Rat Complement C4/C4 ELISA Kit- High Sensitivity

Key Features

	Save Time	Pre-coated 96 well plate
	Quick Start	Kit includes all necessary reagents
	Publication Ready	Reproducible and reliable results

Overview


Product Name:	Rat C4 (Complement Component 4) ELISA Kit
Product Code:	RTFI00613
Size:	96 Assays
Alias:	C4
Detection Method:	Sandwich ELISA, Double Antibody
Reactivity	Rat
Sensitivity:	0.469ng/ml
Range:	0.781-50ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Additional Information

Recovery

Matrices listed below were spiked with certain level of Rat C4 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat C4 in samples.


Matrix	Recovery Range (%)	Average (%)
serum (n=5)	93-105	99
EDTA plasma (n=5)	88-102	96
UFH plasma (n=5)	86-102	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat C4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.


Sample	1:2	1:4	1:8
Serum (n=5)	87-104%	88-97%	85-101%
EDTA plasma (n=5)	87-94%	83-93%	84-100%
UFH Plasma (n=5)	81-98%	85-97%	80-98%

Intra Assay

CV < 8%

Inter Assay

CV < 10%

Kit Components

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8x12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/ -20°C
Sample/Standard Dlution Buffer	20ml	4°C
Biotin-labeled Antibody (Concentrated)	120ul	4°C (Protection from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate (SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protection from light)
Stop Solution	10ml	4°C
Wash Buffer (25X)	30ml	4°C
Plate Sealer	5	-

Other materials required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Protocol

*Note: Protocols are specific to each batch/lot. For the exact instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Procedure
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Sample Preparation

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

Complement C4 Background

What is C4 Complement?

Complement Component 4 (C4) is a protein that plays a crucial role in the immune system's complement system. The complement system is a complex network of proteins that helps the body's defense against pathogens, promotes inflammation, and facilitates the removal of damaged cells or immune complexes. C4 is an important component of this system, and its functions include opsonization, immune complex clearance, and initiation of the classical complement pathway.

C4 Structure

Structurally, C4 consists of an α-chain and a β-chain. Each chain is encoded by a separate gene. The α-chain contains a thioester domain, while the β-chain consists of a C-terminal globular domain. The two chains are connected by a short peptide linker. C4 undergoes various post-translational modifications, including glycosylation, which can influence its function and interactions with other complement components.

C4 Function

The primary function of C4 is to act as an opsonin, which means it enhances the phagocytosis of pathogens by marking them for recognition and destruction by phagocytic cells. C4 binds to the surface of pathogens or immune complexes and facilitates their recognition by other complement proteins, leading to the formation of membrane attack complexes that can destroy the target cell.

C4 also plays a crucial role in the clearance of immune complexes, which are formed when antibodies bind to antigens. It binds to immune complexes and facilitates their removal by phagocytic cells or by binding to complement receptors present on erythrocytes, which transport the complexes to the liver and spleen for further processing.

C4 Clinical Significance

Deficiencies or abnormalities in C4 can lead to autoimmune diseases such as systemic lupus erythematosus (SLE). Low levels of C4 are commonly observed in patients with SLE, as the complement system is overactive and consumes C4 rapidly. Monitoring C4 levels can be useful in diagnosing and monitoring disease activity in SLE patients.

Rat Complement C4 ELISA Kit FAQs

Q: What is the purpose of the Rat C4 ELISA kit?

The Rat C4 ELISA Kit is specifically designed to measure and quantify the levels of complement component 4 (C4) in mouse samples. C4 is an essential protein involved in the immune system's complement system. This kit provides a reliable and accurate method for researchers to assess C4 levels, aiding in the investigation of complement system function, autoimmune diseases, and inflammatory disorders in mouse models.

Q: What type of samples can be used with the Rat C4 ELISA kit?

The Rat C4 ELISA Kit is designed to work with a variety of sample types, including serum, plasma, cell culture supernatants, and other biological fluids. It provides researchers with flexibility in sample selection to suit their experimental requirements.

Q: Can the Rat C4 ELISA Kit be used for diagnostic purposes in humans?

No, the Rat C4 ELISA Kit is intended for research purposes only and is not validated or approved for diagnostic use in humans. It is specifically designed for the quantification of rat complement component 4 (C4) in experimental settings and should not be used for clinical or diagnostic purposes.

Q: Is the Rat C4 ELISA Kit specific to rat samples only?

Yes, the Rat C4 ELISA Kit is specifically designed and optimized for the detection of Rat complement component 4 (C4). It is not recommended for use with samples from other species.

Q: Where can I find additional technical support or assistance with the UNC13A ELISA kit?

For any technical inquiries or assistance regarding the kit, you can reach out to our team. They will be available to answer your questions and provide the necessary guidance to ensure a successful experiment.

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Rat Complement C4 / C4 ELISA Kit

Description