Human HSP90 alpha Immunoassay Kit

Intended Use

Human Enzyme Immunoassay test kit is intended for the quantitative determination of human HSP90 alpha concentration in human serum, plasma, cell lysate.

Background

HSP90 is a composite name for a large group of Heat Shock Proteins whose molecular weights average 90 kDa. HSPs functions primarily as a molecular chaperone, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific degradative pathways. HSP90 is a central component in a number of basic cellular processes including hormone signaling and cell cycle control. HSP90 is ubiquitously expressed, highly conserved and accounts for 1‑2% of the total cellular protein. Recently introduced, standardized nomenclature has divided the 17 identified HSP90 genes into three related and one unrelated classes, HSP90AA, HSP90AB, HSP90BB, and TRAP, respectively.

There are two existing isoforms, Heat Shock Protein 90 alpha (HSP90 alpha) and Heat Shock Protein 90 beta (HSP90 beta), with 76% homology. HSP90 has emerged as a potential target for cancer therapy as many tumor cells such as non-small cell lung cancer, esophageal squamous cell carcinoma, pancreatic carcinoma and malignant melanoma overexpress HSP90alpha. The activity of many oncogenic proteins depend on HSP90 and anti HSP90alpha drugs have become an exciting area in the search for cancer therapies and other diseases.

Assay Principle

An anti-human HSP90 alpha coating antibody is adsorbed onto micro-wells. Human HSP90 alpha present in the sample or standard binds to antibodies adsorbed to the micro-wells. Following incubation unbound biological components are removed during a wash step and a biotin-conjugated anti-human HSP90 alpha antibody is added and binds to human HSP90 alpha captured by the first antibody. Following incubation unbound biotin-conjugated anti-human HSP90 alpha antibody is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti- human HSP90 alpha antibody. Following incubation unbound Streptavidin-HRP is removed during a wash step, and substrate solution TMB is added to the wells. TMB is  incubated for 20 minutes, resulting in the development of a blue color. The color development is stopped with the addition of 2N HCl Stop Solution, and the color is changed to yellow and measured spectrophotometrically at 450 nm. The concentration of HSP90 alpha is directly proportional to the color intensity of the test sample.

Sample Collection and Storage

Cell lysate- Remove cell culture medium and wash cells with PBS twice. Lyse cells with appropriate lysis buffer like RIPA buffer. Assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles.

Serum - Allow blood samples to clot for 2 hours at room temperature before centrifuging for 20 minutes at 2000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles.

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge for 20 minutes at 2000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20 °C. Avoid repeated freeze-thaw cycles.

Material and Components

 Materials provided with the test kits

1. Human HSP90 alpha antibody-coated microtiter plate with 96 wells.
2. Reference standard set, contains 0, 0.156, 0.312, 0.625, 1.25, 2.50, 5.00 and 10.0 ng/ml HSP90 alpha, in liquid form (ready to use) or lyophilized form.
3. Biotin-Conjugated anti-human HSP90 alpha detection antibody, 1 vial
4. Detection antibody diluent, 12 ml.
5. Sample dilution buffer, 15 ml.
6. Streptavidin-HRP, 1 vial.
7. Streptavidin-HRP diluent, 12 ml.
8. TMB Substrate, 12 ml.
9. Stop Solution, 12 ml.
10. Wash Buffer Concentrate (20X), 25ml, 2 bottles.
11. Adhesive Films

Reagent Preparation

1. All reagent should be brought to room temperature (18-22^oC) before use.
2. If reference standards are lyophilized, reconstitute each standard with 0.5 ml distilled water. Allow the reconstituted material to stand for at least 20 minutes. Reconstituted standards should be sealed and stored at 2-8°C.
3. Pour entire contents (25 ml x 2 ) of the Wash Buffer Concentrate (20x) into a clean 1000 ml graduated cylinder. Bring to final volume of 1000 ml with glass-distilled or deionized water. Mix gently to avoid foaming.
4. Biotinylated detection antibody: Make a 1:100 dilution of the concentrated Biotin-Conjugate solution with Detection antibody diluent in a clean plastic tube as needed
5. Streptavidin-HRP: Make dilution of the concentrated Streptavidin-HRP solution with Streptavidin-HRP diluent in a clean plastic tube as needed.

Assay Procedure

1. Predilute your samples before starting with the test procedure. Dilute serum and plasma samples 1:25 with Sample Diluent according to the following scheme: 10 μl Sample +240 μl Sample Diluent
2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Each sample, standard, blank and optional control sample should be assayed in duplicate. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2°-8°C sealed tightly.
3. Wash the microwell strips twice with approximately 400 µl Wash Buffer per well with thorough aspiration of microwell contents between washes. Allow the Wash Buffer to sit in the wells for about 10 – 15 seconds before aspiration. Take care not to scratch the surface of the microwells. After the last wash step, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess Wash Buffer. Use the microwell strips immediately after washing. Alternatively microwell strips can be placed upside down on a wet absorbent paper for not longer than 15 minutes. Do not allow wells to dry.
4. Add the standard to the designated standard wells in duplicate.
5. Add 100 µl of Sample Diluent in duplicate to the blank wells.
6. Add 100 µl of each prediluted sample in duplicate to the sample wells.
7. Cover with an adhesive film and incubate at room temperature (18 to 25°C) for 2 hours, on a microplate shaker set at 400 rpm.
8. Prepare Biotin-Conjugate (see Preparation of Biotin-Conjugate 9.3).
9. Remove adhesive film and empty wells. Wash microwell strips 5 times. Proceed immediately to the next step.
10. Add 100 µl of diluted Biotin-detection antibody to all wells, including the blank wells.
11. Cover with an adhesive film and incubate at room temperature (18 to 25°C) for 1 hour, on a microplate shaker set at 400 rpm.
12. Prepare Streptavidin-HRP (see Preparation of Streptavidin-HRP 9.4).
13. Remove adhesive film and empty wells. Wash microwell strips 5 times according to point c of the test protocol. Proceed immediately to the next step.
14. Add 100 µl of diluted Streptavidin-HRP to all wells, including the blank wells.
15. Cover with an adhesive film and incubate at room temperature (18 to 25°C) for 30 minutes, on a microplate shaker set at 400 rpm.
16. Remove adhesive film and empty wells. Wash microwell strips 5 times. Proceed immediately to the next step.
17. Pipette 100 µl of TMB Substrate Solution to all wells.
18. Incubate the microwell strips at room temperature (18° to 25°C) for 30 minutes. Avoid direct exposure to intense light.
    The color development on the plate should be monitored and the substrate reaction stopped (see next point of this protocol) before positive wells are no longer properly recordable. Determination of the ideal time period for color development has to be done individually for each assay.
    It is recommended to add the stop solution when the highest standard has developed a dark blue color. Alternatively the color development can be monitored by the ELISA reader at 620 nm. The substrate reaction should be stopped as soon as Standard 1 has reached an OD of 0.9 – 0.95.
19. Stop the enzyme reaction by quickly pipetting 100 µl of Stop Solution into each well. It is important that the Stop Solution is spread quickly and uniformly throughout the microwells to completely inactivate the enzyme. Results must be read immediately after the Stop Solution is added or within one hour if the microwell strips are stored at 2 - 8°C in the dark.
20. Read absorbance of each microwell on a spectro-photometer using 450 nm as the primary wave length

 Important Note: The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

 Note: If instructions of this protocol have been followed, serum and plasma samples have been diluted 1:25 and the concentration read from the standard curve must be multiplied by the dilution factor (x25).

 Note: In case of incubation without shaking the obtained O.D. values may be lower than indicated below. Nevertheless the results are still valid.

 Results

• Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 per cent of the mean value.
• Create a standard curve by plotting the mean absorbance for each standard concentration on the ordinate against the human HSP90 alpha concentration on the abscissa. Draw a best fit curve through the points of the graph (a 5- parameter curve fit is recommended).
• To determine the concentration of circulating human HSP90 alpha for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding human HSP90 alpha concentration.
• If instructions in this protocol have been followed, samples have been diluted 1:25 (10 µl sample + 240 µl Sample Diluent) and the concentration read from the standard curve must be multiplied by the dilution factor (x 25).
• It is suggested that each testing facility establishes a control sample of known human HSP90 alpha concentration and runs this additional control with each assay. If the values obtained are not within the expected range of the control, the assay results may be invalid.
• A representative standard curve is shown below This curve cannot be used to derive test results. Each laboratory must prepare a standard curve for each group of microwell strips assayed. Representative standard curve for human HSP90 alpha ELISA. Human HSP90 alpha was diluted in serial 2-fold steps in Sample Diluent. Do not use this standard curve to derive test results. A standard curve must be run for each group of microwell strips assayed.

 Sample values and sensitivity

Panels of 40 serum as well as 16 plasma samples (EDTA, citrate, heparin), from randomly selected healthy donors (males and females) were tested for human HSP90 alpha.

Performance

 I. Precision:

 1) Intra-assay

 2) Inter-assay

Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 8 serum and plasma samples containing different concentrations of human HSP90 alpha. 2 standard curves were run on each plate. Data below show the mean human HSP90 alpha concentration and the

coefficient of variation calculated on 18 determinations of each sample (see Table 4). The calculated overall inter- assay coefficient of variation was 10.0 %

The mean human HSP90 alpha concentration and the coefficient of variation of each sample

 II. Linearity

Serum, plasma (EDTA, citrate, heparin), cell lysate samples with different levels of human HSP90 alpha were analyzed at serial 2 fold dilutions with 4 replicates each.

 III. Recovery

The spike recovery was evaluated by spiking 3 levels of human HSP90 alpha into serum, plasma (heparin, citrate) and cell lysate samples. Recoveries were determined with 2 replicates each. The amount of endogenous human HSP90 alpha in unspiked samples was subtracted from the spike values.

IV. Cross-reactivity

The assay detects both natural and recombinant human HSP90 alpha. The cross reactivity and interference of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into a human HSP90 alpha positive sample.

There was no cross reactivity or interference detected.

Precautions for Use

• All reagents should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statement(s) for specific advice.
• Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.
• Do not mix or substitute reagents with those from other lots or other sources.
• Do not use kit reagents beyond expiration date on label.
• Do not expose kit reagents to strong light during storage or incubation.
• Do not pipette by mouth.
• Do not eat or smoke in areas where kit reagents or samples are handled.
• Avoid contact of skin or mucous membranes with kit reagents or specimens.
• Rubber or disposable latex gloves should be worn while handling kit reagents or specimens.
• Avoid contact of substrate solution with oxidizing agents and metal.
• Avoid splashing or generation of aerosols.
• In order to avoid microbial contamination or cross-contamination of reagents or specimens which may invalidate the test use disposable pipette tips and/or pipettes.
• Use clean, dedicated reagent trays for dispensing the conjugate and substrate reagent.
• Exposure to acid inactivates the conjugate.
• Glass-distilled water or deionized water must be used for reagent preparation.
• Substrate solution must be at room temperature prior to use.
• Decontaminate and dispose specimens and all potentially contaminated materials as they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C.
• Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0% sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.

Storage

 Store standards and the detection antibody at -20°C. The remaining kit reagents can be stored between 2°C and 8°C. Expiry of the kit and reagents is stated on labels. Expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, this reagent is not contaminated by the first handling.