NAME: AMANDA RAMLAKHAN

ID: 00049631

CLASS: IMMUNOLOGY LAB

LECTURER: DR. S.SUEPAUL

LAB NUMBER 2

LAB TITLE: HUMAN COMPLEMENT C3 ‘NL’ BINDARID RADIAL

IMMUNODIFFUSION KIT ASSAY.

OBJECTIVES:

INTRODUCTION:

COMPLEMENT C3 Also known as: C1; C1q; C2; C3; C4; CH50; CH100 (among others)

Formal name: Complement Activity; Complement Component; Total Complement; Total

Hemolytic Complement Activity

Related tests: ESR; C - reactive protein; Rheumatoid Factor; ANA; Autoantibodies

Complement component 3, often simply called C3, is a protein of the immune system. It plays a

central role in the complement system and contributes to innate immunity. In humans it is

encoded on chromosome 19 by a gene called C3. C3 plays a central role in the activation of
complement system. Its activation is required for both classical and alternative complement

activation pathways. People with C3 deficiency are susceptible to bacterial infection.

One form of C3-convertase, also known as C4b2a, is formed by a heterodimer of activated forms

of C4 and C2. It catalyzes the proteolytic cleavage of C3 into C3a and C3b, generated during

activation through the classical pathway as well as the lectin pathway. C3a is an anaphylotoxin

and the precursor of some cytokines such as ASP, and C3b serves as an opsonizing agent. Factor

I can cleave C3b into C3c and C3d, the latter of which plays a role in enhancing B cell

responses. In the alternative complement pathway, C3 is cleaved by C3bBb, another form of C3-

convertase composed of activated forms of C3 (C3b) and factor B (Bb). Once C3 is activated to

C3b, it exposes a reactive thioester that allows the peptide to covalently attach to any surface that

can provide a nucleophile such as a primary amine or a hydroxyl group. Activated C3 can then

interact with factor B. Factor B is then activated by factor D, to form Bb. The resultant complex,

C3bBb, is called the alternative pathway (AP) C3 convertase. C3bBb is deactivated in steps.

First, the proteolytic component of the convertase, Bb, is removed by complement regulatory

proteins having decay-accelerating factor (DAF) activity. Next, C3b is broken down

progressively to first iC3b, then C3c + C3dg, and then finally C3d. Factor I is the protease that

performs these cuts but it requires the help of another protein to supply cofactor activity. This

test measures the amount of C3 proteins in your blood. These proteins are part of your

complement system, which plays an important role in your immune system. Its job is to help kill

disease-causing bacteria and viruses. It also responds to such invaders with inflammation that

protects your body from disease.

Complement component C3 is the most important and abundant protein in the complement

system. It is placed on microbes to destroy them. By measuring complement C3 levels,

especially in how they compare with other parts of the complement system, your doctor can

diagnose and monitor treatment of certain diseases. One of the diseases that commonly involves

abnormal C3 is systemic lupus erythematous, or lupus, an autoimmune disorder. You may have

this test if your doctor suspects you have an autoimmune disorder, especially lupus. Symptoms

of lupus may include:

 Rash in the shape of a butterfly across your cheeks

 Mouth ulcers

 Hair loss

 Joint pain and swelling

You may also have the test if you get repeated bacterial infections. And if you have already been

diagnosed with an autoimmune disease, you may have this test to monitor its

progress. Complement testing may be used to:

 Help diagnose the cause of recurrent microbial infections (such as Streptococcus

pneumonia, Neisseria meningitides, and Neisseria gonorrhea), angioedema, or

inflammation.

 Help diagnose and monitor the activity and treatment of acute or chronic autoimmune

diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis.

 Monitor immune complex-related diseases and conditions such as glomerulonephritis (a

kidney disorder), serum sickness, and vasculitis.

Many things may affect your lab test results. These include the method each lab uses to do the

test. Even if your test results are different from the normal value, you may not have a problem.
To learn what the results mean for you, talk with your health care provider. The normal range for

a complement C3 blood test is 70 to 160 milligrams per deciliter (mg/dL), or 0.7 to 1.6 grams per

liter (g/L).

Your complement levels will often shoot up dramatically just after an infection or injury. When

your complement system is activated in response to ongoing disease such as lupus, levels usually

go down. You can inherit a deficiency in your complement C3, but it's much more common to

acquire a deficiency.  If only your C3 complement level is low and all other complement

components are normal, it's usually because of an inherited component deficiency. This makes it

more likely that you will develop certain autoimmune disorders. More often, you will have

lowered levels of several complement components at once. This is the result of an acquired

disease. If your C3 and C4 levels are reduced, this may be a sign that you have lupus. Usually

your total complement level is also slightly lower in this situation. Low C3 and C4 levels may

also be a sign of alcoholic liver disease, but this is less common.

Other conditions sometimes linked to low C3 levels include:

 C3 deficiency, a condition characterized by recurrent bacterial infections

 Different forms of kidney disease

If you are being treated for a disease like lupus and your complement C3 and C4 levels go up, it

is usually a sign that your treatment is working. 

Radial immunodiffusion (or Mancini method, Mancini immunodiffusion, single radial

immunodiffusion assay) is immunodiffusion technique used in immunology to determine the

quantity of an antigen by measuring the diameters of circles of precipitin complexes surrounding

samples of the antigen that mark the boundary between the antigen and an antibody suspended in
a medium, such as an agar gel. The diameters of the circles increase with time as the antigen

diffuses into the medium, reacts with the antibody, and forms insoluble precipitin complexes.

Antigen-antibody complexes are small and soluble when in antigen excess. Therefore,

precipitation near the center of the circle is usually less dense than it is near the circle's outer

edge, where antigen is less concentrated. The quantity and concentration of insoluble antigen-

antibody complexes at the outer edge of the circle increases with time. Therefore, the clarity and

density of the outer edge increases with time. Expansion of the circle reaches an end point and

stops when antigen and antibody reach equivalence. However, the clarity and density of the outer

edge may continue to increase after the circle stops expanding.

For most antigens, the area and the square of the diameter of the circle at the circle's end point

are directly proportional to the quantity of antigen and are inversely proportional to the

concentration of antibody. Therefore, a graph that compares the quantities or concentrations of

antigen in the original samples with the areas or the squares of the diameters of the precipitin

circles on linear scales will usually be a straight line when all circles have reached their end

points (equivalence method).Circles created by small quantities of antigen reach their end points

before large quantities do. Therefore, if areas or diameters of circles are measured while some,

but not all, circles have stopped expanding, such a graph will be straight in the portion that

contains the smaller quantities or concentrations of antigen and will be curved in the portion that

contains the larger quantities or concentrations.

While circles are still expanding, a graph that compares the quantities or concentrations of the

antigen on a logarithmic scale with the diameters or areas of the circles on a linear scale may be

a straight line (kinetic method). However, circles of the precipitate are smaller and less distinct

during expansion than they are after expansion has ended. Further, temperature affects the rate of
expansion, but does not affect the size of a circle at its end point. In addition, the range of circle

diameters for the same quantities or concentrations of antigen is smaller while some circles are

enlarging than they are after all circles have reached their end points. Therefore, measurements

of the sizes of circles and of graphs produced from such measurements are often less accurate

when circles are expanding than they are after expansion has ended. For that reason, it is often

more desirable to take measurements after all circles have reached their end points than it is to

take measurements while some or all circles are still expanding.

Measurements of large circles are more accurate than are those of small circles. It is therefore

often desirable to adjust the concentration of antibody and the quantity of antigen to assure that

precipitin rings will be large.

PRINCIPLE:

The method involves antigen diffusing radially from a cylindrical well through an agarose gel

containing an appropriate mono-specific antibody. Antigen-antibody complexes are formed

which, under the right conditions, will form a ring precipitin ring. The ring size will increase

until equilibrium is reached between the square of the ring diameter and the antigen

concentration. By measuring the ring diameters produced by a number of samples of known

concentration, a calibration curve may be constructed. The concentration of the antigen in an

unknown sample may then be determined by measuring the ring diameter produced by that

sample and reading of the calibrator curve.

LAB PARTNERS:

AMANDA RAMLAKHAN

ARIANNA MANODATH

REHANNA BASDEO

REAGENTS:

 RID plates (supplied in foil pouches). These contain monospecific antibody to C3 or C4

in agarose gel. Up to fourteen samples can be run per plate (including calibrators).

Preservatives: 0.099% sodium azide, 0.1% E-amino-n-caproic acid (EACA), 0.01%

thiomersal, 0.01% benzamine.

 Calibrators. These are supplied in stabilized liquid form as a set of three high, medium

and low concentrations of c3 and c4. The concentrations given on the vial labels have

been obtained by comparison with the CRM470 international reference material.

 Control serum. This is supplied in stabilized liquid form. The expected concentrations for

c3 and c4 are marked on the vial label.

MATERIALS:

 RID plates

 Calibrators

 Saline

 micropipette
  Jeweler’s eye piece

SPECIMEN COLLECTION

The c3 RID plates contain antibodies to C3c, which will react with both c3 and the degradation

product C3c. they therefore measure the combined c3 and C3c concentration, which is

satisfactory for most purposes. If intact c3 alone is to be measured, then plasma. HAVE TO

WRITE MOREEEE

STORAGE AND STABILITY:

The unopened kits should be stored at 2-8 c and can be used until the expiry date given on the kit

box label. DO NOT FREEZE. The expiry dates of individual components are given on the

component labels. RID plates should be stored at 2-8 C and are damaged by temperature

extremes. Freezing will destroy the gel, therefore RID plates should be kept away from cooling

elements in refrigerators. High temperatures should also be avoided as this will result in moisture

loss from the gel, affecting performance. Unopened plates should be stored flat and upside down

to prevent condensation accumulating in the wells. Handle plates with care to prevent gel

damage.

Unopened calibrators and controls should be stored 2-8 C. once opened they are stable for at

least one week at 2-8C, but for longer storage they should be aliquoted and frozen.
SAFETY:

 This product contains sodium azide. Disposal of reagents into sinks with copper or lead

plumbing should be followed with plenty of water to prevent formation of potentially

explosive metallic azides.

 Take care when handling materials and samples of human origin. Since no test method

can offer complete assurance that infectious agents are absent, consider all clinical

specimens, controls, and calibrators potentially infectious. Handle specimens, solid and

liquid waste, and test components in accordance with local regulations or other safety

hazard guidelines.

 Inspect the packaging for signs of damage.

 Be careful when opening the outer packaging with a sharp instrument so as to avoid

damage to the individual product packaging.

 Avoid agitation, which may cause foaming or the formation of bubbles.

QUALITY CONTROL:

As appropriate, refer to your laboratory standard operating procedure(s) and/or quality assurance

plan for additional quality control requirements and potential corrective actions.

• Three levels of quality control are to be run every 24 hours.

• If more frequent control monitoring is required, follow the established quality control

procedures for your laboratory.

• If quality control results do not meet the acceptance criteria defined by your laboratory, patient

values may be suspect. Follow the established quality control procedures for your laboratory.

Recalibration may be necessary.

• Review quality control results and acceptance criteria following a change of reagent or

calibrator lot.

PROCEDURE:

 Procedure one, two or three was selected.

 Condensation was allowed to evaporate from the RID plates.

 Calibrators and controls were applied to the plated using a micropipette in 5 microliters.

 The lid was replaced and left for some time.

 Then the ring diameters was measures using a jeweler’s eye piece.

 All measurements obtained were recorded and squared in order to plot a calibration curve
RESULTS:

NUMBER SAMPLE DIAMTER DIAMETER CONCENTRATIO

,mm Squared, mm² N

Mg/L
1 Low 3.7 13.67 155

calibrator
2 Medium 7 49 930

calibrator
3 High 8 64 1550

calibrator
4 Control serum 6.5 42.25 1312
5 5.5 30.25 500
6 6.2 38.44 720
7 6.5 42.25 840
8 7 49 1040
9 7.6 57.76 1320
10 8 64 1550
11 8.2 68.89 1720
12 7.5 72.25 1860
13 9.2 84.64 2380
14 9.2 84.64 2380

DISCUSSION:

Complement C3 radial immunodiffusion lab was done using three calibrators and a control.

These reagents were micro pipetted into a RID plate. A jeweler’s eye piece was used to measure

each circle in the RID plate resulting in various diameters.

In this lab we used saline as the only dilution factor however no serum was added. Normally in a

serum lab the complement c3 is tested to see whether it has increased or decreased. Decreased
complement levels also are associated with an increased risk of developing an autoimmune

disease. Both C3 and C4 levels are typically depressed in SLE while C3 alone is low in

septicemia and infections caused by fungi or parasites such as malaria. Complement protein

levels are usually increased, along with other unrelated proteins called acute phase reactants,

during acute or chronic inflammation. These all usually return to normal when the underlying

condition is resolved. However, complement proteins are rarely measured in these conditions,

compared to the widely ordered C-reactive protein (CRP).