Alternative Complement Pathway ELISA Kit [Human]

Product Type

Kit

Description

The complement system plays an essential role in chronic, autoimmune and infectious disease. There are three pathways of complement activation, namely the classical, the alternative and the recently discovered MBL pathway. Impaired complement activity causes humans to become susceptible to repetitive fulminant or severe infections and may contribute to development of autoimmune disease. Inappropriate activation of complement contributes to chronic inflammation and tissue injury.

Applications

The kit is an enzyme-linked immunoassay (ELISA), used to determine the functional complement pathway in human serum.

Principle

The Complement assay combines principles of the hemolytic assay for complement activation with the use of labeled antibodies specific for neoantigen produced as a result of complement activation. The amount of neoantigen generated is proportional to the functional activity of complement pathways. In the Complement system AP kit the wells of the microtitre strips are coated with specific activators of the alternative pathway. Subject serum is diluted in diluent containing specific blocker to ensure that only the alternative pathway is activated. During the incubation of the diluted subject serum in the wells, complement is activated by the specific coating. The wells are then washed and C5b-9 is detected with a specific alkaline phosphatase labelled antibody to the neoantigen expressed during MAC formation. After a further washing step, detection of specific antibodies is obtained by incubation with alkaline phosphatase substrate solution. The amount of complement activation correlates with the colour intensity and is measured in terms of absorbance (optical density (OD)).

Detection Method

Enzyme immunoassay (ELISA) technique

Sample

Human serum.

Sample Volume

100 L of 1:18 diluted serum / determination.

Species Reactivity

Human

Protocol Length

1 hour incubation (37°C) + 30 min. (RT) + 30 min. (RT) = 2 hours total incubation time.

Components

- One frame with red coloured break-apart wells (12x8) sealed in a foil pack with a desiccation sachet. The wells are coated with LPS.
- 35 mL Diluent AP (Dil AP), labelled red.
- 13 mL conjugate containing alkaline phosphatase-labelled antibodies to C5b-9 (blue colour).
- 13 mL Substrate solution ready to use.
- 30 mL wash solution 30x concentrated.
- 0,2 mL negative control (NC) containing human serum (to be diluted as for a subject serum sample).
- 0,2 mL positive control (PC) containing freezed dried human serum

Requires

-Microplate reader with filter 405 nm.
- Precision pipettes with disposable tips.
- Washer for strips, absorbent tissue, tubes and a timer.

Handling Advice

All reagents in the kit are ready for use except washing solution and controls. The reagents should be
stored at 2-8°C except the positive control. The positive control must be stored at -20°C.

Storage

The reagents should be stored at 2-8° C except the positive control. The positive control must be stored at -20°C.

Research Use

For research use only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. Hycult Biotech is not responsible for any patent infringements that might result with the use of or derivation of this product.

Figure 1 AP assay.

Table 1.

Table 2 Sera with known complement deficiencies were tested in the assay and the following results were obtained. All deficient sera were detected in the assay and gave values below 5 %.

Table 3. Inter-assay precision was determined by testing three samples in duplicate. Results were obtained for six different runs.

Table 4. Intra-assay precision was determined by testing one sample in 40 wells.

Alternative Complement Pathway ELISA Kit [Human] (CAT#: CB-P058-K) Datasheet

Overview

Specification

Images

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