rev: June 30, 2004
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Anti-C3A-desArg ELISA-For in vitro research use only-not for use in diagnostic procedures
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Kit Size: 12 x 8 determination
For In Vitro Research use only
C3a enzyme immunoassay (ELISA) detects complement component C3a-desArg highly
selectively using monoclonal antibodies. C3a is generated during activation
of the complement system via the classical or the alternative pathway. The
anaphylatoxin C3a itself is very short-lived and in the serum is cleaved
immediately into the more stable C3a-desArg. Therefore, quantitation of
C3a-desArg allows reliable measurement of the level of (un)specific complement
(over-)activation in the human circulation.
Advantages of the Test Procedure:
--Monoclonal antibodies are used for the dtermination and, thus an optimum standardization and reproducibility is guaranteed.
--8-well microtiter strips are used to allow optimal user flexibility.
--Micromethod, i.e. only 10 ul EDTA plasma is required per patient.
--Test results are available within approx. 2-3 hours.
--The test can be started immediately with the diluted sample (EDTA plasma), i.e. pretreatment of serum to remove native C3 is not required.
The microtiter strips included in the kit are coated with a monoclonal antibody specific for C3a-desArg.
Plasma samples are diluted 1:100 (100 ul/well) and incubated for one hour at room temperature (RT; 18-25'C).
After rinsing off the unbound native C3, peroxidase-con-ugated mouse monoclonal antibody is used for the detection of C3a.
Excess conjugate is removed through a washing step and the amount of C3a-desArg in the plasma sample is quantified using the peroxidase reaction.
-12 x 8-well microtiter strips, coated with monoclonal antibody to C3a.
H 6 vials human C3a-desArg standard (1100 ng/ml),lyoph.*
N 2 vials negative control (human: C3a-desArg content < 150 ng/ml lyoph.*
WB 1 bottle wash buffer concentrate (20x), 20 ml****
SB 2 bottles sample buffer concentrate (10x), 2 x 20 ml****
C 1 vial mab anti-C3a peroxidase conjugate (50x), 0.30 ml **/****
S 1 bottle substrate solution ** (TMB, ready-to-use),11ml
SS 1 bottle stop solution (1NH2SO4), 13ml***
* The sers have been tested for AIDS (HIV I+II), hepatitis B and syphilis and are negative. However, all human blood products should be considered to be potentially infectious.
** Light-sensitive; do not expose to bright light for pro-longed period of time!
*** Avoid skin contact, contains diluted acid!
**** Toxic, contains 0.01% thimerosal!
INSTRUCTIONS FOR USE:
Prepare buffer solutions only with distilled water.
Do not let the wells dry out during the test procedure. The use of the plastic
cover foil or a moist chamber is recommended.
Use sterile disposable plastics for pipetting.
Observe all pertinent precautions concerning the handling of potentially infectious material.
For In Vitro Use Only
1. The standard and control contain human plasma which have been tested for hepatitis B surface antigen and HIV antibodies and was found to be negative. As no known test offers complete assurance that hepatitis B or other infectious agents are absent, the controls should be considered as potentially infectious and should be handled with the same precautions as any other potentially bio-hazardous material.
2. Do not pipette by mouth.
3. Do not smoke, eat, drink or apply cosmetics in areas where kits or serum samples are handled.
4. Any skin complants, cuts, abrasions and other skin lesions should be suitably protected.
5. The kit Conjugate, Sample Buffer and Wash Buffer Concentrate contain thimerosl, which contains mercury and is toxic. When disposing of these reagents, observe authority regulations.
6. The Stop Solution contains sulfuric acid. Avoid contact with skin, eyes and mucous membranes. Accidental spillage should be mopped up with copious amounts of water. If contact with skin or eyes occurs, irrigate with water and seek medical attention immediately.
Sample collection is critical. Care must be taken to avoid C3a generation in the sample.
All operations should be carried out at 2-8'C. Blood samples should be collected with disodium EDTA as anti-coagulant and should be centrifuged immediately for 15 min at 2000x g at 2-8'C. Plasma should be isolated and assayed immediately or stored in aliquots at -70'C. The entire operations must be completed within 30 min.
Consequently, only EDTA plasma has to be used for the measurements which
is wither freshly collected or which has been stored prior at -70'C. Samples
stored at 2-8'C for longer periods of time are obsolete. A correct sample
collection is mandatory for the determination of C3a.
The surface of various plastic materials can also induce complement activation, we, therefore, suggest to use glass tubes for all samples.
Allow kit components to reach R.T.
SB Sample Buffer
To prepare ready-to-use sample buffer (SB), dilute the buffer 1:10 with distilled water. Example: empty the contents of one bottle SB into a glass cylinder, add distilled water to make up 200 ml and mix well by stirring. The buffer is now ready-to-use and 6 weeks stable at 2-8'C.
For preparing the ready-to-use wash buffer (WB), dilute 1:20 with distilled
water. Example: empty the contents of the bottle WB into a glass cylinder,
add distilled water to make up 400 ml and mix well by stirring. The buffer
is now ready-to-use and minimum 6 weeks stable at 2-8'C.
a) Dissolve the positive standard H in 1 ml ready-to-use sample buffer SB,
mix well and incubate for minimum 5 min at RT. Prepare the standard dilutions
H/2. H/4 and H/8 with H.Example: take 500 ul sample buffer and mix
with 500 ul H (=H/2); take 500 ul sample buffer and mix with 500
ul H/2 (=H/4); etc.
b) Dissolve negative control plasma N in 1 ml ready-to-use sample buffer SB, mix well and incubate for minimum 5 min. The negative control is now ready-to-use. For further test runs, aliquot the negative control and store at -20'C.
Anti-C3a Peroxidase Conjugate
The conjugate concentrate has to be diluted 1:50 in ready-to-use sample buffer
(SB). As the diluted solution is not stable, it is recommended to always
only prepare the required conjugate dilution.
The Substrate Solution is provided in ready-to-use form. If stored property at 2-8'C and not exposed to bright light for prolonged periods of time, the solutions is stable until the expiration date printed on the label.
If required, let plasma sample thaw and keep on ice.Dilute plasma samples 1:100 (10 ul for 1 ml) in sample buffer.
* Sample Incubation: According to the pipetting scheme, pipette 100 ul each of ready-to-use sample buffer (blank), standard, control and diluted patient samples (1:100)into the individual wells and incubate 1 h at RT. We recommend double determinations.
* Washing: Empty microtiter strips and pipette 200 ul each of ready-to-use wash buffer into the wells. After max. 1 min., empty wells again and repeat this washing step another twice. Remove excess liquid by tapping the strips on absorbing paper.
* Conjugate Incubation: Pipette 100 ul of the diluted conjugate solution into each well and incubate 1 h at RT.
* Washing: Empty microtiter strips and carry out washing steps as described above.
* Substrate Incubation: Pipette 100 ul substrate solution into the wells and incubate 10 to 15 min.
* Stopping Reaction: Pipette 100 ul SS into the wells and maintain same sequence as before when adding the substrate solution.
Measuring the Absorbance (A)
Measure the Absorbance with a microtiter plate reader at 450 nm wavelength and a reference wavelength of 550 nm (single-beam equipment may also be used). Well A1 (substrate control) serves a BLANK.
If in the individual wells an A of >2.0 is measured, the sample has to be diluted further and measured again.
Calculation of Results
Plot the net absorbance values for each C3a standard concentration on the
y-axis for each corresponding C3a concentration on the x-axis (see: Example
of Standard Curve).
The C3a concentration in each unknown sample can be determined directly by reading the C3a concentration corresponding to the absorbance value (i.e. the dilution factor has already been included in the concentration values of the x-axis). If a dilution greater than x100 has been chosen, only the additional dilution factor has to be included for calculation!
From a standard collective of normals, (20 blood donors) a C3a-desArg content of 150 ng/ml plasma was determined. Titers are elevated if plasma samples are >200 ng/ml.
1. Burger, R.: Zllow,G., Bader,A.,: and Naser, W. (1988). The C terminus of the anaphylatoxin C3a generated upon complement activation represents a neoantigenic deter minant with diagnostic potential. The J.Immunol., 141: 553-558.
2. Mollnes,T.E.; Garred,P.,and Bergseth G.(1988); Effect of time, temperature and anticoagulants on in vitro complement activation: Consequences for collection and preservation of samples to be examined for complement activation. Clin. exp. Immunology 73, 484-488.
3. Hadding, U., und FaBbender, B. (1988). Das Komplementsystem: Ein Funktionstrager fur Infektabwehr und Immunregulation. Naturwissenschaften 75. 544-550.
4. Hugll,T.E.,and Chenoweth,D.E. (1980): Biologically acitve peptides of complement techniques and significance of C3a and C5a measurements. In: Laboratory and Research Methods in Biology and Medicine. R.M. Nakamure, W.R. Dito, and E.S. Tucher,eds Alan r. Liss, New York.p,443.
5. Hakin, R.M. Cardiovascular Pathology 2(3). Suppl.187S- 197S (1993).
6. Burger R. and Zilow G. (1993) Complement-derived anaphylatoxins in natural immunity. In: The Natural Immune System, Humoral Factors, E. Sim ed., IRL Press,Oxford- New York - Tokyo. pp. 209-232.
Pipetting Scheme for C3a ELISA
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