Evaluation of Clinical Relevance of Serum VILIP-I Values in Patients with Cerebrovascular Accident Diagnosis by a new ELISA Method

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Name	VILIP-1 Human ELISA
Cat. No.	RD191119200R RUO
Other names	Hippocalcin-like protein 3, HLP3, VILIP, VLP-1, VSNL1, VISL1, VILIP-1
Product category	Neural tissue damage markers, Oncology
Assay format	Sandwich ELISA, Biotin-labelled antibody
Calibration range	0.1 - 5 ng/ml
Limit of detection	0.027 ng/ml
Applications	Cerebrospinal fluid, Plasma, Serum, Tissue extract
Sample requirements	100 ul/well
Storage/Shipping	2 - 8°C

Introduction

The diagnostics of central nervous system diseases is often difficult (particularly for first-line physicians – cerebrovascular accidents, Alzheimer disease, central nervous system traumas etc.). It is a serious health and social problem. For example, cerebrovascular accidents account for the third most frequent cause of death, with cardiovascular system diseases and tumour diseases being the two most frequent causes of death.
There is no ideal biomarker for central cerebral system damage, which could be examined in peripheral blood and which would be as efficient as, for example, cardiac troponins or natriuretic peptides in myocardial failure¹. However, intensive effort to search for it is in progress.
It was ascertained a short time ago that there are more than 29 genes the products of which can be found in the central nervous system in a higher concentration¹. One of them is VILIP-I (Visin like protein I), which is a potential and very promising biomarker for CNS damage (it is a cytoplasmatic protein with a low relative molecular weight, which is almost absolutely specific of CNS; cytoplasmatic protein occurs in brain in a high concentration independent of the location and type of cells) ^2,5. VILIP-I is a member of the family of neuronal calcium sensor proteins (there are VILIP-1/2/3). VILIP-I takes effect through increasing cAMP in cells; however, the functional role of VILIP-I is not quite clear, and VILIP-I is likely to be a signal protein (there are speculations that VILIP-I leads to the increase in insulin secretion in pancreas.; VILIP-I increases the expression of nicotinic acetylcholine receptors in brain, and generally, it takes effect in the organism as „tumour suppressor“ by inhibiting the proliferation, adhesion and invasivity of cells) ^2–7.
So far, no study that would evaluate VILIP-I concentration in individuals with cerebrovascular accident has been conducted. What is available is only the description of several in-house diagnostic kits for the determination of concentration of this protein, however not by means of the ELISA method. Our objective was to develop, validate and test clinically an ELISA kit for specific determination of serum concentration of human VILIP-I.

Key words: Vilip-I, cerebrovascular accident, ELISA, brain damage

Experimental section

Preparation of recombinant human VILIP-I

The mRNA sequence of VILIP-I gene was obtained from the RefSeq database (Accession Number NM_001442). The relevant sequence was synthesised and optimised for E.coli. The synthetic gene was cloned into the restriction sites of the pRSET expression vector (Invitrogen) with subsequent transformation of the bacterial strain E.coli BL21DE3. The production strain was cultivated at 37° C, and recombinant protein expression was induced by isopropyl β-D-1-thiogalactopy­ranoside (IPTG, Sigma). After the production culture had been disrupted by ultrasound, recombinant VILIP-I (Fig.1) was isolated from the supernatant by gel chromatography.

Fig.1 Protein purity was verified by electrophoresis (12 % homogenous gel, SDS PAGE, method: Lammli, gel colouration: Coomassie blue. In the left lane, there is a standard prepared from proteins sized 14, 21, 31, 45, 66 and 97 kDa; in the right lane, there is isolated recombinant human VILIP-I (reduced and heated sample and non-reduced and non-heated sample, concentration 5 mg/lane). Protein purity is higher than 98 %.

The protein was dialyzed into 50mM NaH²PO⁴ ambiance (pH 7,2), the protein purity was analysed by electrophoresis in polyacrilamide gel in the presence of sodium dodecyl sulphate (12% homogenous gel, SDS PAGE), and protein concentration was determined by the bicinchoninic acid method (BCA method, Sigma, catalogue number BCA1–1KT).

Development of sandwich ELISA assay

The values of VILIP-I serum and tissue concentration were not known, therefore our focus was on developing a sandwich ELISA assay which, with the use of biotin-marked detection antibodies, represents a highly sensitive and specific method. For ELISA determination, we employed specific polyclonal rabbit antibodies against human VILIP-I (Biovendor laboratorní medicína a.s.).
On the microtitration plate (NUNC, Maxicorp), 0,1 ng of antibody/well was bound in 0,1 M carbonate buffer pH 0,9 (incubation 12 h at 4°C), and after the binding solution had been aspirated, 0,2 ml/well of TBS solution (0,05 M Tris, 0,15 M NaCl, pH 7,2), 0,5 % BSA (bovine serum albumin) and 4 % sucrose were dispensed into the plate, and the plate was incubated 30 minutes at laboratory temperature to block the unused binding spots on the surface of the well. After the blocking solution had been aspirated (Columbus washer, Tecan), 0,1 ml of the relevant standard or 3×-diluted serum sample were dispensed into the plate. Buffer with 1,4 % casein in 0,15 M PBS (0,12 M NaCl, 0,03 M sodium phosphate, pH 7,3) was used for dilution, and all measurements were taken two times. Subsequently, the plate was incubated 1 hour at 250C. After the plate had been washed by washing solution 5× (TBS, 0,05% Tween 20, pH 7,2), 0,1 ml of biotin-marked rabbit polyclonal antibody IgG (Biovendor; the kit from Pierce company was employed for the process) was dispensed into all wells of the plate, and the plate was incubated 1 hour at 25°C. After the plate had been washed 5×, 0,1 ml of streptavidin-horseradish peroxidase conjugate (Amdex) was dispensed into all wells of the plate, and the plate was incubated 1 hour at 25°C. After the plate had been washed by washing solution, 0,1 ml of TMB substrate (1,2 mM tetramethylben­zidine containing 3 mM of hydrogen peroxide, KPL, catalogue number 52–00–01) was dispensed into all wells of the plate, and the reaction mixture was incubated 10 minutes at 25°C. The reaction was stopped by adding 0,1 M of sulphuric acid solution (0,1 ml/well), and the resulting yellow colouration (product) was measured photometrically at the wavelength of 450 nm. The intensity of yellow colouring is directly proportional to the content of analyte in the sample (ELISA reader Biotek EL808).
The VILIP-I values in unknown samples were determined using a calibration curve (Fig.2) that was prepared by plotting absorbance values of standards against their known concentration.

Fig.2. Standard VILIP-I ELISA Curve. Calibration curve prepared by plotting absorbance values of standards (A) against their known concentration (measured at 450 nm; ELISA reader Biotek EL808) was constructed to determine VILIP-I values in unknown samples.

The dilution solution for standards, samples, biotin-marked antibody and streptavidin-horseradish peroxidase conjugate was TBS solution, 0,2 % BSA, 0,01 % thimerosal.
In the test, a set of standards 6, 3, 1,5, 0,6, 0,15, 0,06 and 0,03 μg 1^-1 in buffer with casein (see above) was used. The set of standards was prepared by diluting recombinant human VILIP-I.
Serum samples were diluted 10× by the pattern: 1 part of sample + 9 parts of dilution solution.
All combinations resulting from the possibility of using rabbit specific antibodies in the sandwich ELISA assay (data are not presented) were tested in the above-described way. All combinations provided comparable results; therefore the most cost-effective variant was selected. Moreover, this variant minimizes potential cross-reactivities: rabbit specific antibody was bound onto the plate, and biotin-marked rabbit specific antibody was used for detection.

Clinical testing of ELISA assay

Ten individuals with cerebrovascular accident were examined. A group of 10 persons without glucose metabolism disorder and without neurological or malignant disease were adopted as control group. In all individuals, lumbar punction with the analysis of cells, the examination of hemato-liquor barrier, local immunoreaction and inflammation symptoms were carried out by means of specific proteins (indexes of albumin, immunoglobulines, ApoAI, ApoB a CRP in serum and liquor). In all individuals, also brain CT examination was carried out, or this examination was supplemented by the nuclear magnetic resonance examination respectively. In all persons, serum VILIP-I concentration was determined.

Results

Functional characteristics of ELISA assay

In the ELISA assay, no cross-reactivity in the serum of the following animals was found: rabbit, goat, sheep, pig, mouse, horse, hamster, hen, bovine and rat. So, the results of the assay suggest its specificity for human VILIP-I.
The precision and accuracy of the method was also tested to verify the functionality of VILIP-I ELISA. The accuracy of the method was verified by the standard addition method, and recovery expressed as the ratio of obtained/expected value of VILIP-I concentration was established. Serum samples from 2 patients (0,8 and 0,3 μg 1^-1) were enriched by +0,5, +1 and +2 μg 1^-1 of VILIP-I. The average recovery value was 98 %. Another two serum samples (2,1 a 1,8 μg 1^-1 ) were tested in the linearity test. These samples were serially diluted 10×, 20×, 40× and 80×, with the average recovery value being 93 %.
The precision of the method was tested as repeatability of results in 3 serum samples and expressed as both variation coefficient in series (n=8) and reproducibility between series of measurements (n=3). In all cases, the variation coefficient value (CV) was < 10 %.
The determinability limit of the method representing the lowest determinable VILIP-I concentration was 0,01 μg 1^-1 (this values is the expression of VILIP-I concentration corresponding with absorbance calculated according to the formula: average absorbance value of the blank (n=8) + 3× standard deviation of blank average). The detection limit (CV < 10 %) was 0,1 μg 1^-1.

Clinical testing of ELISA

Clinical testing of VILIP-I determination suggested that VILIP-I values were higher in individuals with cerebrovascular accident (n=10) than in persons without neurological disorder of central nervous system (all individuals with CMP had VILIP-I values > 0,05 μg 1^-1; P<0,01), individuals without CNS disorder had values < 0,05 μg 1^-1 (sensitivity 85,7 %, specificity 100 %).
Our study confirmed the assumption that patients with cerebrovascular accident have higher VILIP-I values than persons without neurological disorder. Hence, the first results support the hypothesis published a short time ago that VILIP-I might be a highly efficient serum biomarker for the presence of cerebrovascular accident.

Conclusions

A diagnostic kit (ELISA) for determination of VILIP-I serum concentration has been designed and validated. The basic analytical characteristics of the assay satisfy the conditions for its employment in clinical biochemistry laboratories. Moreover, external validation to obtain the CE mark (IVD) has been completed.

References

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