the tube to dislodge the red blood cell button

Note: If the tubes are shaken too roughly false _ reactions can occur

Clumping

Interpretations

_ of the red blood cells is positive

smooth, uniform

Interpretations

A _, _ appearance of red blood cell suspension is negative

endpoint

Interpretations

The last tube showing agglutination is the _ of the test

titer

Interpretations

The _ is reported out as the reciprocal of the last dilution showing a positive result