CN103558176B - A kind of bean plant agglutinin blood coagulation activity quantitative detecting method - Google Patents

Abstract

The invention discloses a kind of bean plant agglutinin blood coagulation activity quantitative detecting method, belong to plant component detection technique field.The method has carried out cell quantification to detection red cell suspension, utilize the bean plant agglutinin of concentration known to draw blood coagulation activity concentration standard curve as standard items simultaneously, and then calculate bean plant agglutinin blood coagulation activity concentration, thus bean plant agglutinin blood coagulation activity is carried out quantitatively.

Description

A quantitative detection method for coagulation activity of kidney bean phytohemagglutinin

technical field

The invention belongs to the technical field of plant component detection, and in particular relates to a quantitative detection method for coagulation activity of kidney bean lectin.

Background technique

The lectins contained in kidney beans are one of the poisonous substances that cause food poisoning in kidney beans, especially the pods of fresh kidney beans, which often cause poisoning incidents due to improper cooking. Kidney bean poisoning is multifactorial, and the most important factor is that the lectin in kidney bean can recognize and specifically combine with the specific glycosyl groups on the surface of red blood cells to form many crossed "bridges" on the surface of red blood cells, causing the red blood cells to agglutinate, which in turn causes The overall response of the body. The content of lectin in different bean varieties is not the same, so the hemagglutination method can be used to detect the lectin activity in the bean, which provides a basis for guiding people to choose the planting and use of the bean varieties with lower lectin activity.

Erythrocyte agglutination is currently the most widely used detection method for lectins. This method uses natural, enzyme-treated, glutaraldehyde-fixed red blood cells from animals such as rabbits, sheep, and humans to prepare a certain concentration of red blood cell suspension. After standing for a period of time under certain conditions, the activity is judged by visual observation and colorimetry.

Existing methods for measuring lectin activity are mainly divided into two categories: one is qualitative analysis for observing the hemagglutination effect, such as V-shaped microplate method, flat plate method, test tube method, etc.; the other is for spectrophotometric analysis. A semi-quantitative analytical method that takes into account the absorbance at a specific wavelength measured by a microplate reader. These methods have the following disadvantages: (1) only qualitative or semi-quantitative determinations can be made; (2) there are different methods for preparing standard red blood cell suspensions, which are mostly expressed by volume percentage, and the reproducibility is poor; (3) the randomness of determining the reading end time There is a large degree of variability, and some results are observed with the naked eye, which is highly subjective, resulting in large error and low accuracy; (4) There are various measurement methods, and the measured results are very different, so they cannot be compared.

Contents of the invention

The object of the present invention is to provide a method for high-throughput quantitative detection of coagulation activity of bean lectin in order to solve the above problems.

The technical scheme adopted in the present invention is:

A method for quantitative detection of coagulation activity of kidney bean lectin, said method comprising the following steps:

(1) Cell quantification of the red blood cell suspension used for detection;

(2) Use the known concentration of bean phytogelatin as a standard to draw a standard curve of coagulation activity concentration;

(3) Calculate the coagulation activity concentration of the bean lectin.

Further, the step (1) is specifically:

(A) Collect fresh rabbit blood and place it in 5%-10% sodium citrate anticoagulant, mix it with 4 times the volume of Alsever’s solution, and store it in a 4°C refrigerator for later use;

(B) Slowly mix the fresh rabbit blood obtained in step (A) with normal saline at a ratio of 5:8, centrifuge at 800-1000 rpm for 10 minutes, and collect the precipitate;

(C) Repeat the above steps to wash the erythrocytes until the color of the supernatant is almost transparent, and prepare a 20% erythrocyte suspension according to the volume of erythrocytes.

(D) Dilute the erythrocyte suspension obtained in step (C) with normal saline, count erythrocytes, adjust the concentration of erythrocytes to 5×10 ⁷ cells/ml-5×10 ⁸ cells/ml, and store in a 4°C refrigerator for later use.

Preferably, the fresh rabbit blood obtained in the step (A) shall not be stored for more than 14 days for future use.

Further, the step (2) is specifically:

(a) On a microtiter plate, double-dilute six dilutions of a known concentration of bean lectin standard;

(b) Add the quantified red blood cell suspension to each well of the above-mentioned microplate, mix well, and let it stand for 10 minutes, read the absorbance value at 450nm with a microplate reader, and draw the standard curve of the hemagglutinin activity concentration of kidney bean lectin.

Furthermore, in the step (a), the concentration of the common bean lectin standard is 1 mg/ml, and the six dilutions of double dilution are 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml ml, 0.0625mg/ml and 0.03125mg/ml.

Further, the step (3) is specifically:

(I) Wash and dry fresh kidney bean plants, weigh them, put them in a mortar, add physiological saline with a mass volume ratio of 1:1, and grind them on ice;

(II) centrifuge the bean plant obtained in step (I) at 4°C at 8000rpm for 10min, and take the supernatant;

(Ⅲ) After standing still the supernatant obtained in step (II) for 5 minutes, take the supernatant again to obtain the extract of kidney bean lectin;

(IV) Dilute the kidney bean lectin extract obtained in step (III) twice to three dilutions on the microplate;

(Ⅴ) Add the erythrocyte suspension obtained in step (1) to each well of the above microplate plate, mix well, and let it stand for 10 minutes, read the absorbance value at 450nm on a microplate reader, and substitute this value into step (2) to get In the formula of the standard curve of the coagulation activity concentration of the kidney bean lectin, the blood coagulation activity concentration of the kidney bean lectin was calculated.

Preferably, said bean plants are bean pods or grains.

Preferably, the microtiter plate is 96 wells.

The present invention has the following advantages:

The detection method has simple steps, and the method can be used for high-throughput quantitative detection of lectin coagulation activity on kidney bean plants; the prepared red blood cell suspension is subjected to cell quantification, which can ensure the relative consistency of the measurement results between batches; simultaneous detection The results are more accurate, the error is small, and the measured results can be compared with each other.

Description of drawings

Fig. 1 is a curve diagram of the determination of the standard bean lectin in the embodiment.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

(1) Preparation of standard red blood cell suspension and determination of standard curve:

1. Collect 2ml of fresh rabbit blood by heart blood collection, put it in 300ul of 8% sodium citrate anticoagulant, mix it with 8ml of Alsever's solution, and store it in a 4°C refrigerator for later use (the red blood cells are normal within 2 weeks, and the red blood cells are normal within 2 weeks. week, red blood cells appear hemolysis).

2. Take 2.5ml of fresh rabbit blood preserved in Alsever’s solution, slowly mix it with normal saline at a ratio of 5:8, centrifuge at 800-1000rpm for 10 minutes, and collect the red blood cell pellet.

3. After repeating the above steps to wash the red blood cells five times, the color of the supernatant is completely transparent, and then according to the volume of the red blood cell sedimentation, make a 20% red blood cell suspension.

4. Take 100ul of the above-mentioned erythrocyte suspension and dilute it 10 times with normal saline, take 100ul of the diluted erythrocyte suspension, and then dilute it 10 times, then take 15ul of the erythrocyte suspension diluted twice to count the number of erythrocytes, and adjust the concentration of erythrocytes to 5×10 ⁷ cells/ml-5×10 ⁸ cells/ml.

5. Add 100ul of 1mg/ml kidney bean lectin standard on the 96-well ELISA plate, and make a double dilution of 6 dilutions, drop different concentrations (5×10 ⁷ /ml, 1×10 ⁸ /ml, 2×10 ⁸ cells/ml, 4×10 ⁸ cells/ml) 10ul red blood cell suspension, observe the coagulation situation, and measure the absorbance at 450nm.

Referring to Figure 1, the results show that when the concentration of red blood cells is 2×10 ⁸ cells/ml, the blood coagulation reaction speed is moderate, and the linear correlation coefficient of the standard curve based on the absorbance value at 450nm is greater than 0.99.

(2) Preparation of phase bean lectin extract

1. Wash and dry fresh bean pods or beans, weigh them, put them in a mortar, add physiological saline with a mass volume ratio of 1:1, and grind them on ice.

2. Centrifuge at 8000r/min for 10min at 4°C, and take the supernatant.

3. After standing still for 5 minutes, take the supernatant again, which is the extract of kidney bean lectin.

(3) Determination of active concentration of bean lectins

1. On a 96-well microtiter plate, add 58 different varieties of kidney bean pod extracts

100ul.

2. Add 10ul of ² ×108/ml erythrocyte suspension to each well, mix well, let it stand for 10 minutes, read the absorbance value at 450nm on a microplate reader, and substitute this value into the formula of the standard curve for the kidney bean plant The formula for calculating the concentration of lectin coagulation activity is shown in Figure 1.

For the 15 kinds of common bean pod extracts with high content of lectins that exceed the range of the standard curve, dilute 3 concentration gradients twice with normal saline, 100ul per well, and then add 2× ¹⁰⁸ pcs/ml to each well 10ul of the red blood cell suspension, mixed evenly, let stand for 10 minutes, read the absorbance value at 450nm in a microplate reader, take the absorbance value within the standard curve and substitute it into the formula of the standard curve to calculate the hemagglutinin activity concentration of kidney bean lectin.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be Modifications or equivalent replacements of the technical solutions without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the scope of the claims of the present invention.

Claims (7)

1. a method for quantitative detection of coagulation activity of kidney bean lectin, is characterized in that, described method comprises the following steps: (1) Carry out cell quantification to the red blood cell suspension used for detection; (2) Use the common bean phytogelatin of known concentration as a standard to draw a standard curve of coagulation activity concentration; (3) Calculate the coagulation activity concentration of the bean lectin; Described step (1) is specifically: (A) Collect fresh rabbit blood and place it in 5%-10% sodium citrate anticoagulant, mix it with 4 times the volume of Arbor's solution, and store it in a refrigerator at 4°C for later use; (B) Slowly mix the fresh rabbit blood obtained in step (A) with normal saline at a ratio of 5:8, centrifuge at 800-1000 rpm for 10 minutes, and collect the precipitate; (C) Repeat the above steps to wash the erythrocytes until the color of the supernatant is almost transparent, and prepare a 20% erythrocyte suspension according to the volume of erythrocytes; (D) Dilute the erythrocyte suspension obtained in step (C) with normal saline, count erythrocytes, adjust the concentration of erythrocytes to 5×10 ⁷ cells/ml-5×10 ⁸ cells/ml, and store in a 4°C refrigerator for later use. 2. The method for quantitative detection of coagulation activity of kidney bean lectin according to claim 1, characterized in that the fresh rabbit blood obtained in the step (A) should not be stored for more than 14 days. 3. The method for quantitative detection of coagulation activity of kidney bean lectin according to claim 1, wherein said step (2) is specifically: (a) on the microplate, the kidney bean lectin standard substance of known concentration is double-diluted six dilutions; (b) Add quantitative erythrocyte suspension to each well of the above-mentioned microplate, mix well, and let stand for 10 minutes, read the absorbance value at 450nm in a microplate reader, and draw a standard curve of the hemagglutinin activity concentration of kidney bean lectin. 4. The method for quantitative detection of hemagglutination activity of kidney bean lectin according to claim 3, characterized in that, in the step (a), the concentration of the kidney bean lectin standard substance is 1 mg/ml, double dilution six dilutions The concentration is 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.125mg/ml, 0.0625mg/ml and 0.03125mg/ml. 5. The method for quantitative detection of coagulation activity of kidney bean lectin according to claim 1, wherein said step (3) is specifically: (I) Wash and dry the fresh kidney bean plants, weigh them, place them in a mortar, add physiological saline with a mass volume ratio of 1:1, and grind them on ice; (II) Centrifuge the kidney bean plant obtained in step (I) at 4° C. at 8000 rpm for 10 min, and take the supernatant; (Ⅲ) After the supernatant obtained in step (II) was left to stand for 5 minutes, the supernatant was taken again to obtain the kidney bean lectin extract; (Ⅳ) On the microplate, the phase (Ⅲ) obtained phase lectin extract solution is diluted twice to three dilutions; (V) Add the erythrocyte suspension obtained in step (1) to each well of the above-mentioned microplate plate, mix well, and let stand for 10 minutes, read the absorbance value at 450nm in a microplate reader, and substitute this value into step (2) to obtain In the formula of the standard curve of the coagulation activity concentration of the kidney bean lectin, the blood coagulation activity concentration of the kidney bean lectin was calculated. 6 . The method for quantitative detection of hemagglutination activity of bean lectins according to claim 5 , wherein the bean plants are bean pods or grains. 7 . 7. The method for quantitative detection of coagulation activity of bean lectin according to claim 3 or 5, characterized in that the enzyme plate is 96 holes.