Biochemistry laboratory work (Практикум по биохимии). Методическое пособие

The most common reagents recently used in ELISA are substrates that form products that can be detected by the fluorometric method. When a molecule absorbs a photon molecule transitions from the ground electron state to an excited state. The excited molecule can return to the ground state, and the excess energy is converted into heat, the opposite process of the electron going back to the ground level,and a quantum of light is released, which is called fluorescence. The fluorometric method is 10 to 100 times sensitivity than the photometric method.

Enzymatic reactions, the energy of which is realized in the form of light emission - bioand chemiluminescence - are also used as detection systems in ELISA. The rate of such reactions is monitored by the glow intensity of the reaction system, which is registered with a luminometer. The bioluminescence reaction is catalyzed by firefly and bacterial luciferases, and the chemiluminescence reaction (oxidation of cyclic hydrazides by hydrogen peroxide) by horseradish peroxidase.

In recent years, electrochemical methods of determining the activity of enzymes used as tags in ELISA have become widespread. Such probes make it possible to determine the rate of enzymatic reactions in turbid media и convenient for to create flow-through immunoassay cells.

The most common reagents recently used in ELISA are substrates that form products that can be detected by the fluorometric method.

Thyroid stimulating hormone (TSH) is produced by the pituitary gland and regulates the production of thyroid hormones (thyroxine and triiodothyronine) through a "feedback system" that helps to maintain a stable concentration of these hormones in the blood. A decrease in the thyroid hormone concentration leads to an increase in thyroid hormone secretion and stimulates thyroid hormone production, and vice versa, an increase in thyroxine and triiodothyronine hormone secretion leads to a decrease in thyroid hormone production. Thyroid hormones are the main regulators of energy consumption in the body, and maintaining their

11

concentration at the required level is essential for the normal activity of almost all organs and systems.

Hypophysial dysfunction can cause high or low levels of TSH. When its concentration is increased, thyroid hormones are released into the bloodstream in abnormal amounts, causing hyperthyroidism. When TTH concentrations decrease, thyroid hormone production also decreases and symptoms of hypothyroidism develop.

Abnormal thyroid hormone production may be caused by hypothalamic diseases, which start producing increased or decreased amounts of thyroleiberin, a regulator of thyroid hormone secretion by the pituitary gland. Diseases of the thyroid gland, accompanied by abnormal secretion of thyroid hormones, may indirectly (via a feedback mechanism) affect secretion of thyroid hormone, causing a decrease or increase of its concentration in the blood. Thus, the TSH test is one of the most important hormone tests.

What is the study used for?

•To determine the condition of the thyroid gland, indirect assessment of thyroid hormone production.

•For monitoring the therapy of thyroid disorders.

•For diagnosis of thyroid disorders in newborns.

•For diagnosing female infertility and monitoring its treatment.

When the study is necessary?

When the thyroid gland is enlarged, as well as with symptoms of hyperand hypothyroidism:

oHyperthyroidism symptoms:

rapid heart rate,

increased anxiety,

weight loss,

insomnia,

tremors,

12

weakness, rapid fatigue,

diarrhea,

intolerance to light,

decreased visual acuity,

eyes puffiness and dryness,

hyperthermia.

oHypothyroidism symptoms:

skin dryness,

constipation,

cold intolerance,

swelling,

hair loss,

weakness, rapid fatigue,

menstruation cycle disorders.

At regular intervals the test may be prescribed to monitor the effectiveness of thyroid disease therapy. Thyroid levels are often evaluated in newborns considered to be at risk for thyroid disease.

Equipment and materials:

1)vertical scan spectrophotometer;

2)a thermostatically controlled shaker;

3)Plate with immobilized monoclonal antibodies to TTH (ТТГ- IFA-BEST kit produced by Vector Best, Inc);

4)film for plate sealing — 3 pcs;

5)plastic tub — 4 pcs;

6)tips for pipettes;

7)Automatic pipettes of variable volume for 2-20, 20-100, 1001000 microns;

8)multichannel automatic pipette;

9)scissors;

10)glass test-tubes with capacity of 10 ml;

11)distilled water;

12)rubber gloves;

13)laboratory filter paper;

14)rubber backing paper;

13

15)cotton swabs;

16)Vacutainer K3 EDTA;

17)disposable needles for blood sampling;

18)adapter-needle holder.

Reagents*:

1)calibration samples containing known amounts of TSH: 0; 5.6; 16.7; 50; 150; 300 pg/ml;

2)a control sample containing TSH;

3)conjugate Concentrate #1 — biotinylated antibodies to TSH;

4)conjugate Concentrate #2 — streptavidin horseradish peroxidase;

5)conjugate #2 dilution solution;

6)sample dilution solution;

7)solution 1 containing saline and Tween;

8)tetramethylbenzidine (TMB) concentrate;

9)substrate buffer solution;

10)stop reagent;

11)ethanol 96%.

* All reagents were manufactured by «Vector-BEST».

Working scheme

Attention! Work shall be carried out wearing single-use gloves.

1.Wipe the workplace, pipettes and gloves with a cotton swab moistened with 70% ethanol.

2.Draw blood by venipuncture up to a risk of 9 ml.

3.After gentle agitation centrifuge tube with blood for 10 min at 3000g.

4.Collect plasma in a new tube.

5.Add 100 µl of sample dilution solution (SE) to all wells of the plate with immobilized monoclonal antibodies to TTH using a multichannel pipette.

14

6.Add 100 µl of calibration and control samples to each well A-1, B-1, C-1, D-1, E-1, F-1, G-1 using a single channel pipette and

100µl of test samples to the others.

7.Cut off the sticky tape to the required size. Seal the strips by pressing firmly against the tape and place in a shaker.

8.Incubate at 700 rpm at 37° C for 2 h.

9.Prepare working solution of conjugate No.1 10 min before incubation as follows: Add 1 ml of Conjugate Solution 1 and 50 µl of Conjugate Concentrate No.1 to a test tube 1 per strip and mix thoroughly.

10.At the end of incubation remove the foil, shake out the wells, remove the remaining solution by tapping the inverted plate on a rubber plate covered with filter paper. Wash the wells of the plate wells 5 times with solution 1 using a multichannel pipette as follows: add 300 µl of solution 1 to each empty well, incubate 1 min on a shaker with shaking at 700 rpm, then shake off the wells and remove the remaining solution by vigorous tapping the plate on a rubber plate covered with absorbent paper.

11.Add 100 µl of working solution of conjugate No.1 to each well by multichannel pipetting, cover wells with foil and incubate at 37° C at 700 rpm for 1 h.

12.Prepare working solution of conjugate No.2 10 min before the end of incubation as follows: Add 1 ml of Conjugate No.2 dilution solution and 50 µl of Conjugate No.2 concentrate to the tube on a strip and mix thoroughly.

13.At the end of incubation wash the microplate wells 5 times as described in 10.

14.Add 100 µl of working solution of conjugate No.2 to each well by multichannel pipetting, cover wells with foil and place in a shaker and incubate for 30 min at 700 rpm at 37°C.

15

15.Prepare tetramethylbenzidine solution 10 min before incubation as follows: add 1 ml of Substrate Buffer Solution 1 and

70µl of tetramethylbenzidine concentrate to the tube on the basis of 1 strip, mix well and keep in the dark until use. Separate tubs and pipette tips must be used to prepare the tetramethylbenzidine solution.

16.At the end of incubation wash the strips 5 times as described in item 10.

17.Add 100 µl of tetramethylbenzidine solution to each well using a multichannel pipette and incubate in the dark for 30 min at room temperature.

18.Stop the reaction by adding 100 µl of stop reagent to each well using a multichannel pipette, blot the upper surface of the plate with filter paper and measure optical density at 450 nm for the next 5 min.

19.To determine the concentration of TTH in the test samples, a calibration curve must be plotted in the following coordinates: abscissa axis — TTH concentration (pkg/ml); ordinate axis — sample optical density value. The control sample is used to check the accuracy and reliability of the results. If the TTH concentration value calculated from the calibration chart in the control sample falls within the limits specified on the label, the obtained values of TTH concentrations in the samples are considered reliable.

Laboratory work №3: Genomic DNA isolation and preparation of DNA for PCR

At present, due to the intensive study of DNA, effective methods of its extraction and purification from proteins, lipids, lipoproteins, glycoproteins, proteoglycans, and other biopolymers have been developed. These are well known methods such as nucleic acid extraction using centrifugation in the CsCl2 density gradient, fractionation followed by alcohol resuspension (phenolic extraction

16

method, extraction with chloroform, acidic extraction, extraction with a mixture (phenol : Chloroform), protein extraction by mixture (ethanol : chloroform : methanol), extraction by mixture (phenol : ethanol), extraction by mixture (chloroform : isopropanol), and methods of homogeneous DNA extraction by mixture (phenol : guanidine) followed by phase separation. The length of the isolated product can be up to hundreds of thousands of bp, but the output of DNA is 10%. Extraction of lower molecular weight nucleic acids (over 100 bp) is characterized by an increase in extraction efficiency (up to 50-70 %). Disadvantages of this method are the duration of nucleic acid isolation (at least 3.5 h due to DNA resuspension), inconvenience of working with phenol and chloroform, as well as large losses of the isolated product during interphase formation. Moreover, DNA isolated by this method often contains impurities that prevent adequate determination of DNA concentration in the sample and effective work of polymerase (decrease in the number of enzyme turns) during DNA analysis by PCR.

Nucleic acid isolation by sorption onto silicate carriers from solutions of chaotropic salts (fine glass (MS), GF/F (GF/C) filters), using standard reagent kits (RNeasy Mini Kit; Qiagen Inc., Hilden, Germany; 5 Prime-3 Prime, Inc., Boulder, Colorado; QIAamp Blood Kit (QIAGEN, UK)) is widespread. The most common method of DNA isolation is fractionation followed by ethyl alcohol resuspension of DNA. The advantages of this method include the possibility of isolating both RNA and singleand double-stranded DNA. In addition, this method is time-consuming, multistage and labor-intensive, and DNA isolated by this method also contains impurities that prevent adequate determination of their concentration in the sample and effective PCR, so the method of isolation using homogeneous extraction of nucleic acids with phenolguanidine mixture has not found wide application. Significant disadvantages of this method are DNA fragmentation during DNA isolation (the size of isolated molecule does not exceed 40 bp) and inability to isolate DNA less than 100 bp long. Due to the high productivity, the method of nucleic acid sorption on silicate carriers in the presence of chaotropic salts is widely used in various fields of medicine and science, agriculture.

The objective of the work is to isolate and prepare for real-time PCR of genomic DNA from blood leukocytes.

17

Equipment and materials:

1)centrifuge;

2)shaker;

3)spectrophotometer;

4)automatic pipettes of variable volume for 1-10, 10-100 and 1001000 µl;

5)tips for pipettes;

6)disposable tubes for 1,5 and 2 ml;

7)microcolumns;

8)gloves;

9)cotton swabs.

Reagents:

1)lymphocyte separation medium (LSM) («MP Biomedicals», USA)

2)RNase A solution at a concentration of 1 mg/ml;

3)sorption solution*;

4)PBS (10 mM phosphate buffer, 0.15 M NaCl, pH 7.5);

5)lysis buffer (0.6% Nonidet P40, 50 mM Tris-HCl, 0.15 M NaCl, 5 mM NaF, 1 mM PMSF, 2 mM EDTA, 1 mM Na3MoO4);

6)washing solution 1*;

7)washing solution 2*;

8)3M sodium acetate, pH 5.0;

9)distilled water;

10)70% ethanol.

* Solutions produced by BioSilica Ltd.

Working scheme

Attention! Work must be performed with single-use gloves.

1. Preparing for work

Before use the sorption solution should be heated for 10 min at 5556°C until the salts are completely dissolved. The solution should not be heated above 65°C for extended periods of time.

18

2. Blood preparation

1.Draw blood by venipuncture up to a risk of 9 ml.

2.After gentle agitation centrifuge tube with blood for 10 min at 3000g.

3.Remove plasma from the tube.

4.Gently layer cell fraction of blood on equal amount of LSM.

5.Centrifuge at 1200 g for 40 min at 4 ̊.C

6.Carefully take the leukocyte fraction and put in to other tube.

7.Add 9 amounts of PBS, centrifuge at 1200 g for 10 min.

8.Carefully remove the supernatant completely using a pipette without damaging the precipitate.

3. Сell lysis

1. Suspend cells in 70 µl PBS, add 210 µl Lysis Buffer, mix thoroughly by pipetting and incubate for 2 min.

2. Separate the nuclei by centrifugation at 13,000 rpm for 10 min. Carefully remove the supernatant without damaging the precipitate using a pipette. Suspend the nuclei in 100 µl distilled water.

4. DNA extraction by sorption on a glass fiber carrier

1. Add 200 µl of sorption solution to a test tube with resuspended nuclei, incubate at room temperature for 20-30 min with constant stirring on a shaker.

2. Insert the filter column into a 2 ml tube. Add 100 µl of Washing Solution 1 to the filter column followed by the extracted sample. Centrifuge for 1 min at 1,000 rpm. If a solution remains above the filter, centrifuge an additional 1 min at 5,000 rpm.

3. Apply 300 µl of Washing Solution 1 to the filter and centrifuge 1 min at 5,000 rpm. Remove the filtrate by pipetting as follows:

- remove the microcolumn from the 2 ml tube;

19

-hold the microcolumn in your hand and remove the solution from a 2 ml tube using a pipette, using separate tips for each sample without touching the walls of the tube;

-return the microcolumn to the 2 ml tube.

4. Apply another 300 µl of Solution 1, centrifuge at 13,000g for 1 min.

5. Remove the solution in the same manner as the previous steps.

6. Apply 500 µl of Washing Solution 2 to the filter and centrifuge for 1 min at 13,000 rpm. Remove the filtrate as described in step 7.

7. Repeat step 10.

8. Centrifuge the microcolumn tube for 1 min at 13,000 rpm to remove residual solution.

9. Remove the microcolumn and place it in a new 1.5 ml tube. Place new microcolumn tubes on a rack. (Micro-columns must be VERTICAL!).

10.Add 150 µl of distilled water to the filter. Care must be taken to ensure that a drop of added water falls exactly on the center of the filter.

11.Incubate the microcolumn for 5 min at room temperature, centrifuge for 1 min at 1,000 rpm and then 1 min at 13,000 rpm.

12.Remove the filter column and transfer the isolated DNA sample to a new tube. The resulting solution contains purified DNA.

5. Preparing the DNA for PCR

To construct a calibration curve, you need to spectrophotometrically determine the concentration of DNA in the solution that does not contain RNA.

20