CN1260492A - Tumor prognosis evaluation reagent kit and its preparation technology - Google Patents

Abstract

The prognosis estimation kit for tumor includes positive contrast (VEGF), normal saline, antigen-antibody label, seal glue, enzyme labelling plate buffer solution, monoclonal antibody or polyclonal antibody of VEGF, zymolyte solution, termination solution and washing solution. At the same time it also provides the preparation process of monoclonal antibody of VEGF and other buffer solution and reagent, can be used for simple and quick prognosis estimation of tumor.

Description

Tumor prognosis evaluation reagent kit and preparation technology thereof

The present invention relates to a kind of tumor prognosis evaluation reagent kit and preparation technology thereof.

The growth of normal structure medium vessels remains static, and only occurs adjustable, of short duration propagation in the organ of injured or cyclic regeneration; But in the tumor tissues, angiogenic growth is uncontrolled, continues to carry out.Tumour cell can produce a series of cell factors that promote the vascularization function that have under certain condition, as VEGF, and bFGF, ANG etc.In tumor vascular growth, play an important role.VEGF is by combine different its biologically active that is situated between with the vascular endothelial cell surface receptor, by plasminogen activation activator and fibrinolytic system, and degraded basilar memebrane composition, thus the stimulating endothelial cell migration, immerse, form tubular structure.

The generation of tumour, development, transfer must depend on the generation of new vessels, in tumor vascular forming process, the growth factors such as VEGF relevant with vascularization begins to express, after tumour is subjected to the chemotherapy and radiation effect, tumour cell is suppressed or damages together with the tumor vascular endothelial cell growth, and the vegf expression amount descends or do not express.The height of vegf expression amount is relevant with tumor size and growth conditions, and the knurl body is big, the tumor tissues vegf expression amount height fast of growing; And the knurl body is little, and the slow tumor tissues vegf expression amount of silk ribbon attached to an official seal or a medal of growing is low.And the knurl body is constant relatively, and it is relative constant to breed the tumor tissues VEGF suitable with apoptosis speed.Gross tumor volume, the speed of growth are subjected to the influence of factors such as patient's state of an illness, methods of treatment, result of treatment, and the active situation and the fluctuation of these genes of monitoring cancer patient change the variation tendency that can indicate the state of an illness, and be significant.This kit provides the dynamic observing of vascularization gene molecule level of tumour patient for the clinician, for result of treatment, the PD of tumour patient, prognosis changes that basis for estimation is provided.

The object of the present invention is to provide the preparation technology of VEGF monoclonal antibody and other buffering agent in a kind of tumor prognosis evaluation ELISA kit and the kit thereof, reagent, can be used for the prognosis evaluation of tumour fast simply.

The every box of this kit is made up of following material:

Bag is cushioned 1 bottle of liquid

1 bottle of positive control (VEGF)

1 bottle of negative control (physiological saline)

1 bottle of the monoclonal antibody of VEGF or polyclonal antibody

1 bottle of two anti-label

1 bottle of enzyme substrate solution

1 bottle of stop buffer

1 bottle of cleansing solution

1 of sealing compound

1 of ELISA Plate

Wherein, positive control (VEGF), negative control (physiological saline), two anti-labels, sealing compound, ELISA Plate are existing material, and bag is cushioned the Monclone antibody of liquid, VEGF or restrains gallery antibody, enzyme substrate solution, stop buffer, cleansing solution for being prepared from by technology of the present invention more.

Preparation technology of the present invention realizes by following steps:

1. MONOCLONAL ANTIBODIES SPECIFIC FOR

1.1 in mouse immune BAL B/C mouse 6-8 week, add Fu Shi with 50-100ug VEGF

The Freund's complete adjuvant lumbar injection is behind the At intervals of two to three weeks, with same dosage VEGF abdomen

Chamber booster immunization 2-3 time, 3-4 days extracting spleen cells merge after the last immunity.

1.2 antigen VEGF, research institute provides by the auspicious gloomy genetically engineered drug in Shaanxi.

1.3 Fusion of Cells, immune mouse spleen cell and Sp2/0 cell mix in 10: 1 ratio

Close, under the effect of 50%PEG (MW=1500), carry out Fusion of Cells, use ELISA

The indirect method preliminary screening goes out positive colony, again with in and inhibition test turn out to be sun

After the sex clone, clone 2-3 time, filter out the secretory antibody sun through limiting dilution assay

The property single clone, stable going down to posterity 3 months, preparation ascites and liquid nitrogen cryopreservation.

1.4 the evaluation of monoclonal antibody

1.4.1 hybridoma cell line chromosome analysis: the hybridoma in the growth period of taking the logarithm

Handle with colchicine, through Giemsa dyeing, the microscopy counting.

1.4.2Ig class and IgG subclass are measured: with the anti-mouse Ig Asia of U.S. Sigma company

Class antiserum and the Hybridoma Cell Culture supernatant that is concentrated into former volume 1/20

Liquid is made the two-way immunodiffusion of agar.

1.4.3 the specific mensuration of monoclonal antibody: VEGF is carried out electrotransfer, then

Carry out ELISA dyeing respectively with each strain monoclonal antibody.

1.4.4 the monoclonal antibody specific activity is measured: the albumen of each strain monoclonal antibody is carried out quantitatively,

Measure the titre of each strain then by different dilutabilitys.

2. polyclone and clone ELISA measure the VEGF in the urine sample

2.1 the compound method of various damping fluids and reagent

A. bag is cushioned liquid: the Na of 0.05M PH9.6 ₂CO ₃-NaHCO ₃

Na ₂CO ₃(MW 105.99) 1.59 grams

NaHCO ₃(MW 84.01) 2.93 grams

Distill water-soluble to 1000ml

B. the PBS-Tween 20 of cleansing solution: PH 7.4

NaCl (MW 58.44) 8.0 grams

KH ₂PO ₄(MW 136.09) 0.2 gram

Na ₂HPO ₄12 H ₂O (MW 358.14) 2.9 grams

(perhaps NaHPO ₄1.14 gram)

KCl (MW 74.56) 0.2 gram

Distill water-soluble to 1000ml

Tween?20?0.5ml

C. enzyme labeling thing dilution: add sheep blood serum in above-mentioned cleansing solution, concentration is 2%.

D. enzyme substrate solution: o-phenylenediamine (O.P.D)-H ₂O ₂

(1) phosphoric acid-citrate buffer solution PH5.0

①0.2M?Na ₂HPO ₄

Get Na ₂HPO ₄12 H ₂O 71.63 grams are water-soluble to 1000ml with distillation.

2. 0.1M citric acid solution

Get citric acid 19.2 grams, water-soluble with distillation to 1000ml, get 1. liquid

25.7ml, 2. liquid 24.3ml, adding distil water 50ml again.

(2)O.P.D-H ₂O ₂

O-phenylenediamine (O.P.D) 10mg

Phosphoric acid-citrate buffer solution 25ml

3%H ₂O ₂(analyzing pure) 0.4ml

E. stop buffer 2MH ₂SO ₄

The concentrated sulphuric acid (95-98%) 22.2ml

Distilled water 177.3ml

(timing slowly splashes into the concentrated sulphuric acid in the distilled water, and the limit edged shakes up.)

2.2 operation steps

Get monoclonal or polyclonal antibody, the parallel bag of normal mice IgG (all being diluted to 10mg/ml) is by 40 orifice plates (by monoclonal or polyclonal antibody, the B package is by normal mice IgG as the A package), 0.1ml/ wet 4 ℃ of refrigerator overnight of box are put in the hole, use cleansing solution wash plate 3 times, each 3 minutes, dry.Add tested antigen 0.1ml/ hole in the hole of parallel bag quilt, hatched 1 hour for 37 ℃, the same washing 3 times dries.Add monoclonal or polyclonal antibody (being diluted to 10mg/ml) again in each hole, hatched 1 hour for 37 ℃ in the 0.1ml/ hole, and the same washing 3 times dries, and adds enzyme target two anti-(being diluted to 1: 4000) in each hole, 0.1

Add enzyme substrate solution 0.1ml/ hole, 37 ℃ of lucifuges were hatched 20 minutes.At each

Add 2M H in the hole ₂SO ₄0.025ml/ the hole, cessation reaction is surveyed with the enzyme joint inspection

Instrument is measured the absorbance of the 490nm in each hole.

2.3 the result judges

The OD ratio calculation:

Embodiment: one, kit is formed:

96 person-portions, 48 person-portions, 20 person-portions

Bag is cushioned liquid bottle 10ml 5ml 2.5ml

Positive control (VEGF) bottle 1ml 0.5ml 0.25ml

Negative control (physiological saline) bottle 1ml 0.5ml 0.25ml

The monoclonal antibody of VEGF

Or polyclonal antibody bottle 10ml 5ml 5ml

Two anti-label bottle 10ml 5ml 5ml

Enzyme substrate solution bottle 6ml 3ml 3ml

Stop buffer bottle 6ml 3ml 3ml

Washing liquid bottle 25ml 12.5ml 6ml

Seal 10 10 1 of blob of viscose

ELISA Plate piece 96 orifice plates 48 orifice plates 24 orifice plates two, specimen collection

1. urina sanguinis, dehydration and a large amount of transfusion patient should not be adopted urine.

2. best fresh urine sample can be preserved 48 hours for 4 ℃, can preserve for 4 weeks for-20 ℃.

3. urine can not multigelation.

Three, reagent is stored

1.4 ℃ preservation, the term of validity are 4-6 month.

2. coating buffer must cover tightly, 4 ℃ of preservations, for a long time need not, should more renew coating buffer.Four, detect step

1. taking-up ELISA Plate, every hole add 2 of coating buffers (wherein 3 holes do not add, directly add sun,

Each 2 of negative controls, a hole gives over to blank).

2 add patient's urine 10ul, with the coating buffer mixing.Add a cover and seal film, put wet box, 4 ℃

Spend the night or 37 ℃ more than 3.5-4 hour.

3 then take out, and inhale and remove coating buffer, and every hole adds cleansing solution 200ul, washes 3 times, and is each

Add leave standstill 1 minute after the washing lotion after, get rid of only, pat dry surplus liquid.

4 add one anti-2,37 ℃ of wet boxes 60 minutes.

5 with step 3, washes 3 times.

6 add two anti-2,37 ℃ of wet boxes 60 minutes.

7 with step 3, washes 3 times.

8 every holes drip substrate A and B each 1 (blank is not dripped), and mixing is put wet box 37

℃ * 30 minutes (or room temperature 45 minutes) lucifuges.Then, every hole adds stop buffer 1

Drip, survey the OD value at microplate reader 490mm place.Five, judged result

The OD ratio calculation:

Six, clinical assessment reference

1 sample OD ratio to be checked 〉=2.1 show vascularization peptide gene table

Active.

2 sample OD ratios to be checked are suspicious between 1.5-2.0.

3 sample OD ratios to be checked are below 1.5, and the expression of expression vascularization peptide gene is in quiet

The phase of ending.

Claims (3)

1, tumor prognosis evaluation ELISA kit comprises positive control (VEGF), physiological saline, two anti-labels, sealing compound, ELISA Plate, it is characterized in that, wherein also comprise the monoclonal antibody or polyclonal antibody, enzyme substrate solution, stop buffer and the cleansing solution that are cushioned liquid, VEGF.

2, kit according to claim 1 is characterized in that, said bag is cushioned the Na that liquid is 0.05M, PH9.6 ₂CO ₃-NaHCO ₃, said enzyme substrate solution is o-phenylenediamine (O.P.D)-H ₂O ₂, said stop buffer 2MH ₂SO ₄, said cleansing solution is the PBS-Tween 20 of PH 7.4.

3, kit according to claim 1 is characterized in that, the monoclonal antibody of said VEGF adopts following method preparation:

(1) in mouse immune BAL B/C mouse 6-8 week, adds Fu Shi with 50-100ug VEGF

The Freund's complete adjuvant lumbar injection is behind the At intervals of two to three weeks, with same dosage VEGF abdomen

Chamber booster immunization 2-3 time, 3-4 days extracting spleen cells merge after the last immunity;

(2) Fusion of Cells, immune mouse spleen cell and Sp2/0 cell mix in 10: 1 ratio

This mixing under the effect of 50%PEG (MW=1500), is carried out Fusion of Cells,

Go out positive colony with ELISA indirect method preliminary screening, in using again and inhibition test

After turning out to be positive colony, clone 2-3 time, filter out branch through limiting dilution assay

Secrete the single clone of antibody positive, stable going down to posterity 3 months, preparation ascites and liquid

Nitrogen is frozen.