Name: measuring the 50% haemolytic complement (ch<sub>50</sub>) activity of serum
Uploaded: 2010-03-29T18:05:00
Description: Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

<p class="jove_content">The author would like to thank Miss Lora Matthews for performing the technique and Mr Paul Shepherd for cinematography. This project was funded by the University of South Australia, Laboratory Medicine program.</p>

<p class="jove_title">Preparation of 5x Veronal Buffered Saline (VBS)</p>
<ol>
	<li>To prepare the Veronal Buffered Saline (VBS), three separate solutions need to be prepared.</li>
	<li>Prepare solution 1 by dissolving 21.25gm of NaCl and 0.94gm of Sodium Barbitone in 350ml of distilled water. The final concentrations of NaCl and Sodium Barbitone are 1.02M and 13mM respectively.</li>
	<li>Prepare solution 2 by dissolving 1.44gm of Barbitone in 125ml of hot distilled water. The final concentration of Barbitone is 62.5mM.</li>
	<li>Prepare soluti...

<ol>
	<li>Goldsby, R. A., Kindt, T. J., Osborne, B. A., Kuby, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> <em style='font-style: italic'>Immunology</em>. , (2003).</li></ol>

<p class="jove_content">The CH<sub>50</sub> assay is subject to many interferences. Some SRBC are more fragile than others, resulting in spontaneous haemolysis that is unrelated to complement activity.  The affinity of the rabbit antibody varies from lot to lot and from one manufacturer to another; this affects the amount of antibody that binds to the SRBC.  Also, the process of sensitising SRBC with antibody results in cells with differing amounts of antibody coating the SRBC.  Specimen collection and storage are an important potential source ...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Sheep red blood cells</td>
<td>&nbsp;</td>
<td>CSL</td>
<td>2490201</td>
<td>Use at 1% final</td>
</tr>
<tr>
<td>Serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Human serum</td>
</tr>
<tr>
<td>Rabbit Anti-Sheep Haemolytic serum (RBCs), Unconjugated</td>
<td>&nbsp;</td>
<td>AdB Serotec</td>
<td>C12HSB</td>
<td>&nbsp;</td>
<tr>
<td>96 well flat bottom plate</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>83.1839</td>
<td>&nbsp;</td>
<tr>
<td>Veronal buffered saline</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Use at 1x final</td>
</tr>
<tr>
<td>Waterbath</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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<td>Plate reader</td>
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<td>&nbsp;</td>
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<td>37&deg;C room/incubator</td>
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measuring the 50% haemolytic complement (ch<sub>50</sub>) activity of serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH<sub>50</sub> is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH<sub>50</sub> tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin).  When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results.  If a complement component is absent, the CH<sub>50</sub> level will be zero; if one or more components of the classical pathway are decreased, the CH<sub>50</sub> will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution.  After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.

Watch this Scientific Journal Video about Measuring the 50% Haemolytic Complement CH50 Activity of Serum at JoVE.com

Measuring the 50% Haemolytic Complement CH50 Activity of Serum

<p class="jove_content">The classical pathway is activated by antibody and culminates in target cell lysis. The CH<sub>50</sub> assay provides a measure of the complement activity of a serum sample. This video demonstrates the steps involved in determining the CH<sub>50</sub> of a serum sample, the calculations and interpreting of results.</p>

Measuring the 50% Haemolytic Complement (CH<sub>50</sub>) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual ...

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