Complement assay experiment

Summary

Complement assay is the measurement of total complement hemolytic activity and is used to detect various acute inflammatory conditions.

Operation method

Hemolytic test (CH50) method

Materials and Instruments

Serum
Phosphate buffer Physiological saline NaCl Na2HPO4 KH2PO4 Magnesium sulfate solution
Horizontal centrifuge Water bath Spectrophotometer

Move

I. Preparation of materials

1. Concentration of 1 × 10 g / ml of sheep red blood cells preparation(1) Take diluted and washed sheep erythrocytes and prepare a slightly thicker cell suspension than 5%.(2) Take 1 ml of 50% cell suspension and add it to 14 ml of distilled water to measure . Find the number of milliliters of diluent to be added to a given volume of 5% cell suspension by the formula below.
Vi is the volume of 50% cell suspension prepared, Vf is the number of ml of dilution that should be added to a given amount of 5% cell suspension: Ds is the _OD540 value of a known standardized 1×100 cells/ml suspension - 0.385.2. Sensitized sheep cellsTake sheep erythrocyte suspension (1 x ^102/ml ) and add equal amount of hemolysin (2 U/ml). Mix thoroughly. Place in a 37℃ water bath for 30 minutes, shaking every 10 minutes to avoid cell decline that is ^5×108/ml sensitized sheep red blood cells.
II. Operational steps

1. Determination of complement CH50 units(1) Add samples according to the table below:
(2) Mix the tubes thoroughly. Place in a 37°C water bath for 60 minutes. (Shake 1 to 2 times to avoid sinking of red blood cells.)
(3) Immediately after removal from the water bath, each tube was centrifuged at 2000 rpm for 10 minutes, and the supernatant from each tube was transferred to another test tube.
(4) Determination of the OD value of the supernatant of each tube (wavelength = 540 nm)
2. Calculation of CH50 units of complement
(1) Subtract the OD value of the red blood cell control tube (tube 7) from the OD values of the measurement tubes (tubes 1 to 5) and the hemolysis control tube (tube 6). Subtract the OD value of serum chromatography calculated according to the actual amount of serum to be measured per tube (80/800 x the actual amount of the tube x the OD value of tube 8). This is the corrected OD value of the assay tube. Then calculate the hemolysis rate of each tube (y value)

Then calculate the y/(1-y) value for each tube.
(2) In the double logarithmic coordinate paper, y/(1-y) value for the horizontal coordinate, serum dosage for the vertical coordinate, get a straight line. As 50% hemolysis (y = 0.5) of the y / (1 ─ y) value is equal to 1. Can be equal to 1 by the horizontal coordinates of a point in the straight line to obtain the corresponding intercept in the vertical coordinates, which produces 50% hemolysis of the serum to be measured in the actual dosage.
(3) Finally, the total complement activity per milliliter of serum to be tested is calculated according to the following formula and expressed in units per milliliter.

Common Problems

The 50% hemolysis test to determine the amount of complement in the serum is expressed by the following formula:

The formula can be further justified from the Von Krogh equation:

Assuming that X = amount of fresh serum added

y=percentage of hemolysis in decimals

V=slope

k=constant

Transformed into logarithms, the above equation becomes

When 50% hemolysis is used as an endpoint

Substituting the above equation

Since -log1 = 0, , i.e., it means that 50% of the hemolytic units of complement is the amount of fresh serum added.

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