Acid-labile subunit (ALS) ELISA Kit

Sample

Serum and plasma

Species Reactivity

Human

Detection Method

sELISA

Intended Use

This enzyme immunoassay kit is suited for measuring ALS in human serum or EDTA-/heparin /citrate plasma for scientific purposes.

Contents of Kit

1. Microtiter plate: ready for use, coated with rabbit-anti-ALS-antibody. Wells are separately breakable. (8x12) wells
2. Sample Buffer (PP): ready for use, red colored, please use for the reconstitution of Standards and Controls KS1/KS2 and for the dilution of Samples and Controls KS1/KS2. 1 x 125 mL
3. Standards (A-F): lyophilized, (native human ALS), concentrations are given on vial labels and on quality certificate in ng/mL. 6 x 1 mL
4. Control Serum 1 (KS1): lyophilized, (human serum), concentration is given on quality certificate in ng/mL. 1 x 250 μL
5. Control Serum 2 (KS2): lyophilized, (human serum), concentration is given on quality certificate in ng/mL. 1 x 250 μL
6. Dilution Buffer (VP): ready for use, please use for the dilution of Antibody Conjugate. 1 x 7 mL
7. Antibody Conjugate (AK): 50-fold concentrate, contains the biotinylated anti-human rabbit ALS Antibody. Dilute before use 1:50 in Dilution Buffer and use 50 μl for each well in the assay. 1 x 140 μL
Attention: Please dilute Antibody Conjugate freshly according to daily requirements.
8. Enzyme Conjugate (EK): ready for use, contains HRP (Horseradish-Peroxidase)-labeled Streptavidin. 1 x 12 mL
9. Washing Buffer (WP): 20-fold concentrated solution, dilute 1:20 in A.dest. or in deionized Water. 1 x 50 mL
10. Substrate (S): ready for use, horseradish-peroxidase-(HRP) substrate, stabilised Tetramethylbencidine. 1 x 12 mL
11. Stopping Solution (SL): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
12. Sealing Tape: for covering the microtiter plate. 2

Storage

Store the kit at 2-8°C after receipt until its expiry date. The lyophilized reagents should be stored at –20 °C after reconstitution. Avoid repeated thawing and freezing.
The shelf life of the components after initial opening is warranted for 4 weeks, store the unused strips and microtiter wells airtight together with the desiccant at 2-8°C in the clip-lock bag, use in the frame provided. The reconstituted components standards A-F and Control Sera KS1 and KS2 must be stored at –20°C (max. 4 weeks). For further use, thaw quickly but gently (avoid temperature increase above room temperature and avoid excessive vortexing). Up to 3 of the freeze-thaw cycles did not influence the assay. The 1:20 diluted Washing Buffer WP is 4 weeks stable at 2-8°C

Performance Characteristics

Calibration - Traceability
No international standard or reference preparation of ALS is available. Initially, the ALS ELISA Kit was calibrated against a human serum standard. In a second step the test system was recalibrated with eukaryotic, recombinant ALS. The recombinant ALS was measured in three different lots. A comparison of the measured results is shown in Figure below. The analysis revealed a factor of 5 to transfer Units in mass units (ng/mL). According to the function y = 4,997x, the factor of 5 is used in the conversion of Units (mU/mL) in mass units (ng/mL): 1 Unit ALS ≈ 5 ng rec. ALS

Figure. Assay Calibration, relation of Units and mass units.
A previously conducted comparative analysis of serum samples demonstrates that the ALS ELISA Kit measures comparable results referring to an in-house assay used by an academic group.

Figure. Comparative analysis of a competitive immunoassay with the ALS ELISA Kit (serum samples: n=25).

Precision

Intra-Assay Variance
Two samples have been measured 22 times in the same assay. The measured coefficient of variation (CV) is 6.7% on average.

Inter-Assay Variance
Serum samples were measured in independent assays. On average the coefficient of variation was 8.96% (SD 6.11).

Sensitivity

Sensitivity was assessed by measuring the blank and calculating the theoretical concentration of the blank + 2SD. In three measurements a range of with a mean sensitivity of 0.53 ng/mL was detected.

General Description

The Insulin-like Growth Factors (IGF) – I and II are bound to specific binding proteins in circulation (IGFBP). Until today seven different proteins have been identified: IGFBP-1 to 7. IGF bioavailability, transport and storage is regulated and facilitated by these binding proteins which are expressed differentially according physiological and developmental requirements. The most abundant IGFBP in circulation is IGFBP-3. Together with IGFBP-5 it is able to form the so called ternary complex with IGF and the acid-labile subunit (ALS). In the circulation nearly all IGF is bound in this ternary complex and thus not able to cross the endothelial barrier. Only very small amounts of IGF or IGFBP-3 exist outside this complex. The acid-labile subunit is an important part of the IGF-storage mechanism in circulation. In ALS deficiency or in ALS knock-out mice the concentrations of IGF and IGFBP-3 in the circulation are significantly decreased thus resulting in impaired growth.
The acid-labile subunit is synthesized as propeptide of 605 amino acids. The signal peptide, necessary for ALS secretion (AA 1-27) cleaved off enduring the transport process (Swiss-Prot P35858 Version 82). Thus the mature protein consists of 578 amino acids and contains about 20 leucin rich sequence repeats. Beside the leucin-rich repeats several potential N-linked glycosylation sides are described. Miller BS et al. were able to demonstrate that incomplete glycolsylation of IGFs, ALS and IGFBP-3 results in a decreased serum concentration of these proteins. Oral mannose therapy resulted in a partial normalization of the glycosylation pattern and went along with improved growth. Mutations in or the complete knock out of the ALS gene result in IGF / IGFBP-3 deficiency and therewith in disturbance of growth. Beside growth also other endocrine axes may be involved. In primary ALS deficiency hyperinsulinemia could be observed. Further, the ALS-IGF-IGFBP-system seems to be of relevance in coronary disease.
The results of this test system can be used as supplementary information in GH-diagnostics together with IGF-I and IGFBP-3 measurements. Thus, it is of use in evaluation of GH-deficiency and excess.
The first ALS immunoassay was described by Baxter RC in 1990. By this in-house radioimmunoassay it was shown that ALS is present in high concentrations in serum (50 μg/mL) of healthy humans. But not detectable in other body fluids like amniotic fluid, cerebrospinal fluid or seminal plasma – in spite of the fact that these body fluids contain high levels of IGFBP-3.

Standard Curve

The following data is for demonstration only and cannot be used in place of data generation at the time of assay.


Note: The exemplary shown standard curve cannot be used for calculation of your test results. You have to establish a standard curve for each test you conduct!