CN1188235A - Anti-human IV type collagen enzyme linked immunological quantitative determining kit and preparing method - Google Patents

Abstract

The present invention relates to an antihuman IV type collagen protease linked immunoquantitative assay kit and its preparation method. It consists of enzyme scale plate and testing reagent, and uses human placenta to extract IV collagen, and adopts cell engineering--hybridoma technology to prepare monoclonal antibody, and uses enzyme linked immunosorbent principle to coat the monoclonal antibody on enzyme scale plate, and adopts double antibody sandwich method to quantitatively determine IV type collagen content in human serum. Said invention can be used for diagnosis and treatment of fibrosis of liver and judgement after disease, and possesses good sensitivity, specificity and reproducibility and accuracy, so that it can meet requirement for clinical examination.

Description

Anti-human IV type collagen enzyme linked immunological quantitative determining kit and method for making

The present invention relates to the anti-human IV type collagen enzyme linked immunological quantitative determining kit, be used for a variety of causes and cause the diagnosis of diseases such as liver fibrosis, diabetes and silicosis and the monitoring of the course of disease thereof.

The invention still further relates to the method for making and the assay method of this kit.

Liver fibrosis is the liver chronic injury that is caused by multiple reason, composition abnormal deposition between the liver cell expolasm, the syndrome that causes liver function damage, along with going deep into of liver fibrosis research, constantly disclosed collagen structure, function and pathological internal relation, especially more deep to the research of IV collagen type (english abbreviation IV-CL), more valuable in diagnosis, therefore detect of the diagnosis of the content of the IV collagen type in the blood to liver fibrosis, result of treatment and more after judgement significant, at present in treatment and diagnosis and course of disease monitoring, most ways that adopt livers to pierce through, patient's misery not only, and in therapeutic process, can not regularly carry out, therefore, damaged patient's physique, also bother very much simultaneously, the kit that a kind of import is arranged, it promptly is coated on anti-human IV type collagen aminoterminal 7S monoclonal antibody on the small bead, adopts the operation of double-antibody sandwich two step method again, this kit costs an arm and a leg, troublesome poeration.

Purpose of the present invention adopts a kind of double-antibody sandwich single stage method on the anti-human IV type collagen monoclonal antibody coated elisa plate to measure the anti-human IV type collagen enzyme linked immunological quantitative determining kit of IV collagen type content in the human serum for the shortcoming that overcomes above-mentioned prior art with not enough just, thereby can judge and monitor the course of disease rapidly, accurately.

The present invention also provides the method for making and the method for quantitatively determining of this kit.

The objective of the invention is to realize by following technical proposal:

Anti-human IV type collagen enzyme linked immunological is measured kit, it is characterized in that it by the ELISA Plate of anti-human IV type collagen protein monoclonal antibody bag quilt with measure reagent and forms, and mensuration reagent is:

The standard antigen dried frozen aquatic products:

The enzyme labelled antibody bond,

The dilution phosphate buffer,

Washing lotion tween phosphate buffer,

Colour developing liquid A tetramethyl benzidine, the B hydrogen peroxide,

Stop buffer 2M sulfuric acid solution.

The method for making of kit of the present invention comprises the preparation of standard antigen, MONOCLONAL ANTIBODIES SPECIFIC FOR, the making of the ELISA Plate of monoclonal antibody bag quilt, the preparation of enzyme labelled antibody bond and the preparation of reagent.

Standard antigen extracts preparation from people's placenta, it cleans back slurrying with placenta, extracts for several times with neutral salt, through pepsin digestion, sodium chloride precipitation, and cross ion-exchange chromatography CM-24 fibre columns, adopt sodium chloride linear gradient elution IV Collagen Type VI promptly to get standard antigen.

The making of standard song when these product are used to measure, can with the standard antigen dried frozen aquatic products with phosphate buffer dilution variable concentrations be 1000,500,250,125,62,31,16,0ng/ml, eight points are as the horizontal ordinate of curve, with A450OD mean value is ordinate, uses the logarithmic paper curve plotting.

The making of the ELISA Plate of the monoclonal antibody bag quilt of kit of the present invention is that the dilution of monoclonal anti body and function phosphate buffer is added in the ELISA Plate hole, every hole can add 100 μ l, absorption is spent the night, wash plate with the tween phosphate buffer, sealed 1 hour 37 ℃ of temperature with ox, horse serum confining liquid again, dry, dry up, be about to monoclonal antibody and be coated on the ELISA Plate.

Monoclonal Antibody is that antigen that the personnel selection placenta extracts the IV collagen type carries out getting mouse boosting cell behind the immune BaCB/ mouse and the myeloma cell is merged cultivation, through screening and cloning, select the strain of specific anti-human IV Collagen Type VI monoclonal cell to inject mouse peritoneal and prepare ascites, obtain monoclonal ascites, obtain monoclonal antibody with the caprylic acid deposition and purification again.

It is that horseradish peroxidase is dissolved in the acetate buffer solution for the preparation of enzyme labelled antibody bond, adds NaIO ₄Solution, ethylene glycol and purifying anti-human IV type collagen spend the night, and add NaHB ₄Solution promptly obtains the enzyme labelled antibody bond with ammonium sulfate precipitation, centrifugal, desalination, adds equal-volume glycerine and preserves, and selects suitable dilutability during use.

Washing lotion is the tween phosphate buffer, is used to wash plate, can preserve 6 months at 2-8 ℃.

Colour developing liquid is used for colour developing, preserves 6 months at 2-8 ℃.

Stop buffer 2M sulfuric acid solution was preserved 6 months at 2-8 ℃.

IV collagen type monoclonal antibody can be aminoterminal 7S, carboxyl NC ₁, the triple helical center, IV Collagen Type VI whole antibody wherein any one.

The assay method of kit is characterized in that it is undertaken by following step successively:

A) on ELISA Plate, add dilution in the hole,

B) add the human serum sample again, in 37 ℃ of water-baths, left standstill 10-20 minute,

C) add the enzyme labelled antibody bond again, in 37 ℃ of water-baths, left standstill 60 minutes;

D) wash plate with washing lotion and dry, dry up;

E) add colour developing liquid A, B successively, in 37 ℃ of water-baths, left standstill 5-10 minute;

F) add stop buffer, finish reaction;

G) survey OD450 value, result of calculation.

Animal used as test BACB/C mouse body weight 12-14g of the present invention is provided by Beijing Experimental Animal Center, and ELISA Plate is import and homemade two kinds, and microplate reader is BIO-RAD.

The clinical practice result:

1, kit clinical practice result:

Detect clinical samples 436 examples, normal healthy controls 162 examples wherein, hepatitis 36 examples, diabetic's 92 examples, liver cancer patient 77 examples, cirrhosis 69 examples the results are shown in Table (1):

Table 1 kit clinical practice result: group example number average value standard deviation surpasses the positive common practice of the upper limit and counts * and percent thereof

X SD X ± SD normal healthy controls 162 44.96 59.4 10 6.2% diabetes groups 92 225.9 49 53% hepatitis groups 36 237.9 20 55.5% liver cirrhosis group 69 527.88 54 78.3% liver cancer groups 77 325..6 55 71.4%

The result shows that each patient all is significantly higher than normal healthy controls group (P＜0.01), and liver cirrhosis group is significantly higher than other group (P＜0.01) in each patient's group.

2,18 examples are made a definite diagnosis AML patients serum IV Collagen Type VI measurement result through pathology and are seen Table 2

Table 2 patients with alcoholic liver disease hepatocirrhosis measurement result group number of cases average surpasses ULN example number and slight AML 3 69.4 0 0% alcoholic hepatitis 2 1050.7 2 100% of percentage (merging IIBV infects) alcoholic fibrosis II 5 148.6 1 20% alcoholic fibrosis III 4 143.6 2 50% alcoholic fibrosis IV 4 176.4 4 100% thereof

As can be seen from Table 2, II level fiberization has 20% patient to be higher than normally in 13 routine patients with liver fibrosis, has 50% to be higher than normally among the III level fiberization patient, and 100% is higher than normally among the IV level fiberization patient; The alcoholic hepatitis patients serum IV-CL concentration that merges the HBV infection in 2 examples also all is higher than normally, and the result shows that serum I V-CL level and degree of hepatic fibrosis are proportionate, and closely related with necrosis of liver cells and cell infiltration degree.

3,126 routine hepatopath IV Collagen Type VI testing results see Table:

(1), 126 routine hepatopath's cause of disease check results see Table (3)

Table 3 hepatopathy cause of disease check result

The example number hepatitis B third liver second+third viral hepatitis type E virus hepatitis, 110 88 16 51 alcoholic hepatitis, 12 viral+alcohol hepatopathys 4211

Check result shows virus hepatitis 110 examples, alcoholic hepatitis 12 examples, and 4 examples are Combination hepatitis.

(2) the .IV Collagen Type VI is to the diagnostic value of liver fibrosis more than 2 grades: to above-mentioned 126 just hepatopath's liver puncture tissue specimen carry out the Electronic Speculum pathologic finding.Adopt multiple serum I V-CL to measure to patients with liver fibrosis serum specimen more than 2 grades, determination data is handled its dividing value of back, susceptibility, specificity and accuracy the results list (4):

Table 4 hepatocirrhosis is to the diagnostic value index dividing value sensitiveness % specificity % accuracy %PCIII 600ng/ml 81.6 81.8 81.7HA-Ch 80ng/ml 81.3 77.8 80.2HA 20ng/ml 76.9 78.6 77.5TIMP 240ng/ml 69.2 81.9 73.4PIIIP 0.9u/ml 71.4 75.0 72.6IV-Ch 150ng/ml 71.4 72.7 71.8LN 2.4u/ml 70.1 73.9 71.3IV-C 120ng/ml 65.2 65.8 65.3PH 20ng/ml 70.0 55.6 65.3 of liver fibrosis more than 2 grades

The result points out that kit is 71.4% to the diagnostic sensitivity of liver fibrosis more than 2 grades in the table 4, and specificity is 72.7%, and accuracy is 71.8%.

5.IV Collagen Type VI the results are shown in Table 5 to the diagnostic value of liver fibrosis more than 3 grades:

Table 5 hepatocirrhosis is to the diagnostic value PCIII 700ng/ml 60.0 65.2 63.1HA-Ch 220ng/ml 78.3 80.0 79.3HA 40ng/ml 77.8 80.6 79.5TIMP 280ng/ml 68.1 70.0 72.2PIIIP 1.0u/ml 73.9 68.7 70.8IV-Ch 180ng/ml 74.2 61.7 66.7LN 2.6u/ml 60.0 64.0 62.4IV-C 200ng/ml 71.1 87.3 80.8PH 35ng/ml 75.0 60.0 66.0 of liver fibrosis more than 3 grades

The result points out that kit is 74.2% to the diagnostic sensitivity of liver fibrosis more than 3 grades in the table 5, and specificity is 61.7%, and accuracy is 66.7%.

Compared with the prior art the technology of the present invention has following advantage and effect:

A) this kit can be used for clinical detection liver, pulmonary fibrosis kidney trouble and diabetes, especially the diagnostic and therapeutic effect of liver fibrosis and judgement is after being ill had important practical significance;

B) have higher sensitivity, specificity is good, and repeatability reaches the clinical detection requirement;

C) use method of operating, quick and precisely, low price easily popularizes;

Further specify the technology of the present invention below in conjunction with embodiment:

The anti-human IV type collagen albumen monoclonal antibody bag of kit by plate with rolling over bar shaped 96 hole ELISA Plate, with monoclonal anti soma bag by on each bottom surface, hole, one in every box, mark 2 bottles of former dried frozen aquatic productses, every bottle of 20ml/ bottle of sample diluting liquid, 1 bottle of enzyme antibody bond, one bottle of bond dilution 12ml, one bottle of washing lotion, colour developing liquid A, two bottles of B, every bottle of 6ml, 1 bottle of stop buffer, every bottle of 6ml, during mensuration, make to add in 8 holes of typical curve the standard antigen dried frozen aquatic products with diluted on ELISA Plate, concentration is 1000 successively, 500,250,125,63,31,16,0ng/ml, every hole adds 50 μ l, put in the water-bath, placed 10 minutes, every hole adds the enzyme labelled antibody bond 100 μ l with 50 times of bond diluted again, in water-bath, placed 1 hour, washing lotion in the bottle for handling liquid toilet or cosmetic substance is added 20 times of distillation dilutions, wash plate, add colour developing liquid A again in each hole, each 50 μ l of B placed in water-bath 10 minutes, add stop buffer 50 μ l, cessation reaction is surveyed each hole and is surveyed OD450 value, curve plotting with microplate reader, on same plate, measure the IV Collagen Type VI in the human blood, get human blood, system serum, increase serum sample diluting liquid 50 μ l in the hole earlier, increase serum 50 μ l again, in water-bath, placed 10 minutes for 37 ℃, add above-mentioned dilution enzyme labelled antibody bond 100 μ l, continue in water-bath 37 ℃ and placed 60 minutes, wash plate and drying with washing lotion, dry up, add obvious liquid A successively, B, 37 ℃ of placements added stop buffer in 10 minutes in water-bath, cessation reaction is surveyed A450OD value, result of calculation with microplate reader.

The working range 16-1000ng/l of kit typical curve, sensitivity is 16ng/l.

Claims (4)

1, anti-human IV type collagen enzyme linked immunological is measured kit, it is characterized in that it by the ELISA Plate of anti-human IV type collagen protein monoclonal antibody bag quilt with measure reagent and forms, and mensuration reagent is:

The standard antigen dried frozen aquatic products:

The enzyme labelled antibody bond,

The dilution phosphate buffer,

Washing lotion tween phosphate buffer,

Colour developing liquid A tetramethyl benzidine, the B hydrogen peroxide,

Stop buffer 2M sulfuric acid solution.

2, kit method for making as claimed in claim 1 comprises the preparation of standard antigen, the making of the ELISA Plate of monoclonal antibody bag quilt, and the preparation of enzyme labelled antibody bond and the preparation of reagent is characterized in that:

A) MONOCLONAL ANTIBODIES SPECIFIC FOR, be that the IV collagen type antigen that people's placenta extracts is carried out immune BACB/C mouse, the splenocyte and the myeloma cell that get immune mouse are again merged, cultivate, screening and cloning, select the strain of specific anti-human IV Collagen Type VI monoclonal cell to inject mouse peritoneal and prepare ascites, obtain monoclonal antibody ascites and obtain monoclonal antibody with the caprylic acid purifying;

B) making of the ELISA Plate of monoclonal antibody bag quilt splashes in each hole of ELISA Plate the absorption of spending the night after monoclonal anti body and function carbonate buffer solution released, wash plate with washing lotion, seal 37 ℃ of temperature with ox, horse serum confining liquid again, dry, dry up, promptly make the ELISA Plate of monoclonal antibody bag quilt.

3, method for making according to claim 2 is characterized in that IV collagen type monoclonal antibody can be aminoterminal 7S, carboxyl NC ₁, the triple helical center, IV Collagen Type VI whole antibody wherein any one.

4, the assay method of kit as claimed in claim 1 is characterized in that it is undertaken by following step successively:

A) on ELISA Plate, add dilution in the hole,

B) add the human serum sample again, in 37 ℃ of water-baths, left standstill 10-20 minute,

C) add the enzyme labelled antibody bond again, in 37 ℃ of water-baths, left standstill 60 minutes;

D) wash plate with washing lotion and dry, dry up;

E) add colour developing liquid A, B successively, in 37 ℃ of water-baths, left standstill 5-10 minute;

F) add stop buffer, finish reaction;

G) survey OD450 value, result of calculation.