CN1215083C - Monoclonal antibody for resisting human liver cancer and its application - Google Patents

Abstract

The present invention provides a symbolic antigen specific monoclonal antibody MF4C4 for resisting human liver cancer, which has the characteristic of strong specificity for combining with a liver cancer antigen. The present invention provides MF4C4 immune globulin, a fragment thereof, an immune coupling substance and a medical composition containing the immune globulin, or the fragment or the immune coupling substance, and also provides a detecting reagent kit for detecting liver cancer.

Description

Human liver cancer monoclonal antibody and application

Technical field

The present invention relates to oncology and medical field.More specifically, the present invention relates to the monoclonal antibody specific and the application thereof of anti-human liver cancer.

Background technology

Primary hepatocarcinoma (HCC) is one of common malignancy, mortality ratio height, serious harm human beings'health and life.At present, with regard to world wide, its sickness rate is in rising trend, and as a kind of disease of high mortality, the cause of disease of liver cancer, generation, evolution, diagnosis, treatment all more and more cause people's attention.Wherein, the early diagnosis of liver cancer for the patient lapse to and prognosis all has crucial meaning.

At present, the mark of diagnosing cancer of liver is first elected AFP, and the serodiagnosis that is used for liver cancer has been continued to use for many years, is used for the gene diagnosis of liver cancer blood transfer recently again.But this method still has problems on specificity and susceptibility.With regard to specificity, for HCC patient, the factor of disturbing diagnostic accuracy mainly is because can detect AFP albumen and mRNA in patient's blood of part with liver cirrhosis or chronic hepatitis.Authority's statistics shows that the Serum AFP positive rate is 19.8% in acute icterohepatitisshock, and chronic persistent hepatitis is 19.9%, and chronic active hepatitis is 34.4%, and posthepatitic cirrhosis can reach 44.6%, and the serum concentration of AFP of above-mentioned non-cancer hepatopathy is many between 25-200ng/ml.The Northern hybridization and the immunohistochemical methods result of the other hepatic tissue of contrast liver cancer and cancer often find that the other hepatic tissue of cancer has the weak positive expression of AFP, therefore often has the proteic false positive results of Serum AFP.When these patients cause part of hepatocytes to come off into blood as inflammation, necrosis for a certain reason, also can cause the false positive results of AFP mRNA in the blood.One group of Matsmnura etc. studies show that in 15% liver cirrhosis patient and 12% chronic hepatitis patient can detect the AFP mRNA positive.In another group experiment, Jiang etc. then detect the 6 routine AFP mRNA positives in 11 routine chronic hepatitis patients, detect the 1 routine AFP mRNA positive among the 2 routine acute hepatitis patients.With regard to susceptibility, about 1/3 HCC patient, its Serum AFP feminine gender (＜20ng/ml), the positive rate of AFP is also lower in the HCC that breaks up (I level) and differentiation extreme difference (IV level), therefore can not detect effectively these tumour patients.This just illustrates that AFP exists suitable rate of missed diagnosis and misdiagnosis rate in diagnosing cancer of liver, so the early diagnostic rate that will improve liver cancer needs the higher and stronger new mark of specificity of susceptibility.

Because the present detection of high specificity and/or the effective ways of treatment liver cancer of still lacking, therefore, this area presses for exploitation specific anti-human liver cancer monoclonal antibody, and the effective ways that detect and treat liver cancer.

Summary of the invention

Purpose of the present invention provides a kind of specific anti-human liver cancer significant antigenic monoclonal antibody.

Another object of the present invention provides a kind of test kit of specific detection people's liver cancer.

Another object of the present invention provides a kind of therapeutical agent of specific treatment people liver cancer.

In a first aspect of the present invention, a kind of immunoglobulin (Ig) is provided, it is characterized in that it is incorporated into MXR7 albumen specifically.Preferably, described immunoglobulin (Ig) is incorporated into the MXR7 albumen of having removed the N end (for example, having lacked 50-100 amino acid of N end, preferably the MXR7 albumen behind 50-80 amino acid) specifically

More preferably, described immunoglobulin (Ig) is characterized in that, described MXR7 albumen has the aminoacid sequence shown in the SEQ ID NO:2, or has lacked 55 amino acid whose MXR7 albumen of N end.

In an example of the present invention, described immunoglobulin (Ig) is a monoclonal antibody.

In another example of the present invention, described immunoglobulin (Ig) is MF4C4 by mouse hybridoma cell, and CCTCC No.C200111 produces.

In a second aspect of the present invention, a kind of immune conjugate is provided, this immune conjugate contains the coupling part that is incorporated into the proteic immunoglobulin (Ig) of MXR7 specifically and is selected from down the group: medicine, toxin, cytokine, radionuclide, enzyme.

In a third aspect of the present invention, a kind of hybridoma that produces monoclonal antibody is provided, it is that mouse hybridoma cell is MF4C4, CCTCC No.:C200111.

In a fourth aspect of the present invention, a kind of test kit that detects liver cancer is provided, it contains above-mentioned immunoglobulin (Ig) or immune conjugate, or its active fragments.

In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains above-mentioned immunoglobulin (Ig) or above-mentioned immune conjugate, and pharmaceutically acceptable carrier.

Description of drawings

Fig. 1: use monoclonal antibody MF4C4, detect the proteic expression of people embryo liver cell MXR7 by the western blotting.

Fig. 2: use monoclonal antibody MF4C4, detect the expression of MXR7 in the liver cancer tissue by the immunohistochemical methods method.

Embodiment

The inventor after years of research and found that MXR7 gene and liver cancer have very close getting in touch, and can be used as the responsive marker gene of liver cancer-specific.Find on the basis at this, separate obtaining the MXR7 gene from placenta cdna library, express MXR7 albumen, and succeed in developing a kind of authentic monoclonal antibody, be i.e. anti-human liver cancer monoclonal antibody MF4C4.This antibody confirms good stability and liver cancer antigen bonded high specificity after deliberation.

MXR7 is that people such as Lage in 1996 are by differential hybridization method separating clone.The same period, the inventor obtains the MXR7 gene from the separation of liver cancer cDNA library when research liver cancer-specific sensitivity marker gene.This full length gene cDNA sequence is 2263bp, be positioned human chromosome Xq26, form by 8 exons, the heparitin sulfate proteoglycan that coding is made up of 580 amino acid, molecular weight 66kDa, (glycosyl-phosphatidylinositol, GPI) anchor is connected in cytolemma, belongs to proteoglycan family by glycosyl-phosphatidyl inositol.MXR7 gene great expression when the embryo is organized in mesoderm, synthon fetus liver, lungs and kidney, low expression level in placenta tissue, then do not express in other tissue, being considered to participate in the growth period form takes place and adjusting and controlling growth, in case form is finished, to transcribe to be to bear and regulate, the wean back just can not detect.

Studies show that the generation of MXR7 and liver cancer development has substantial connection, not only liver cancer higher recall rate arranged in early days, and along with progression of HCC, its recall rate also increases thereupon.The inventor detects has MXR7 to express in 76.7% (23/30) liver cancer tissue, wherein there is the weak positive expression of MXR7 in 13.3% (4/30) cancer beside organism, and MXR7 expresses negative patient in the other 7 routine liver cancer tissues, and the MXR7 of its cancer beside organism also expresses feminine gender.Importantly in Serum AFP male patient liver cancer, 88% express MXR7 mRNA, and can find still that in patient's liver cancer of Serum AFP feminine gender 73% is MXR7 mRNA and expresses positive.In diameter＜3cm liver cancer, MXR7 mRNA positive expression rate is 77%, is significantly higher than AFPmRNA expression rate 41% in serum afp rate of rise 43% and the liver cancer.MXR7 does not all detect the MXR7 expression in adenoma of liver, cholangiocellular carcinoma, metastatic liver cancer and 12 kinds of common solid tumors and 21 non-liver cancer clones, show that MXR7 is the gene of specifically expressing in the liver cancer, its specificity is apparently higher than the AFP that still is used at present the diagnosing cancer of liver mark, and more responsive than AFP, recall rate is higher, and higher recall rate is especially arranged in the liver cancer patient of AFP feminine gender.Therefore MXR7 is a new liver cancer marker, can work in coordination with the early diagnosis that is used for liver cancer with the AFP complementation.

On the other hand, because the expression level of MXR7 in the malignant progression of liver cancer increase gradually, and may become the important target gene of the prognosis or anti-hepatoma Metastasis and the recurrence that are used to judge liver cancer.

The MXR7 gene is one of two genes relevant with X chromosome of finding in liver cancer at present.Liver cancer has fairly obvious familial aggregation and genetic predisposition, and male sex's sickness rate is 10 times of women approximately, and epidemiology survey has proved that mother suffers from liver cancer the hereditation of filial generation is suffered from the influence of liver cancer to filial generation much larger than father.The whether key-gene of the liver cancer susceptibility heredity research of still needing of MXR7 gene.

The relation of MXR7 and liver cancer is very close, and the molecule mechanism of its effect is still unclear, remains further to be studied.The MXR7 gene is at tire liver high level expression, and the adult closes, and recovers during liver cancer, and is very similar to the spatial and temporal distributions of AFP genetic expression, but MXR7 mRNA expression ratio AFP is more responsive, and positive rate is higher.This gene is connected in cell surface with the GPI anchor, is the coreceptor (co-receptor) of IGF-2.Therefore can come off into blood the same with other member of this family can detect in liver cancer patient blood, with this as the serodiagnostic specificity marker thing of early hepatocarcinoma.

On above-mentioned research basis, the inventor utilizes hybridoma technology to prepare the monoclonal antibody MF4C4 of anti-people MXR7.

MF4C4 monoclonal antibody of the present invention or its fragment can be used for the localized diagnosing image of radioactivity of liver cancer, also can be used for immunotherapy, thereby for example make its tumour cell that can directly lead by directly or indirectly MF4C4 monoclonal antibody or its fragment being linked to each other with chemotherapeutic or radiotherapeutic agents.

The present invention includes: have the corresponding aminoacid sequence of MF4C4 monoclonal antibody monoclonal antibody, have the monoclonal antibody of MF4C4 monoclonal antibody variable region chain, and other protein or protein conjugate and fusion expressed product with these chains.Particularly, the present invention includes and have the hypervariable region of containing (complementary determining region, any protein of light chain CDR) and heavy chain or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product), as long as identical or at least 90% homology of hypervariable region of this hypervariable region and light chain of the present invention and heavy chain, preferably at least 95% homology.As is known to the person skilled in the art, immune conjugate and fusion expressed product comprise: medicine, toxin, cytokine (cytokine), radionuclide, enzyme and other diagnosis or treatment molecule and MF4C4 monoclonal antibody or its fragment bonded and the conjugate that forms.The present invention also comprises and MF4C4 monoclonal antibody or its fragment bonded cell surface marker thing or antigen.

As used herein, " immunotoxin " refers to target cell is had the material of specificity avidity and lethality, and for example immune conjugate and fusion expressed product comprise: medicine, toxin, cytokine (cytokine), radionuclide or other treatment molecule and MF4C4 monoclonal antibody or its fragment bonded and the conjugate that forms.Concrete example for example has ¹³¹I-MF4C4, MTX-MF4C4 conjugate etc.

For MF4C4 monoclonal antibody heavy chain of the present invention and sequence of light chain, can measure with ordinary method.The hypervariable region of MF4C4 monoclonal antibody V chain or complementary determining region (complementarity determining region, CDR) interesting especially, because relate to conjugated antigen to small part in them.Therefore, the present invention includes those the light chain immunoglobulins and the molecule of weight chain variable chain, as long as its CDR and MF4C4 monoclonal antibody CDR have the homology of (preferably more than 95%) more than 90% with band CDR.

The present invention not only comprises complete monoclonal antibody, also comprises having immunocompetent antibody fragment, as Fab or (Fab ') 2 fragments; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody, as have the murine antibody binding specificity but keep antibody from people's antibody moiety.

The present invention also provides coding liver cancer significant antigenic cDNA.The antigenic Nucleotide full length sequence of MF4C4 monoclonal antibody of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be according to the significant antigenic nucleotide sequence of liver cancer, especially open reading frame sequence designs primer, prepares template by ordinary method well known by persons skilled in the art, obtains relevant sequence by amplification.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.

The present invention also provides above-mentioned immunoglobulin (Ig) or its segmental dna molecular.The sequence of these dna moleculars can as above be used routine techniques, and utilizing mouse hybridoma cell is that MF4C4 (CCTCC No.:C200111) obtains.In addition, also the encoding sequence of light chain and heavy chain can be merged, form single-chain antibody.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.

The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used to transform appropriate host cell, so that it can marking protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces: the bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS7,293 cells or Bowes melanoma cells etc.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgGl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, handle the combination of (salt analysis method), centrifugal, the broken bacterium of infiltration, supersound process, super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods with protein precipitant.

In addition, the present invention also provides a kind of test kit that detects liver cancer, and it contains above-mentioned immunoglobulin (Ig) or immune conjugate, or its active fragments.A kind of people's diagnosing cancer of liver test kit has been finished clinical experiment 193 examples, detects positive rate up to 74%.

The present invention also provides a kind of pharmaceutical composition, and it contains above-mentioned immunoglobulin (Ig) or immune conjugate, and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intraperitoneal, intravenously or topical.

Pharmaceutical composition of the present invention can be directly used in the treatment of liver cancer.In addition, also can use the other treatment agent simultaneously, as IFN-α, IFN-β, TNF-α etc.

Pharmaceutical composition of the present invention contains above-mentioned immunoglobulin (Ig) of the present invention of safe and effective amount or above-mentioned immune conjugate and pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.

When making pharmaceutical composition, be that MF4C4 immune conjugate with safe and effective amount is applied to Mammals, wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.

The present invention not only provides the hybridoma cell line that produces the high anti-people MXR7 monoclonal antibody of tiring, and also provides monoclonal antibody to detect the proteic content of MXR7 in the clinical patient serum.According to literature search, the present international and domestic report that does not all have identical work.Monoclonal antibody of the present invention can successfully be applied to the serodiagnosis (clinical being suitable for) of primary hepatocyte hepatocarcinoma, and is particularly extremely meaningful with the prognosis of judging hepatocarcinoma patient to the differential diagnosis of α-FP negative HCC.Antibody of the present invention can also be applied to immunotherapy of hepatocellular carcinoma, improves the complex therapy curative effect of liver cancer.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The acquisition of people MXR7 cDNA encoding sequence

Press the sequence of MXR7 gene, the synthetic primer of reading the frame upstream and downstream that lays respectively at separates the encoding sequence (SEQ ID NO:1) that obtains following MXR7 gene from the placenta cdna library amplification.

1?cagcacgtct?cttgctcctc?agggccactg?ccaggcttgc?cgagtcctgg?gactgctctc

61?gctccggctg?ccactctccc?gcgctctcct?agctccctgc?gaagcaggat?ggccgggacc

121?gtgcgcaccg?cgtgcttggt?ggtggcgatg?ctgctcagct?tggacttccc?gggacaggcg

181?cagcccccgc?cgccgccgcc?ggacgccacc?tgtcaccaag?tccgctcctt?cttccagaga

241?ctgcagcccg?gactcaagtg?ggtgccagaa?actcccgtgc?caggatcaga?tttgcaagta

301?tgtctcccta?agggcccaac?atgctgctca?agaaagatgg?aagaaaaata?ccaactaaca

361?gcacgattga?acatggaaca?gctgcttcag?tctgcaagta?tggagctcaa?gttcttaatt

421?attcagaatg?ctgcggtttt?ccaagaggcc?tttgaaattg?ttgttcgcca?tgccaagaac

481?tacaccaatg?ccatgttcaa?gaacaactac?ccaagcctga?ctccacaagc?ttttgagttt

541?gtgggtgaat?ttttcacaga?tgtgtctctc?tacatcttgg?gttctgacat?caatgtagat

601?gacatggtca?atgaattgtt?tgacagcctg?tttccagtca?tctataccca?gctaatgaac

661?ccaggcctgc?ctgattcagc?cttggacatc?aatgagtgcc?tccgaggagc?aagacgtgac

721?ctgaaagtat?ttgggaattt?ccccaagctt?attatgaccc?aggtttccaa?gtcactgcaa

781?gtcactagga?tcttccttca?ggctctgaat?cttggaattg?aagtgatcaa?cacaactgat

841?cacctgaagt?tcagtaagga?ctgtggccga?atgctcacca?gaatgtggta?ctgctcttac

901?tgccagggac?tggtgatggt?taaaccctgt?ggcggttact?gcaatgtggt?catgcaaggc

961?tgtatggcag?gtgtggtgga?gattgacaag?tactggagag?aatacattct?gtcccttgaa

1021?gaacttgtga?atggcatgta?cagaatctat?gacatggaga?acgtactgct?tggtctcttt

1081?tcaacaatcc?atgattctat?ccagtatgtc?cagaagaatg?caggaaagct?gaccaccact

1141?attggcaagt?tatgtgccca?ttctcaacaa?cgccaatata?gatctgctta?ttatcctgaa

1201?gatctcttta?ttgacaagaa?agtattaaaa?gttgctcatg?tagaacatga?agaaacctta

1261?tccagccgaa?gaagggaact?aattcagaag?ttgaagtctt?tcatcagctt?ctatagtgct

1321?ttgcctggct?acatctgcag?ccatagccct?gtggcggaaa?acgacaccct?ttgctggaat

1381?ggacaagaac?tcgtggagag?atacagccaa?aaggcagcaa?ggaatggaat?gaaaaaccag

1441?ttcaatctcc?atgagctgaa?aatgaagggc?cctgagccag?tggtcagtca?aattattgac

1501?aaactgaagc?acattaacca?gctcctgaga?accatgtcta?tgcccaaagg?tagagttctg

1561?gataaaaacc?tggatgagga?agggtttgaa?agtggagact?gcggtgatga?tgaagatgag

1621?tgcattggag?gctctggtga?tggaatgata?aaagtgaaga?atcagctccg?cttccttgca

1681?gaactggcct?atgatctgga?tgtggatgat?gcgcctggaa?acagtcagca?ggcaactccg

1741?aaggacaacg?agataagcac?ctttcacaac?ctcgggaacg?ttcattcccc?gctgaagctt

1801?ctcaccagca?tggccatctc?ggtggtgtgc?ttcttcttcc?tggtgcactg?act

(SEQ?ID?NO：1)

Total length is 1853bp, wherein ORF is positioned at the 109-1848 position, the heparitin sulfate proteoglycan that coding is made up of 580 amino acid, its aminoacid sequence is shown in SEQ ID NO:2, molecular weight 66 kDa, (glycosyl-phosphatidylinositol, GPI) anchor is connected in cytolemma, belongs to proteoglycan family by glycosyl-phosphatidyl inositol.

MAGTVRTACL?VVAMLLSLDF?PGQAQPPPPP?PDATCHQVRS?FFQRLQPGLK 50

MVPETPVPGS?DLQVCLPKGP?TCCSRKMEEK?YQLTARLNME?QLLQSASMEL 100

KFLIIQNAAV?FQEAFEIVVR?HAKNYTNAMF?KNNYPSLTPQ?AFEFVGEFFT 150

DVSLYILGSD?INVDDMVNEL?FDSLFPVIYT?QLMNPGLPDS?ALDINECLRG 200

ARRDLKVFGN?FPKLIMTQVS?KSLQVTRIFL?QALNLGIEVI?NTTDHLKFSK 250

DCGRMLTRMW?YCSYCQGLYM?VKPCGGYCNY?VMQGCMAGVV?EIDKYWREyI 300

LSLEELVNGM?YRIYDMENYL?LGLFSTIHDS?IQYVQKNAGK?LTTTIGKLCA 350

HSQQRQYRSA?YYPEDLFIDK?KVLKVAHVEH?EETLSSRRRE?LIQKLKSFIS 400

FYSALPGYIC?SHSPVAENDT?LCWNGQELVE?RYSQKAARNG?MKNQFNLHEL 450

KMKGPEPVVS?QIIDKLKHIN?QLLRTMSMPK?GRVLDKNLDE?EGFESGDCGD 500

DEDECIGGSG?DGMIKVKNQL?RFLAELAYDL?DVDDAPGNSQ?QATPKDNEIS 550

TFHNLGNVHS?PLKLLTSMAI?SVVCFFFLVH 580

(SEQ?ID?NO：2)

Wherein, compare with the MXR7 gene of announcing, (amino acid is by M → V) to have the change of a → g at 913 (please determining the position according to new sequence).

Embodiment 2

The expression of people MXR7 protein fragments

The inventor has carried out repeatedly attempting to the expression of MXR7, but finds to remove its N end effective expression and purifying MXR7 albumen.

In this embodiment, with following PCR primer:

Primer 1:5 '-cggaattctgccaggatcagat-3 ' (SEQ ID NO:3)

Primer 2: 5 '-cgctcgagtcagtgcaccag-3 ' (SEQ ID NO:4)

By the PCR method, from people's placenta cDNA storehouse (CLONTECH, Human Placenta cDNA) human cloning MXR7 cDNA (523aa in, disappearance N end 57aa), be connected to 6 * His fusion expression vector pPROEXHT (GIBCO BRL, 10711-018), behind the IPTG abduction delivering, with Ni-NTA agarose affinity chromatography method purifying MXR7 albumen.

Embodiment 3

The preparation of MF4C4 monoclonal antibody and purifying

(1) preparation of hybridoma

MXR7 protein immunization BALB/c mouse with purifying among the embodiment 2: during initial immunity Freund's complete adjuvant is mixed with antigen 1 00 μ l (50-100 μ g) equal-volume, fully emulsified, abdominal injection.After 3 weeks mouse is carried out booster immunization, the antigen amount reduces by half, and uses Freund's incomplete adjuvant instead, and volume injected and method are constant.Treat that mice serum tires to reach and prepare after the requirement to carry out cytogamy that merging first three day impacts immunity (50 μ g antigen) to mouse.

In immune mouse, prepare murine myeloma cell P3X63Ag8.653 (ATCC CRL-1580).

The bone-marrow-derived lymphocyte and the myeloma cell of sensitization are merged, carry out selectivity with the HAT substratum and cultivate (is nurse cell with the Turnover of Mouse Peritoneal Macrophages).

Then, detect the Hybridoma Cell Culture supernatant with the ELISA method: with MXR7 albumen (the 2 μ g/ml) coated elisa plate of purifying, every hole 100 μ l, spend the night by 4 ℃.After the room temperature sealing, add supernatant 100 μ l to be checked, hatch 1h for 37 ℃.Wash 3 times, add ELIAS secondary antibody (anti-mouse IgG-HRP), hatch 45min for 37 ℃.Get rid of ELIAS secondary antibody, wash 3 times, add substrate developer 50 μ l, room temperature left standstill 5 minutes, added stop buffer 50 μ l.The result measures wavelength 450nm value with microplate reader, the OD value be higher than negative control more than 2 times the person can be considered the positive.

Then, (limiting dilution assay is a nurse cell with the Turnover of Mouse Peritoneal Macrophages) cultivated in the positive hybridoma cell cloning of selecting.Cultivate through the cloning of 2-3 wheel, obtain stable can produce the tire hybridoma cell clone of monoclonal antibody of height.With the hybridoma cell clone enlarged culturing, and frozen guarantor's kind.

A kind of positive hybridoma cell behaviour liver cancer hybridoma cell line MF4C4, this hybridoma lies in and is preserved in Chinese typical culture collection center (CCTCC September 5 calendar year 2001, China, the Wuhan City), preserving number is Chinese typical culture collection center (CCTCC, China, the Wuhan City) No.C200111.

(2) preparation of MF4C4 monoclonal antibody and purifying

Above-mentioned hybridoma MF4C4 is seeded to mouse peritoneal, and preparation ascites is extracted monoclonal antibody again from ascites.

The purifying of monoclonal antibody MF4C4: adopt protein A affinity chromatography.At first prepare protein A affinity column, behind PBS balance pillar, get the ascites or the Hybridoma Cell Culture supernatant that contain anti-MXR7 monoclonal antibody and cross post, wash pillar with PBS then, approach zero to the OD value, with glycine-HCl solution (pH2.3) wash-out of 50mmol/L, collect elutriant, measure the OD value of each collection tube, keep the elutriant of peak region, elutriant is used to prepare enzyme labelled antibody after concentrating through dialysing.

Embodiment 4

The evaluation of MF4C4 monoclonal antibody

With the MF4C4 monoclonal antibody of preparation among the embodiment 3, detect the proteic expression of MXR7 in people's embryo liver cell by conventional Western blot hybridization (Western blot) experiment.The result shows that monoclonal antibody combines (Fig. 1) with a protein band specificity of 66KD position.

Use the MF4C4 monoclonal antibody, according to a conventional method the hepatocellular carcinoma tissue slice is carried out immunohistochemical staining.The result as shown in Figure 4, wherein Fig. 4 A-4D is respectively different amplification, the result show in tumour coating district progressivity cancer nests in the endochylema painted obviously.Illustrate that anti-MXR7 monoclonal antibody has specificity and susceptibility preferably.

Embodiment 5

With the MF4C4 monoclonal antibody of preparation among the embodiment 3, detect the proteic content of MXR7 in hepatocarcinoma patient (100 example), non-liver cancer liver and gall diseases patient (43 example) and normal people's (50 example) serum by the indirect competitive ELISA method.The result shows that MXR7 albumen high expression level in the 74% hepatocarcinoma patient serum only have 18.60%MXR7 albumen positive among the non-liver cancer liver and gall diseases patient, and protein expression level is low than hepatocarcinoma patient, and the proteic content of MXR7 is very low in the normal human serum.This this anti-MXR7 monoclonal antibody of explanation has specificity and susceptibility preferably.

Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉king, Red Male

＜120〉human liver cancer monoclonal antibody and application

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<222>(109)..(1848)

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cagcacgtct?cttgctcctc?agggccactg?ccaggcttgc?cgagtcctgg?gactgctctc 60

gctccggctg?ccactctccc?gcgctctcct?agctccctgc?gaagcagg?atg?gcc?ggg 117

Met?Ala?Gly

1

acc?gtg?cgc?acc?gcg?tgc?ttg?gtg?gtg?gcg?atg?ctg?ctc?agc?ttg?gac 165

Thr?Val?Arg?Thr?Ala?Cys?Leu?Val?Val?Ala?Met?Leu?Leu?Ser?Leu?Asp

5 10 15

ttc?ccg?gga?cag?gcg?cag?ccc?ccg?ccg?ccg?ccg?ccg?gac?gcc?acc?tgt 213

Phe?Pro?Gly?Gln?Ala?Gln?Pro?Pro?Pro?Pro?Pro?Pro?Asp?Ala?Thr?Cys

20 25 30 35

cac?caa?gtc?cgc?tcc?ttc?ttc?cag?aga?ctg?cag?ccc?gga?ctc?aag?tgg 261

His?Gln?Val?Arg?Ser?Phe?Phe?Gln?Arg?Leu?Gln?Pro?Gly?Leu?Lys?Trp

40 45 50

gtg?cca?gaa?act?ccc?gtg?cca?gga?tca?gat?ttg?caa?gta?tgt?ctc?cct 309

Val?Pro?Glu?Thr?Pro?Val?Pro?Gly?Ser?Asp?Leu?Gln?Val?Cys?Leu?Pro

55 60 65

aag?ggc?cca?aca?tgc?tgc?tca?aga?aag?atg?gaa?gaa?aaa?tac?caa?cta 357

Lys?Gly?Pro?Thr?Cys?Cys?Ser?Arg?Lys?Met?Glu?Glu?Lys?Tyr?Gln?Leu

70 75 80

aca?gca?cga?ttg?aac?atg?gaa?cag?ctg?ctt?cag?tct?gca?agt?atg?gag 405

Thr?Ala?Arg?Leu?Asn?Met?Glu?Gln?Leu?Leu?Gln?Ser?Ala?Ser?Met?Glu

85 90 95

ctc?aag?ttc?tta?att?att?cag?aat?gct?gcg?gtt?ttc?caa?gag?gcc?ttt 453

Leu?Lys?Phe?Leu?Ile?Ile?Gln?Asn?Ala?Ala?Val?Phe?Gln?Glu?Ala?Phe

100 105 110 115

gaa?att?gtt?gtt?cgc?cat?gcc?aag?aac?tac?acc?aat?gcc?atg?ttc?aag 501

Glu?Ile?Val?Val?Arg?His?Ala?Lys?Asn?Tyr?Thr?Asn?Ala?Met?Phe?Lys

120 125 130

aac?aac?tac?cca?agc?ctg?act?cca?caa?gct?ttt?gag?ttt?gtg?ggt?gaa 549

Asn?Asn?Tyr?Pro?Ser?Leu?Thr?Pro?Gln?Ala?Phe?Glu?Phe?Val?Gly?Glu

135 140 145

ttt?gtc?aca?gat?gtg?tct?ctc?tac?atc?ttg?ggt?tct?gac?atc?aat?gta 597

Phe?Phe?Thr?Asp?Val?Ser?Leu?Tyr?Ile?Leu?Gly?Ser?Asp?Ile?Asn?Val

150 155 160

gat?gac?atg?gtc?aat?gaa?ttg?ttt?gac?agc?ctg?ttt?cca?gtc?atc?tat 645

Asp?Asp?Met?Val?Asn?Glu?Leu?Phe?Asp?Ser?Leu?Phe?Pro?Val?Ile?Tyr

165 170 175

acc?cag?cta?atg?aac?cca?ggc?ctg?cct?gat?tca?gcc?ttg?gac?atc?aat 693

Thr?Gln?Leu?Met?Asn?Pro?Gly?Leu?Pro?Asp?Ser?Ala?Leu?Asp?Ile?Asn

180 185 190 195

gag?tgc?ctc?cga?gga?gca?aga?cgt?gac?ctg?aaa?gta?ttt?ggg?aat?ttc 741

Glu?Cys?Leu?Arg?Gly?Ala?Arg?Arg?Asp?Leu?Lys?Val?Phe?Gly?Asn?Phe

200 205 210

ccc?aag?ctt?att?atg?acc?cag?gtt?tcc?aag?tca?ctg?caa?gtc?act?agg 789

Pro?Lys?Leu?Ile?Met?Thr?Gln?Val?Ser?Lys?Ser?Leu?Gln?Val?Thr?Arg

215 220 225

atc?ttc?ctt?cag?gct?ctg?aat?ctt?gga?att?gaa?gtg?atc?aac?aca?act 837

Ile?Phe?Leu?Gln?Ala?Leu?Asn?Leu?Gly?Ile?Glu?Val?Ile?Asn?Thr?Thr

230 235 240

gat?cac?ctg?aag?ttc?agt?aag?gac?tgt?ggc?cga?atg?ctc?acc?aga?atg 885

Asp?His?Leu?Lys?Phe?Ser?Lys?Asp?Cys?Gly?Arg?Met?Leu?Thr?Arg?Met

245 250 255

tgg?tac?tgc?tct?tac?tgc?cag?gga?ctg?gtg?atg?gtt?aaa?ccc?tgt?ggc 933

Trp?Tyr?Cys?Ser?Tyr?Cys?Gln?Gly?Leu?Val?Met?Val?Lys?Pro?Cys?Gly

260 265 270 275

ggt?tac?tgc?aat?gtg?gtc?atg?caa?ggc?tgt?atg?gca?ggt?gtg?gtg?gag 981

Gly?Tyr?Cys?Asn?Val?Val?Met?Gln?Gly?Cys?Met?Ala?Gly?Val?Val?Glu

280 285 290

att?gac?aag?tac?tgg?aga?gaa?tac?att?ctg?tcc?ctt?gaa?gaa?ctt?gtg 1029

Ile?Asp?Lys?Tyr?Trp?Arg?Glu?Tyr?Ile?Leu?Ser?Leu?Glu?Glu?Leu?Val

295 300 305

aat?ggc?atg?tac?aga?atc?tat?gac?atg?gag?aac?gta?ctg?ctt?ggt?ctc 1077

Asn?Gly?Met?Tyr?Ara?Ile?Tyr?Asp?Met?Glu?Asn?Val?Leu?Leu?Gly?Leu

310 315 320

ttt?tca?aca?atc?cat?gat?tct?atc?cag?tat?gtc?cag?aag?aat?gca?gga 1125

Phe?Ser?Thr?Ile?His?Asp?Ser?Ile?Gln?Tyr?Val?Gln?Lys?Asn?Ala?Gly

325 330 335

aag?ctg?acc?acc?act?att?ggc?aag?tta?tgt?gcc?cat?tct?caa?caa?cgc 1173

Lys?Leu?Thr?Thr?Thr?Ile?Gly?Lys?Leu?Cys?Ala?His?Ser?Gln?Gln?Arg

340 345 350 355

caa?tat?aga?tct?gct?tat?tat?cct?gaa?gat?ctc?ttt?att?gac?aag?aaa 1221

Gln?Tyr?Arg?Ser?Ala?Tyr?Tyr?Pro?Glu?Asp?Leu?Phe?Ile?Asp?Lys?Lys

360 365 370

gta?tta?aaa?gtt?gct?cat?gta?gaa?cat?gaa?gaa?acc?tta?tcc?agc?cga 1269

Val?Leu?Lys?Val?Ala?His?Val?Glu?His?Glu?Glu?Thr?Leu?Ser?Ser?Arg

375 380 385

aga?agg?gaa?cta?att?cag?aag?ttg?aag?tct?ttc?atc?agc?ttc?tat?agt 1317

Arg?Arg?Glu?Leu?Ile?Gln?Lys?Leu?Lys?Ser?Phe?Ile?Ser?Phe?Tyr?Ser

390 395 400

gct?ttg?cct?ggc?tac?atc?tgc?agc?cat?agc?cct?gtg?gcg?gaa?aac?gac 1365

Ala?Leu?Pro?Gly?Tyr?Ile?Cys?Ser?His?Ser?Pro?Val?Ala?Glu?Asn?Asp

405 410 415

acc?ctt?tgc?tgg?aat?gga?caa?gaa?ctc?gtg?gag?aga?tac?agc?caa?aag 1413

Thr?Leu?Cys?Trp?Asn?Gly?Gln?Glu?Leu?Val?Glu?Arg?Tyr?Ser?Gln?Lys

420 425 430 435

gca?gca?agg?aat?gga?atg?aaa?aac?cag?ttc?aat?ctc?cat?gag?ctg?aaa 1461

Ala?Ala?Arg?Asn?Gly?Met?Lys?Asn?Gln?Phe?Asn?Leu?His?Glu?Leu?Lys

440 445 450

atg?aag?ggc?cct?gag?cca?gtg?gtc?agt?caa?att?att?gac?aaa?ctg?aag 1509

Met?Lys?Gly?Pro?Glu?Pro?Val?Val?Ser?Gln?Ile?Ile?Asp?Lys?Leu?Lys

455 460 465

cac?att?aac?cag?ctc?ctg?aga?acc?atg?tct?atg?ccc?aaa?ggt?aga?gtt 1557

His?Ile?Asn?Gln?Leu?Leu?Arg?Thr?Met?Ser?Met?Pro?Lys?Gly?Arg?Val

470 475 480

ctg?gat?aaa?aac?ctg?gat?gag?gaa?ggg?ttt?gaa?agt?gga?gac?tgc?ggt 1605

Leu?Asp?Lys?Asn?Leu?Asp?Glu?Glu?Gly?Phe?Glu?Ser?Gly?Asp?Cys?Gly

485 490 495

gat?gat?gaa?gat?gag?tgc?att?gga?ggc?tct?ggt?gat?gga?atg?ata?aaa 1653

Asp?Asp?Glu?Asp?Glu?Cys?Ile?Gly?Gly?Ser?Gly?Asp?Gly?Met?Ile?Lys

500 505 510 515

gtg?aag?aat?cag?ctc?cgc?ttc?ctt?gca?gaa?ctg?gcc?tat?gat?ctg?gat 1701

Val?Lys?Asn?Gln?Leu?Arg?Phe?Leu?Ala?Glu?Leu?Ala?Tyr?Asp?Leu?Asp

520 525 530

gtg?gat?gat?gcg?cct?gga?aac?agt?cag?cag?gca?act?ccg?aag?gac?aac 1749

Val?Asp?Asp?Ala?Pro?Gly?Asn?Ser?Gln?Gln?Ala?Thr?Pro?Lys?Asp?Asn

535 540 545

gag?ata?agc?acc?ttt?cac?aac?ctc?ggg?aac?gtt?cat?tcc?ccg?ctg?aag 1797

Glu?Ile?Ser?Thr?Phe?His?Asn?Leu?Gly?Asn?Val?His?Ser?Pro?Leu?Lys

550 555 560

ctt?ctc?acc?agc?atg?gcc?atc?tcg?gtg?gtg?tgc?ttc?ttc?ttc?ctg?gtg 1845

Leu?Leu?Tnr?Ser?Met?Ala?Ile?Ser?Val?Val?Cys?Phe?Phe?Phe?Leu?Val

565 570 575

cac?tgact 1853

His

580

<210>?2

<211>?580

<212>?PRT

<213>?Homo?sapiens

<400>?2

Met?Ala?Gly?Thr?Val?Arg?Thr?Ala?Cys?Leu?Val?Val?Ala?Met?Leu?Leu

1 5 10 15

Ser?Leu?Asp?Phe?Pro?Gly?Gln?Ala?Gln?Pro?Pro?Pro?Pro?Pro?Pro?Asp

20 25 30

Ala?Thr?Cys?His?Gln?Val?Arg?Ser?Phe?Phe?Gln?Arg?Leu?Glu?Pro?Gly

35 40 45

Leu?Lys?Trp?Val?Pro?Glu?Thr?Pro?Val?Pro?Gly?Ser?Asp?Leu?Gln?Val

50 55 60

Cys?Leu?Pro?Lys?Gly?Pro?Thr?Cys?Cys?Ser?Arg?Lys?Met?Glu?Glu?Lys

65 70 75 80

Tyr?Gln?Leu?Thr?Ala?Arg?Leu?Asn?Met?Glu?Gln?Leu?Leu?Gln?Ser?Ala

86 90 95

Ser?Met?Glu?Leu?Lys?Phe?Leu?Ile?Ile?Gln?Asn?Ala?Ala?Val?Phe?Gln

100 105 110

Glu?Ala?Phe?Glu?Ile?Val?Val?Arg?His?Ala?Lys?Asn?Tyr?Thr?Asn?Ala

115 120 125

Met?Phe?Lys?Asn?Asn?Tyr?Pro?Ser?Leu?Thr?Pro?Gln?Ala?Phe?Glu?Phe

130 135 140

Val?Gly?Glu?Phe?Phe?Thr?Asp?Val?Ser?Leu?Tyr?Ile?Leu?Gly?Ser?Asp

145 150 155 160

Ile?Asn?Val?Asp?Asp?Met?Val?Asn?Glu?Leu?Phe?Asp?Ser?Leu?Phe?Pro

165 170 175

Val?Ile?Tyr?Thr?Gln?Leu?Met?Asn?Pro?Gly?Leu?Pro?Asp?Ser?Ala?Leu

180 185 190

Asp?Ile?Asn?Glu?Cys?Leu?Arg?Gly?Ala?Arg?Arg?Asp?Leu?Lys?Val?Phe

195 200 205

Gly?Asn?Phe?Pro?Lys?Leu?Ile?Met?Thr?Gln?Val?Ser?Lys?Ser?Leu?Gln

210 215 220

Val?Thr?Arg?Ile?Phe?Leu?Gln?Ala?Leu?Asn?Leu?Gly?Ile?Glu?Vel?Ile

225 230 235 240

Asn?Thr?Thr?Asp?His?Leu?Lys?Phe?Ser?Lys?Asp?Cys?Gly?Arg?Met?Leu

245 250 255

Thr?Arg?Met?Trp?Tyr?Cys?Ser?Tyr?Cys?Gln?Gly?Leu?Val?Met?Val?Lys

260 265 270

Pro?Cys?Gly?Gly?Tyr?Cys?Asn?Val?Val?Met?Gln?Gly?Cys?Met?Ala?Gly

275 280 285

Val?Val?Glu?Ile?Asp?Lys?Tyr?Trp?Arg?Glu?Tyr?Ile?Leu?Ser?Leu?Glu

290 295 300

Glu?Leu?Val?Asn?Gly?Met?Tyr?Arg?Ile?Tyr?Asp?Met?Glu?Asn?Val?Leu

305 310 315 320

Leu?Gly?Leu?Phe?Ser?Thr?Ile?His?Asp?Ser?Ile?Gln?Tyr?Val?Gln?Lys

325 330 335

Asn?Ala?Gly?Lys?Leu?Thr?Thr?Thr?Ile?Gly?Lys?Leu?Cys?Ala?His?Ser

340 345 350

Gln?Gln?Arg?Gln?Tyr?Arg?Ser?Ala?Tyr?Tyr?Pro?Glu?Asp?Leu?Phe?Ile

355 360 365

Asp?Lys?Lys?Val?Leu?Lys?Val?Ala?His?Val?Glu?His?Glu?Glu?Thr?Leu

370 375 380

Ser?Ser?Arg?Arg?Arg?Glu?Leu?Ile?Gln?Lys?Leu?Lys?Ser?Phe?Ile?Ser

385 390 395 400

Phe?Tyr?Ser?Ala?Leu?Pro?Gly?Tyr?Ile?Cys?Ser?His?Ser?Pro?Val?Ala

405 410 415

Glu?Asn?Asp?Thr?Leu?Cys?Trp?Asn?Gly?Gln?Glu?Leu?Val?Glu?Arg?Tyr

420 425 430

Ser?Gln?Lys?Ala?Ala?Arg?Asn?Gly?Met?Lys?Asn?Gln?Phe?Asn?Leu?His

435 440 445

Glu?Leu?Lys?Met?Lys?Gly?Pro?Glu?Pro?Val?Val?Ser?Gln?Ile?Ile?Asp

450 455 460

Lys?Leu?Lys?His?Ile?Asn?Gln?Leu?Leu?Arg?Thr?Met?Ser?Met?Pro?Lys

465 470 475 480

Gly?Arg?Val?Leu?Asp?Lys?Asn?Leu?Asp?Glu?Glu?Gly?Phe?Glu?Ser?Gly

485 490 495

Asp?Cys?Gly?Asp?Asp?Glu?Asp?Glu?Cys?Ile?Gly?Gly?Ser?Gly?Asp?Gly

500 505 510

Met?Ile?Lys?Val?Lys?Asn?Gln?Leu?Arg?Phe?Leu?Ala?Glu?Leu?Ala?Tyr

515 520 525

Asp?Leu?Asp?Val?Asp?Asp?Ala?Pro?Gly?Asn?Ser?Gln?Gln?Ala?Thr?Pro

530 535 540

Lys?Asp?Asn?Glu?Ile?Ser?Thr?Phe?His?Asn?Leu?Gly?Asn?Val?His?Ser

545 550 555 560

Pro?Leu?Lys?Leu?Leu?Thr?Ser?Met?Ala?Ile?Ser?Val?Val?Cys?Phe?Phe

565 570 575

Phe?Leu?Val?His

580

<210>3

<211>22

<212>DNA

＜213〉primer

<400>3

cggaattctg?ccaggatcag?at 22

<210>4

<211>20

<212>DNA

＜213〉primer

<400>4

cgctcgagtc?agtgcaccag 20

Claims (7)

1. an immunoglobulin (Ig) is characterized in that, it is incorporated into MXR7 albumen specifically, and is MF4C4 by mouse hybridoma cell, and CCTCC No.C200111 produces.

2. immunoglobulin (Ig) as claimed in claim 1 is characterized in that, described MXR7 albumen has the aminoacid sequence shown in the SEQ ID NO:2,57 amino acid of perhaps described MXR7 protein delation N end.

3. immunoglobulin (Ig) as claimed in claim 1 is characterized in that it is a monoclonal antibody.

4. an immune conjugate is characterized in that, this immune conjugate contains the coupling part that is incorporated into the described immunoglobulin (Ig) of the proteic claim 1 of MXR7 specifically and is selected from down the group: medicine, toxin, cytokine, radionuclide, enzyme.

5. a hybridoma that produces monoclonal antibody is characterized in that, it is that mouse hybridoma cell is MF4C4, CCTCC No.C200111.

6. a test kit that detects liver cancer is characterized in that, it contains described immunoglobulin (Ig) of claim 1 or the described immune conjugate of claim 4.

7. a pharmaceutical composition is characterized in that, it contains described immunoglobulin (Ig) of claim 1 or the described immune conjugate of claim 4, and pharmaceutically acceptable carrier.

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