5-Hydroxyindoleacetic acid (5-HIAA) ELISA kit instructions

June 04, 2021

5-Hydroxyindoleacetic acid (5-HIAA) ELISA kit instructions
(Germany IBL: RE59131)
1, the scope of application
This ELISA kit can be used for in vitro quantitative detection of 5-HIAA in human urine.
2. Foreword
Primary carcinoid tumors are usually derived from intestinal chromaffin cells in the midgut and are mostly located in the terminal ileum. Carcinoid tumors usually secrete a variety of sputum, and these tumors are generally characterized by an increase in the secretion of 5-HIAA (the final metabolite of serotonin) in the urine. The 5-HIAA traditional detection method produces purple by diazotization of nitroso naphthol. However, it has been shown that many other substances present in the urine interfere with this reaction and result in false positives. Attempts have also been made to overcome this problem by combining ion exchange chromatography with fluorescence analysis. However, these methods are low in sensitivity and time consuming. Recently, the following methods have been used to detect 5-HIAA: high-performance liquid chromatography in the ultraviolet region 5-HIAA combined with fluorescence analysis (or electrochemical analysis). Because of the variety of interfering complexes present in the urine, both methods require solvent extraction. The 5-HIAA enzyme-linked immunosorbent assay is a novel and simple method for the quantitative detection of tumor-like syndrome markers in urine.
3. Experimental principle
This ELISA kit utilizes the principle of competition, in which a biotin-labeled antigen competes with a biotin-labeled antigen for binding to a certain amount of antibody binding site. The amount of biotinylated antigen bound to the antibody is inversely proportional to the concentration of the analyte in the sample. After the reaction system reaches equilibrium, the unbound biotin-labeled antigen is washed away with a washing solution. The amount of the antibody bound to the biotin-labeled antigen was determined by using avidin alkaline phosphatase as a marker and PNPP (p-nitrophenol phosphate) as a substrate. The amount of the unknown can be obtained by comparing the enzyme reactivity of the unknown with the reaction curve of a known standard.
4, kit components
Quantity
Sign
ingredient
1×12×8
MTP
The coated plate is detachable and the micropores are coated with anti-rabbit IgG (sheep, monoclonal).
1×5ml
ANTISERUM
5-HIAA antiserum
Blue, ready to use, contains: antiserum (rabbit), phosphate buffer, stabilizer.
1×5ml
BIOTIN
5-HIAA Biotin
Ready-to-use, blue, contains: phosphate buffer, stabilizer.
1 x 0.2 mL
ENZCONJ CONC
Enzyme Concentrate (100×)
Contains: Alkaline phosphatase-labeled avidin antibody (sheep), Tris buffer, stabilizer
1×7×0.5ml
CAL AG
Standard AG
0; 0.4; 1.0; 2.75; 7.5; 20; 55 mg/L
0; 2.1; 5.25; 14.4; 39.4; 105; 288 μmol/L
Ready to use, contains: 5-HIAA (methylation), stabilizer.
1×2×0.5ml
CONTROL 1+2 LYO
Quality control 1+2, lyophilized powder, containing: human urine (normal and abnormal human urine), please refer to the reagent bottle label for specific concentration and allowable range.
1×2ml
METHYLRAGE
Methylation reagent, yellow, ready to use, containing methylene chloride
1 x 1 mL
HCL
HCl, ready to use, 0.1 M HCl
1 x50ml
ASSAUBUF CONC
Detection buffer, concentrated (10 x)
Contains: phosphate buffer, BSA, stabilizer
1 x4ml
DILREAG
Diluent, ready to use, contains: N,N-dimethylformamide
1 x50ml
WAHSBUF CONC
Washing liquid, concentrated (20 x)
Contains: phosphate buffer, Tween, stabilizer
1×9
PNPP SUBS
PNPP substrate containing: p-nitrophenyl phosphate (PNPP)
1×27ml
PNPP BUF
PNPP substrate buffer, ready to use, contains: diethanolamine, water,
1×15ml
PNPP STOP
PNPP stop solution, ready to use, containing: 1M NaOH, 0.25M EDTA
3×
FOIL
Sticky metal plate
5, the equipment required for the experiment (but not provided by the kit)
Pipette (20; 25; 50; 100; 1000 μL)
Glass test tube (12×75mm)
Vortex mixer
8-channel pipette with reservoir
Washing bottle, automatic or semi-automatic coated board cleaning system
Microplate reader
Double distilled or deionized water
Fume hood
Absorbent paper, sampling tip, timer
6. Preparation instructions before the experiment
For manual operation and automatic operation:
note
The 96-person test reagent can be divided into 3 independent experiments. The following volume is the amount of reagent required for one test with four coated plates (32 servings). If the customer wants to reduce the amount of the standard from 7 to 6, you can remove the standard G. The report range of the experimental results is correspondingly reduced to 3000 μg / L
6.1 Preparation of lyophilized powder or concentrated ingredients
dilution
ingredient
Diluent
proportion
Remarks
Storage
stability
15ml
Detection buffer
150ml
Double distilled water
1:10
Fully mixed
2-8 ° C
2 weeks
Control product
0.5ml
0.1M HCl
Allow to stand for 15 min, avoid bubbles when mixing
≤-20°C
8 weeks
15ml
detergent
300ml
Double distilled water
1:20
Heat to 37 ° C to dissolve all crystals (if necessary) and dissolve thoroughly.
2-8 ° C
4 weeks
60μL
Enzyme complex
60ml
Detection buffer (diluted)
1:101
Temporary configuration, can only be used once, avoiding bubble generation during mixing
18-25 ° C
30min
3
PNPP substrate
8ml
PNPP substrate buffer
Temporary configuration, can only be used once, free of bubbles when mixing
18-25 ° C
10min
6.2 dilution of the sample
The 5-HIAA pus in the sample above the highest standard should be further diluted with the assay buffer after methylation.
7, the first day
7.1 dilution of control products and patient samples (except standard)
Sample preparation will result in a 255-fold dilution. But this has taken this into account in the standardization.
note
Do not methylate the standard, it has been methylated.
1)
20 μL of the control and the sample were separately added to the corresponding glass test tubes.
2)
After completing the next steps, continue to operate in the fume hood.
3)
50 μL of the dilution was added to each tube. Mix on a vortex mixer.
4)
25 μL of methylation reagent was added to each tube. The test tube solution was mixed immediately after the addition of the reagent. Note: The yellow color of the reaction mixture should be stable. If the color disappears quickly, the amount of acid in the sample is too large. In this case, 20 μL of methylation reagent was added to the test tube.
5)
Cover the tube and incubate for 20 min at room temperature (18-25 ° C)
6)
Add 5 ml of diluted assay buffer to each tube. Cover the tube with a stopper and rotate it several times. Mix up to five times manually or with a mixer so that the liquid can mix well.
7)
After completing this step, you can not operate under the fume hood.
8)
50 μL of the supernatant was aspirated and an ELISA experiment was performed immediately. The supernatant was stable for 1 h at room temperature.
8.2 ELISA operation
1)
50 μL of the standard, methylation control and methylated patient samples were added to the corresponding microwells.
2)
50 μL of 5-HIAA biotin was added to each well.
3)
50 μL of 5-HIAA antiserum was added to each well.
4)
Cover the plate and carefully shake the cover plate. Incubate overnight (16-20 h) at 2-8 °C.
8, the next day
1)
The metal plate was removed, the reaction solution in the well was discarded, and the plate was washed 3 times with 250 μL of the washing solution, and patted dry on the absorbent paper.
2)
Add 150 μL of temporarily configured enzyme conjugate to each well
3)
Use a new sticky metal cover. Incubate for 120 min on an orbital shaker (500 rpm) at room temperature (18-25 ° C).
4)
The enzyme conjugate was added to the microwells approximately 10 minutes before the incubation of the genus.
5)
The metal plate was removed, the reaction solution in the well was discarded, and the plate was washed 3 times with 250 μL of the diluted washing solution, and patted dry on the absorbent paper.
6)
When adding the substrate and stop solution, if necessary, an 8-channel pipette can be used for dosing. The substrate and stop solution must be applied at the same time interval to avoid bubble generation.
7)
200 μL of temporarily configured PNPP substrate solution was added to each well.
8)
Incubate for 60 min on an orbital shaker at room temperature (18-25 ° C).
9)
50 μL of PNPP stop solution was added to each well to terminate the reaction. Gently shake the coated plate to mix the ingredients.
10)
The OD value (reference wavelength: 600-650 nm) was measured at 405 nm within 60 min after the addition of the stop solution.
9, reference value
Experimental results cannot be considered as the only factor in medical diagnosis, and medical diagnosis is also associated with other clinical observations and other diagnostic tests.
The following are the normal values ​​of 5-HIAA for the apparently healthy population: (97.5%)

We recommend that each lab set its own range of normal values.
Urine
Mg/24h
Molmol/day
5-HIAA
6 to 10
31.5~52.5
This translation is for reference only, please refer to the original for details.
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