Overview

  • Product name

    Rat Retinol Binding Protein 4 ELISA Kit
    See all Retinol binding Protein 4 kits
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    Overall 5 3.3%
    Inter-assay
    Sample n Mean SD CV%
    Overall 3 2.7%
  • Sample type

    Cell culture supernatant, Urine, Serum, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    3.07 pg/ml
  • Range

    13.3 pg/ml - 850 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 116 109% - 126%
    Urine 104 103% - 105%
    Serum 113 107% - 116%
    Citrate Plasma 100 91% - 106%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Rat
    Does not react with: Sheep, Rabbit, Goat, Cow, Dog, Human, Pig
  • Product overview

    Abcam’s Retinol Binding Protein 4 (RBP4) in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of RBP4 protein in rat serum, plasma, urine, and cell culture supernatant.

    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Notes

    Retinol-Binding Protein 4 (RBP4) is a small, highly conserved member of the lipocalin superfamily involved in the transportation of retinol (Vitamin A) in blood. Synthesized in hepatocytes and adipocytes, RBP4 is also an adipokine involved in insulin resistance in the AG4KO mouse model. Elevated serum levels of RBP4 are seen in obesity and Type 2 diabetes.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate (12 x 8 well strips)

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Rat RBP4 Capture Antibody 1 x 600µl
    10X Rat RBP4 Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    Antibody Diluent 4BC 1 x 6ml
    Plate Seals 1 unit
    Rat RBP4 Lyophilized Recombinant Protein 2 vials
    Sample Diluent NS 1 x 50ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Development Solution 1 x 12ml
  • Research areas

  • Function

    Delivers retinol from the liver stores to the peripheral tissues. In plasma, the RBP-retinol complex interacts with transthyretin, this prevents its loss by filtration through the kidney glomeruli.
  • Involvement in disease

    Retinal dystrophy, iris coloboma, and comedogenic acne syndrome
  • Sequence similarities

    Belongs to the calycin superfamily. Lipocalin family.
  • Cellular localization

    Secreted.
  • Information by UniProt
  • Alternative names

    • Plasma retinol binding protein
    • Plasma retinol-binding protein
    • Plasma retinol-binding protein(1-176)
    • PRBP
    • PRO2222
    • RBP
    • RBP4
    • RDCCAS
    • RET4_HUMAN
    • Retinol binding protein 4
    • Retinol binding protein 4 interstitial
    • Retinol binding protein 4 plasma
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab203362 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.

     

  • Background-subtracted data values (mean +/- SD) are graphed.

  • The concentrations of RBP4 were measured in duplicate and interpolated from the RBP4 standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2).

Protocols

References

ab203362 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

I needed to assay retinol binding protein 4 in serum samples from Meriones unguiculatus however, commercial antibodies are not available. Old research indicates that there can be crossreactivity between rat anti-RBP4 antiserum and gerbil RBP4, and the Abcam customer service rep I discussed this with suggested that due to 98% identity in sequence, the kit may work in this species, so I enrolled in the Abtrial program to test this kit. The kit requires (in rat samples) that serum is diluted 1:64,000-1:640,000 times, so I calculated a dilution factor of 1:100,000x based on my serum retinol concentrations (HPLC determined) in the samples to put RBP concentrations near the middle of the external standardization curve. Obviously, a 5-step serial dilution prior to analysis in 66 samples is not ideal, but was performed using an electronic multichannel pipette for accuracy and efficiency in 96-well plates as 5 x 15 uL sample:135 uL diluent (this allows all dilutions to occur in the provided 50 mL of diluent "NS") with mixing in between steps.

All steps were performed according to the booklet, with samples run at the same time as the standard curve. All samples were within the standard curve, which was determined by 4-factor logistic regression as recommended. One low concentration point did not fit the curve very well but the samples were not near this concentration. We were planning on running a second kit from the same lot if this first one appeared to work as a duplicate to ensure that all samples were run on each plate.

I believe that this kit did not work in our samples as the results do not correlate at all (R-squared of ~0) with serum retinol concentrations in fasted gerbils, a well-known correlation in other rodents and mammals due to the toxicity of free retinol in serum. 11 of the 66 samples indicated that there was less RBP than retinol on a molar basis, half of which with than 50% of retinol, which would be extremely harmful to the animal if true.

It is interesting that the values did fall along the standard curve, indicating that the dilution was adequate, and were varied over a somewhat plausible range ignoring the relation to serum retinol.

I have attached images of the standard curve, a plot of the ELISA values vs serum retinol, and an image of the plate just prior to a final mix and reading at 450nm.

Unfortunately, recombinant gerbil RBP4 is not commercially available, so we could not definitively rule out other sources of error on this experiment.

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Verified customer

Submitted Jun 20 2018

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