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Human Factor H / Complement factor H ELISA Kit

SKU:
HUFI00749
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P08603
Sensitivity:
14.063ng/ml
Range:
23.438-1500ng/ml
ELISA Type:
Sandwich
Synonyms:
CFH, ARMD4, ARMS1, beta-1-H-globulin, CFH, CFHL3, FH, FHL1, HF1, HF2, HUS, adrenomedullin binding protein, age-related maculopathy susceptibility 1, AHUS1, AMBP1, ARMD4, beta-1H, beta-1-H-globulin, CFHL3, complement factor H, factor H, factor H-like
Reactivity:
Human
Research Area:
Immunology
€499
Frequently bought together:

Description

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Human Factor H/Complement factor H ELISA Kit

The Human Factor H (Complement Factor H) ELISA Kit is specifically designed for the precise measurement of complement factor H levels in human serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides accurate and reproducible results, making it an invaluable tool for various research applications.Complement factor H is a key regulator of the alternative pathway of the complement system, which plays a crucial role in immune response and inflammation.

Dysregulation of complement factor H has been implicated in various autoimmune and inflammatory diseases, making it a promising biomarker for studying these conditions and developing targeted therapeutics.Overall, the Human Factor H (Complement Factor H) ELISA Kit offers researchers a reliable method for quantifying complement factor H levels, furthering our understanding of its role in health and disease.

Product Name:Human Factor H / Complement factor H ELISA Kit
Product Code:HUFI00749
Size:96 Assays
Alias:CFH, ARMD4, ARMS1, beta-1-H-globulin, CFH, CFHL3, FH, FHL1, HF1, HF2, HUS, adrenomedullin binding protein, age-related maculopathy susceptibility 1, AHUS1, AMBP1, ARMD4, beta-1H, beta-1-H-globulin, CFHL3, complement factor H, factor H, factor H-like 1, FH, FHL1, H factor 1, H factor 1, complement, H factor 2, complement, HF, HF1ARMS1, HF2, HUSMGC88246
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human CFH concentrations in serum plasma and other biological fluids.
Sensitivity:14.063ng/ml
Range:23.438-1500ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human CFH and the recovery rates were calculated by comparing the measured value to the expected amount of Human CFH in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)87-10094
EDTA plasma(n=5)87-10496
UFH plasma(n=5)89-10297
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CFH and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)86-103%85-94%86-94%
EDTA plasma(n=5)86-98%93-98%82-96%
UFH plasma(n=5)81-99%84-100%81-98%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment 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
UniprotP08603
UniProt Protein Function:CFH: Factor H functions as a cofactor in the inactivation of C3b by factor I and also increases the rate of dissociation of the C3bBb complex (C3 convertase) and the (C3b)NBB complex (C5 convertase) in the alternative complement pathway. Genetic variations in CFH are associated with basal laminar drusen (BLD); also known as drusen of Bruch membrane or cuticular drusen or grouped early adult-onset drusen. Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch membrane. Basal laminar drusen refers to an early adult-onset drusen phenotype that shows a pattern of uniform small, slightly raised yellow subretinal nodules randomly scattered in the macula. In later stages, these drusen often become more numerous, with clustered groups of drusen scattered throughout the retina. In time these small basal laminar drusen may expand and ultimately lead to a serous pigment epithelial detachment of the macula that may result in vision loss. Defects in CFH are the cause of complement factor H deficiency (CFHD). A disorder that can manifest as several different phenotypes, including asymptomatic, recurrent bacterial infections, and renal failure. Laboratory features usually include decreased serum levels of factor H, complement component C3, and a decrease in other terminal complement components, indicating activation of the alternative complement pathway. It is associated with a number of renal diseases with variable clinical presentation and progression, including membranoproliferative glomerulonephritis and atypical hemolytic uremic syndrome. Defects in CFH are a cause of susceptibility to hemolytic uremic syndrome atypical type 1 (AHUS1). An atypical form of hemolytic uremic syndrome. It is a complex genetic disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, renal failure and absence of episodes of enterocolitis and diarrhea. In contrast to typical hemolytic uremic syndrome, atypical forms have a poorer prognosis, with higher death rates and frequent progression to end-stage renal disease. Susceptibility to the development of atypical hemolytic uremic syndrome can be conferred by mutations in various components of or regulatory factors in the complement cascade system. Other genes may play a role in modifying the phenotype. Genetic variation in CFH is associated with age-related macular degeneration type 4 (ARMD4). ARMD is a multifactorial eye disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid (known as drusen) that lie beneath the retinal pigment epithelium and within an elastin- containing structure known as Bruch membrane. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:Protein type:Secreted; Secreted, signal peptide

Chromosomal Location of Human Ortholog: 1q32

Cellular Component: extracellular region; extracellular space

Molecular Function:heparan sulfate proteoglycan binding; heparin binding; protein binding

Biological Process: complement activation; regulation of complement activation

Disease: Basal Laminar Drusen; Complement Factor H Deficiency; Hemolytic Uremic Syndrome, Atypical, Susceptibility To, 1; Macular Degeneration, Age-related, 4

NCBI Summary:This gene is a member of the Regulator of Complement Activation (RCA) gene cluster and encodes a protein with twenty short consensus repeat (SCR) domains. This protein is secreted into the bloodstream and has an essential role in the regulation of complement activation, restricting this innate defense mechanism to microbial infections. Mutations in this gene have been associated with hemolytic-uremic syndrome (HUS) and chronic hypocomplementemic nephropathy. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. [provided by RefSeq, Oct 2011]
UniProt Code:P08603
NCBI GenInfo Identifier:158517847
NCBI Gene ID:3075
NCBI Accession:P08603.4
UniProt Secondary Accession:P08603,P78435, Q14570, Q2TAZ5, Q38G77, Q5TFM3, Q8N708 Q9NU86, A5PL14,
UniProt Related Accession:P08603
Molecular Weight:51,034 Da
NCBI Full Name:Complement factor H
NCBI Synonym Full Names:complement factor H
NCBI Official Symbol:CFH
NCBI Official Synonym Symbols:FH; HF; HF1; HF2; HUS; FHL1; AHUS1; AMBP1; ARMD4; ARMS1; CFHL3
NCBI Protein Information:complement factor H
UniProt Protein Name:Complement factor H
UniProt Synonym Protein Names:H factor 1
Protein Family:Complement factor
UniProt Gene Name:CFH
UniProt Entry Name:CFAH_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct 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.

StepProtocol
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.

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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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.

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