Preparation method of heavy metallic lead resistant monoclonal antibody

Abstract

The invention discloses a making method of heavy-metal lead monoclonal antibody in the biological technical domain, which is characterized by the following: coupling lead ion and carrier protein through p-aminobenzene-diethylenetriamine pentoacetic acid as bifunctional metal chelant; obtaining full antigen; crossing splenocyte and Sp2 / 0 marrow tumour cell to make crossed tumour cell; sieving; obtaining stable strong secretory specificity anti-lead monoclonal monomer to inhibit Pb-DTPA.

Description

(1) Technical field

[0001] The invention relates to the preparation of anti-heavy metal lead monoclonal antibody, which belongs to the field of biotechnology. It is specially used for the preparation of monoclonal antibodies that specifically recognize heavy metal lead, and its high-sensitivity and rapid detection of heavy metal residues in agricultural products and agricultural production environments. It is especially suitable for large-scale sample detection and on-site monitoring. (2) Background technology

[0002] Lead is a heavy metal with high toxicity in environmental pollutants. It is widely distributed in nature and has many industrial uses. With the rapid development of my country's industry and transportation, environmental lead pollution is becoming more and more serious. Because lead is a multi-affinity poison, it is easy to accumulate in certain organs of the human body, causing chronic poisoning and endangering human health. It mainly damages the nervous sys...

Examples

Embodiment 1

[0031] The detection of embodiment 1 lead standard solution

[0032] The indirect competitive ELISA method was used to conduct a preliminary exploration of the detection curve. Methods as below:

[0033] Coating: Dilute the coating antigen bovine serum albumin-p-aminophenyl-diethylenetriaminepentaacetic acid-Pb with 10mM, pH7.4 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer , 50 μl / well was added to a 96-well enzyme-linked microanalysis plate (Corning Co.), and placed at 4°C overnight or at 37°C for 2 hours;

[0034] Washing: wash 5 times with PBST;

[0035] Blocking: gelatin diluted to 1% in PBST, 100 μl / well, 37°C for 1.5 hours;

[0036] Washing: wash 5 times with PBST;

[0037] Sample addition: Dilute lead standard solution (Sigma Co.) stepwise with N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution containing 2mM diethylenetriaminepentaacetic acid (Sigma Co.) into 2, 0.2, 0.02, 0.002, 0.0002, 0.00002, 0.000002mM, the diluted lead solution wa...

Embodiment 2

[0044] Detect lead residues in the tap water sample of embodiment 2

[0045] Bovine serum albumin-p-aminophenyl-diethylenetriaminepentaacetic acid-Pb diluted to 0.5μg / ml coated microanalysis plate, the positive well supernatant was diluted 10 times as the working concentration, 1% gelatin was used as a block, antigen-antibody reaction matrix (HBS) containing 1mM DTPA with a pH value of 5.0 and an ionic strength of 0mol / L, 50μl per well in a microanalysis plate;

[0046] Sample preparation to be tested:

[0047] Tap water sample: Measure 400 μl of three tap water (laboratory), add 0.801 μl of 1000 μg / ml lead standard solution to the three water samples respectively, and prepare a 0.04 mM liquid sample;

[0048] The above samples were detected by the lead ELISA detection method, and the operation was as follows:

[0049] 1) Coating:

[0050] Add bovine serum albumin-p-aminophenyl-diethylenetriaminepentaacetic acid-Pb 0.95μg / ml to each well of the microanalysis plate, add 50μl...

(1) Technical field

[0001] The invention relates to the preparation of anti-heavy metal lead monoclonal antibody, which belongs to the field of biotechnology. It is specially used for the preparation of monoclonal antibodies that specifically recognize heavy metal lead, and its high-sensitivity and rapid detection of heavy metal residues in agricultural products and agricultural production environments. It is especially suitable for large-scale sample detection and on-site monitoring. (2) Background technology

[0002] Lead is a heavy metal with high toxicity in environmental pollutants. It is widely distributed in nature and has many industrial uses. With the rapid development of my country's industry and transportation, environmental lead pollution is becoming more and more serious. Because lead is a multi-affinity poison, it is easy to accumulate in certain organs of the human body, causing chronic poisoning and endangering human health. It mainly damages the nervous sys...

Examples

Embodiment 1

[0031] The detection of embodiment 1 lead standard solution

[0032] The indirect competitive ELISA method was used to conduct a preliminary exploration of the detection curve. Methods as below:

[0033] Coating: Dilute the coating antigen bovine serum albumin-p-aminophenyl-diethylenetriaminepentaacetic acid-Pb with 10mM, pH7.4 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer , 50 μl / well was added to a 96-well enzyme-linked microanalysis plate (Corning Co.), and placed at 4°C overnight or at 37°C for 2 hours;

[0034] Washing: wash 5 times with PBST;

[0035] Blocking: gelatin diluted to 1% in PBST, 100 μl / well, 37°C for 1.5 hours;

[0036] Washing: wash 5 times with PBST;

[0037] Sample addition: Dilute lead standard solution (Sigma Co.) stepwise with N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid buffer solution containing 2mM diethylenetriaminepentaacetic acid (Sigma Co.) into 2, 0.2, 0.02, 0.002, 0.0002, 0.00002, 0.000002mM, the diluted lead solution wa...

Embodiment 2

[0044] Detect lead residues in the tap water sample of embodiment 2

[0045] Bovine serum albumin-p-aminophenyl-diethylenetriaminepentaacetic acid-Pb diluted to 0.5μg / ml coated microanalysis plate, the positive well supernatant was diluted 10 times as the working concentration, 1% gelatin was used as a block, antigen-antibody reaction matrix (HBS) containing 1mM DTPA with a pH value of 5.0 and an ionic strength of 0mol / L, 50μl per well in a microanalysis plate;

[0046] Sample preparation to be tested:

[0047] Tap water sample: Measure 400 μl of three tap water (laboratory), add 0.801 μl of 1000 μg / ml lead standard solution to the three water samples respectively, and prepare a 0.04 mM liquid sample;

[0048] The above samples were detected by the lead ELISA detection method, and the operation was as follows:

[0049] 1) Coating:

[0050] Add bovine serum albumin-p-aminophenyl-diethylenetriaminepentaacetic acid-Pb 0.95μg / ml to each well of the microanalysis plate, add 50μl...