CA1287801C - Method for determining human collagen peptides by way of enzyme immunoassay - Google Patents

Abstract

S P E C I F I C A T I O N

Title:

A METHOD FOR DETERMINING HUMAN COLLAGEN PEPTIDES BY WAY
OF ENZYME IMMUNOASSAY

ABSTRACT OF THE DISCLOSURE

A method for determining human type III, IV and VI
collagen peptides by way of enzyme immunoassay according to the sandwich technique using a monoclonal antibody and a polyclonal antibody each to the human type III, IV or VI collagen peptide, characterized in that the monoclonal antibody to the collagen peptide is used as an antibody to be coated onto a solid phase and/or an antibody to be labeled with an enzyme.
This method can be carried out with a smaller amount of samples in a simple manner to obtain a precise result and is thus useful for diagnosis of hepatic diseases.

Description

~87~1 . i j, BACKGROUND OF THE INVENTION
¦l 1. Field of the Invention:
!~ This invention relates to a method for determining human !I type III, IV and VI collagen peptides, which is useful for easy 5 1 diagnosis of hepatic diseases. More particularly, this ~, invention relates to a method for quantitative determination of the human type III, IV and VI collagen peptides by way of en- ~
zyme immunoassay based on the so-called sandwich method (techniquei) 1~ wherein specific monoclonal and polyclonal antibodies to the 10 l' human type III, IV and VI collagen peptides are used.
Il 2. Description of the Prior Art:
¦~ A radioimmunoassay for the measurement of the human type ¦l III procollagen N-terminal peptide in human blood using a Il polyclonal antibody was already reported, for example, in 15 ¦~ Rohde et al., Eur. J. Clin. Invest. 9, 451-459, (1979).
¦ However, a method for the determination of the human type III, IV and VI collagens themselves in blood has not yet been known, chiefly for the reason that the structure of Il collagens is not so significantly different among animal species 20 1~ as to make it easy to produce an antibody for assay ~from Il other animals and that the solubility of collagens in blood is ¦ too poor to measure the concentration of collagens in blood.
¦l If human collagens per se in blood can be measured exactly, ¦l it will greatly contribute to convenient and easy diagnosis of 25 1~ hepatic diseases. Consequently, there is a great demand for ¦ developing a new method for effectively and precisely determin-ing human collagen peptides in a simple manner, especially in the field of diagnosis of hepatic diseases.

¦ BRIEF SUMMARY OF THE INVENTION

.
., - ~ . . .
:~ -.
.

~1 2~37801 Accordingly, it is an object of the present invention to provide a new method for determining human type III, IV and VI
collagen peptides in a simple manner.
It is another object of the present invention to provide a method for the enzyme immunoassay of human type III, IV and VI collagen peptides applicable to diagnosis of hepatic diseases.
It is still another object of the present invention to provide a method for effectively and precisely determining human type III, IV and VI collagen peptides by way of enzyme immunoassay based on the sandwich technique with a specific monoclonal antibody to these collagen peptides.
Other object, features and advantages of the present invention will become apparent as the description proceeds.
Detailed Description of the Invention As a result of extensive researches made by the present inventors for developing a new simple method for determining the human type III, IV and VI collagen peptides in a specific manner, it has now been found that a precise and rapid determination of the human type III, IV and VI collagen peptides can be carried out with a small amount of samples by way of enzyme immunoassay according to the sandwich technique using a specific monoclonal antibody to each collagen peptide.
In accordance with the present invention, there is provided a method for determining human type III, IV or VI
collagen peptides by way of enzyme sandwich immunoassay which comprises: (a) contacting a serum sample suspected of containing human type III, IV or VI collagen peptides with a ~.

.
. . ,. : .

- - . - ~

128~7801 first monoclonal antibody specific for either of human type III, IV or VI collagen peptides, said monoclonal antibody being bound to a solid phase under conditions which allow formation of a reaction product between said first monoclonal antibody and said corresponding collagen peptide; (b) con1:acting said first reaction product with a second monoclonal antibody specific for either of said human type III, IV or VI
collagen peptides and which is conjugated with an enzyme under conditions which allow formation of a second reaction product between said second monoclonal antibody and said first reaction product, said conjugated monoclonal antibody being different from said monoclonal antibody used in step (a); (c) adding a substrate for said enzyme; and (d) assaying for reaction products of said substrate.

lS The specific monoclonal antibody employed in the method of this inven~ion is an anti-human collagen peptide monoclonal antibody of IgG, IgA and IgM classes, which is obtained by immunizing an animal such as a mouse with human collagen peptides to form a hybridoma from anti-human collagen peptide monoclonal antibody-producing cells of the animal and myeloma cells, cloning the hybridoma and thereafter selecting and cultivating clones capable of producing an anti-human collagen peptide monoclonal antibody having reactivity with any of the human type III, IV and VI collagen peptides. This monoclonal antibody can be purified, if necessary, according to a conventional purifying method by fractionation with a sulfate such as ammonium sulfate followed by column chromatography with DEAE-Sephacel (Pharmacia Fine Chemicals) equilibrated ; ~ ' ~`

, . ~ .
' ~
-lZ~7801 with a buffer of a particular pH value.
The polyclonal antibody employed in the method of this invention is obtained by immunizing an animal such as a rabbit with the human collagen peptides, taking blood from the immunized animal and purifying the resultant anti-serum.
Employed as the antibody to be labeled with an enzyme, especially with a peroxidase in the method of this invention is an IgG fraction obtainable by the fractionation of a material containing antibodies with ammonium sulfate or sodium sulfate and the subsequent purification on a DEAE-cellulose such as DEAE-Sep~acel* column. In case of the polyclonal antibody, it is preferred to carry out further purification on a Sepharose* 4B affinity column because this would enhance * Trade Mark 4a - .: . . .
,: -~Z8~7801 ¦ the specificity. It is also possible to use F(ab')2 obtainable 1', by digestion with pepsin or its reduced product Fab'. Thus, i~, i 1 the present invention includes such an embodi~ent wherein the I monoclonal and polyclonal antibodies used in the method of 5 !~ this invention may be their specific binding sites F(ab')2 or Fab' as such.
~ The solid phase to be coated with the monoclonal or !~ polyclonal antibody should normally be inert to all the ~i substances used for the antigen-antibody reaction including 10 !' a liquid vehicle and is selected from a wide variety of in-t organic and organic inert carrier materials such as glass, ceramics and resinous materials in the form of a plate or sphere. Such solid phase should be homogeneous in quality , and identical in size, or otherwise, the ~uantity of the 15 ll monoclonal or polyclonal antibody coated on the individual ', i solid phase fluctuates, thus resulting in an inaccurate result of measurements. Because of easiness in processing, organic resinous materials such as polystyrene, polyvinyl l~ resin, polyamide resin in the form of a plate or sphere is 20 1I preferable, with polystyrene balls and polyvinyl chloride ;:¦! plates being most preferable. A preferable example of the ~ enzymes utilizable for the labeling of the monoclonal antibody ., ¦' is a peroxidase such as one derived from horse raddish.
Various kinds of buffer solutions can be used for the 25 ~i immunoassay of the present invention to provide a definite pH
value desirable in the system. A buffer substance used for this purpose is selected from various known compounds havinq a buffering action, according to the conditions required in the 'i system. Preferable examples of the buffer substances include 30 ~¦ phosphates, tris-HCl, acetates and amino acids. These .,., ~,.. . . - ......... - . .
- ; ..

.
, , - - . ~. .

Il ~2~37801 substances are used with an acid or sodium chloride at a concentration desired in the system. The operations for the I immunization, chromatography, equilibration, fractionation ¦! and spectrophotometry in the method of this invention can be 5 il, carried out according to the methods known per se for these ¦l purposes.
¦l Accordingly, the method of the present invention is j featured by a solid phase enzyme immunoassay which is unique ¦ in using the monoclonal antibodies to the human type III, IV
10 ~ and VI collagen peptides as an antibody to be coated onto a ¦ solid phase or to be labeled with an enzyme.
The following description refers to Figures 2,~3 and 4. For the sake of convenience all of the drawings will ! be introduced briefly as follows:
15 ~ Fig. 1 shows a standard curve of each human collagen peptide based on the sandwich technique using the purified mouse anti-human collagen peptide monoclonal antibody (IgG type) and the purlfied rabbit anti-human collagen peptide polyclonal antibody. In Fig. 1, the open circles ( -o- ) plot the curve for the human type III collagen, the closed circles ( --- ) for the human type IV collagen, and the solid triangles ( ~~~ ?
, for the human type VI collagen.
Fig. 2 shows a serum level of immunoactive human type III
collagen peptide in normal subjects and in patients with chronic active hepatitis and liver cirrhosis. In Fig. 2, the inset solid bar means an average value, the asterisk ( * ) a statistical significance ( P<0.01 ) of the average when the serum concentration of the human type III collagen peptide iscompared between the normal s~bjects and the patients. Each 30 1I numeral in parentheses means the number of samples and the shaded part means a mean of the normal subjects (M)+2SD.

- .- . ~ . . . :

.

~lZ87801 Fig. 3 shows a serum level of immunoactive human type IV
collagen peptide in normal subjects and in patients with chronic active hepatitis and liver cirrhosis. In Fig. 3, the inset solid bar means an average value. The double asterisk ( ** ) means a statistical significance ( P<0.001 ) of the average when the serum concentration of the human type IV collagen peptide is compared between the normal subjects and the patients.
Each numeral in the parentheses means the number of samples and the shaded part means a mean of the normal subjects (M)+2SD.
Fig. 4 shows a serum level of immunoactive human type VI
collagen peptide in normal subjects and in patients with chronic active hepatitis and liver cirrhosis. In Fig. 4, the inset solid bar means an average value. The asterisk ( * ) means a statistical significance ( P<0.05 ) of the average when the lS -~erum concentration of the human type VI collagen peptide is compared between the normal subjects and the patients. Each numeral in parentheses means the number of samples and the shaded part means a mean of the normal subjects (M)+2SD.
- Our recent immunological tests have revealed that a sig-20 - nificant increase in the human collagen peptide level is observed in tissue or blood of patients suffering from liver fibrosis caused by hepatic diseases such as chronic hepatitis and liver cirrhosis.
As will be shown in the accompanied Figs. 2, 3 and 4, the human type III, IV and VI collagen peptides levels in sera from patients with liver cirrhosis as measured in accord-~; ~ ance with the method this invention are significantly higher ;~ 7 ~::
~4 : , ~ . . . .
. . ~ . .
~ ~ . , " "

~L2~780~

than those of sera from healthy normal subjects. According to the present invention, the measurement of the collagen peptide levels in blood is carried out in a simple manner and enables foreknowing hepatic diseases, especially liver fibrosis, without relying on biopsy which is burdensome on patients.
We have confirmed that fibrosis of hepatic tissues cannot be determined by the conventional liver function tests relying on measurement of the activity of ZTT (zinc sulfate turbidity test), GOT (glutamate-oxaloacetate transaminase), GPT
(glutamate-pyruvate transaminase), ALP (alkaline phosphatase), -LDH (lactate dehydrogenase), y-GTP (y-glutamyl transpeptidase), etc. As well as the method for determining human prolyl 4-hydroxylase by immunoassay previously developed by the present inventors (Japanese Laid-open Patent Appln. No. Sho.
60-204726), therefore, the present invention is very useful in the field of diagnosis of hepatic diseases, since detection of diseases of this type at an early stage can be expected by the measurement of the human type III, IV and VI collagen peptide levels in blood according to the method of this invention and since the diagnosis of fibrosis of hepatic tissues can be made by the method of this invention capable of measur-ing the human type III, IV and VI collagen peptides. In addition, the method itself of this invention is simple and convenient in easiness of the operation and in the use of a smaller amount of samples and a result obtained by the method of this invention is exact and trustworthy. Thus, the method of this invention is extremely useful in the field of diagnosis of hepatic diseases.

8-~j A

~... . - ~ . . .
,.- . . : . :

~ ~7801 PREFERABLE EMBODIMENTS OF THE INVENTION
The present invention will now be illustrated in more detail by way of the following examples, but it is to be construed that the scope of this invention is not limited by these specific exampIes.

8a A~
~ .,~
~.

. .

.

:: .

1, ~L287801 Ll Example 1 ¦¦ Preparation of monoclonal antibodies to human collagen peptides ~a) Purification of human type III, IV and VI collagens as ~, antigen:
S ¦' According to the method of Mayne et al. [Artery, 7, ll, 262-280, (1980)], human placenta was homogenized in 0.5-N
¦i acetic acid, digested with pepsin (1 mg/ml) to make the contained collagens soluble, and incorporated with NaCl so l~ that its final concentration became 2-M to effect precipitation 10 ¦! of the collagens. The precipitate was dissolved in 0.5-N
lll acetic acid from which a fraction containing the type I and III
!~ collagens was obtained through dialysis against a 0.5-N acetic l! acid solution containing 0.7-M NaCl and the supernatant was ,ll then dialyzed against a 0-5-N acetic acid solution containing 1 1.2-M NaCl whereby a fraction containing the type IV and V
1~ collagens was precipitated. Further, the supernatant was ¦, dialyzed against a 0.5-N acetic acid solution containing 1.8-M
,'~ NaC1 to precipitate the type VI collagen. The fraction !l containing the type I and III collagens obtained above was ¦ dissolved in a 50-mM tris-HCl buffer solution (pH 7.4) containing 0.5-M NaC1 and dialyzed sequentially against a 50-mM
¦I tris-HCl buffer solution (pH 7.4) containing 1.7-M NaCl and ¦ a tris-HCl buffer solution (pH 7.4) containing 2.7-M NaCl 1 whereby the type III and I collagens were precipitated, 25 il respectively, and the type III collagen was separated from ¦l the type I collagen. In the same manner as described for the type I and III collagens, the fraction containing the type IV
and V collagens was dissolved in a 50-mM tris-HCl buffer solution (pH 7.4) containing 0.5-M NaCl and dialyzed against 30 !¦ a 50-mM tris-HCl buffer solution (pH 7.4) containing 2.2-M

~e ~

~ ~7801 NaCl whereby the type IV collagen was precipitated and ¦¦ separated from the type V collagen. The quality of the type [I, I~ and VI collagens thus obtained was examined according ¦I t~ Sykes et al. method reported in Biochem. Biophys. Res.
I Commun., 72, 1472-1480 (1976) by subjecting these collagens ¦ to the sodium dodecyl sulfate-polyacrylamide gel electro-¦ phoresis (SDS-PAGE) whereby the purity of these collagens was respectively determined as about 95~.
I (b) Preparation of antibody-producing cells:
i Two 8 weeks old BALB/c female mice were initially injected intraperitoneally with 100 ~g of each of the human type III, -IV and VI collagens in Freund's complete adjuvant. After ¦ the initial administration, the mice were then forced to ¦ receive a booster injection of 100 yg antigen dissolved in a ¦ 50-mM tris-HC1 buffer solution (pH 7.4) containing 0.5-M NaCl, ¦¦ 2 to 4 times at 2 to 4 week intervals. As a final immunization, ¦
¦¦ the mice weré subjected to subsidiary immunization by intra-~¦ venous administration. After 3 days, the mice were kïlled to ¦ extirpate their spleens and the splenocytes were harvested.
~ (c) Cell fusion:
- I Materials and methods used are as follows:
I RPMI 1640 culture medium: RPMI No. 1640 (Difco Lab.) ¦ incorporated with sodium bicarbonate (12 mM), sodium pyruvate (1 mM), L-glutamine (2 m~l), penicillin G potassium (50 u/ml), streptomycin sulfate (50 ~g/ml) and amikacin sulfate (100 ¦ ~g/ml), adjusted at pH 7.2 with dry ice and filtered through a 0.2 ~m Toyo membrane filter for sterilization.
Il NS-l culture medium: the above RPMI 1640 culture medium il incorporated with 15~ (v/v) fetal bovine serum (M.A.
30 ¦I Bioproducts) sterilely filtered.

, ! *Trade Marlc ,' 10 A
- . ~ . . -: - .
', . ' : -` :, , 1 ~l2~37801 ,, ¦ HAT selection medium: the above NS-l culture medium ¦I further incorporated with hypoxyanthine (100 ~M), aminopterine I (0.4 ~M) and thymidine (16 ~M).
Il HT culture medium: the same medium as the above HAT
I with the exception of aminopterine.
PEG*4000 solution: RPMI 1640 culture medium without calf serum, containing 50~ (w/w) polyethylene glycol (PEG 4000, Merck).
I Utilizing 8-azaguanine-resistant myeloma cell line, NS-l 10 ll (P3-NSl-l), cell fusion was effected according to a slightly modified method of Oi and Herzenberg described in "Selected ¦¦ Method in Cellular Immunology" (ed. B. B. Mishell and S. M.
¦I Shiigi, W. H. Freeman and Company, (1980), pp. 351-372).
!, The karyo-splenocytes produced as described in the preceding lS ~ (b) (cell viability 95~) were mixed in a ratio of 5 or 6 to 1 li with mouse myeloma cells (cell viability 95%) for fusion.
¦ Thus, the splenocytes and the myeloma cells were respectively Il washed in RPMI 1640 culture medium, suspended in the same ¦~ medium and mixed together for fusion in the ratio described 20 1~ above. 40 ml of the RPMI 1640 culture medLum was put into a 50 ml conical styrene resin test tube (Iwaki Glass, Japan), ¦I centrifuged at 400 xg for 10 min., and the supernatant was discarded completely by suction. To the precipitated cells !I was added dropwise in 1 min. 1 ml of the PEG 4000 solution 25 ¦~ warmed at 37C with gentle stirring. The cells were re-¦ suspended and dispersed by stirring gently for another minute.

! To the suspension was then added dropwise in 1 min. 1 ml of the RPMI 1640 culture medium warmed at 37C. After repeating l, the same operation once more, the cells were dispersed again, 30 ¦I by adding the same culture medium dropwise in 2 to 3 min.
! I *Trade Mark A

- .
; . .
- . . - , .~ .
: - . . . .
,. ., . -, :, . ::, -1~87&01 j under continuous agitation. ~he dispersion was centrifuged at 400 xg for 10 min., and the supernatant was removed ¦ completely by suction. To the precipitated cells was added ¦ rapidly 10 ml of the NS-l culture medium warmed at 37C, and ¦ big cell clumps were then dispersed by pipetting carefully ¦ with a 10 ml pipette. The dispersion was diluted with 20 ml ¦ of the same culture medium, and distributedinto 96-well micro-plates made of polystyrene (Iwaki Glass) so that 5.9 x 105 cells/0.1 ml of the culture medium might exist in each well.
As a preliminary treatment, the 96-well microplates had been j treated with 0.2 ml of the NS-l culture medium overnight at 37C in a CO2 incubator, and the culture medium had been removed therefrom by suction on actual use. After completion I of the cell fusion, the microplates were incubated at 37C
~ in 7% CO2/93% air under saturated humidity.
(d) Selective proliferation of hybridomas by the aid of a selective culture medium:
On the first day of incubation, two drops (ca. 0.1 ml) of the HAT culture medium were added with a Pasteur pipette.
On the 2nd, 3rd, 5th, 8th and 11th days, the half (0.1 ml) of each culture medium was replaced with new HAT culture medium. On 14th day, each culture was replaced with HT culture ¦ medium. Thereafter, the same procedure was repeated at 3 ~¦ to 4 day intervals. Usually, sufficient growth of hybridoma cells was observed 2 to 3 weeks after the incubation period.
All the wells containing hybridomas were tested for the presence of antibody, using a solid phase-antibody binding - test (ELISA) as described in the subsequent (e). All the cultures positive for antibody production were transferred to 24-well plates made of polystyrene (Iwaki Glass) containing .
.. . : - .

I, ~1 2~7801 I 1 ml of the HT culture medium containing 107 m~use thymocytes ¦ as feeders. The cultures thus treated were incubated, as described in the preceding (c), at 37C in 7% CO2 for about I a week. Once or twice during the incubation period 0.5 ml ¦ of the supernatant of each well was replaced with 0.5 ml of I new HT culture medium. At the time the sufficient growth of ¦ hybridomas was observed, the cultures were screened again ¦ for the positivity of antibody production by ELISA method, ¦ and subjected to a cloning procedure by the limiting dilution ! method as explained in (f) below. The residual culture j after cloning was transferred into a 25 cm3 tissue culture I flask made of polystyrene (Iwaki Glass), for the preparation ¦ of a frozen sample.
I (e) Detection of hybridomas capable of producing anti-human 15 collagen peptide by means of the solidphase-antibody I binding test (ELISA):
¦A method used for the present example was a slight modification of the method of Rennard et al. described in l Anal. Biochem., 104, 205-214, (1980) which is appropriate for the detection of antibody production in hybridoma.
Each well of 96-well microtitration plates (Flow Lab.) was coated with 0.5-1.0 ~g of the human type III, IV or VI collagen, ¦
and the wells remained uncoated were blocked with 1~ bovine serum albumin (BSA). To the wells was added a part of the supernatant from the wells exhibiting hybridoma growth. The incubation was carried out at room temperature for about 1 hr. After the addition of goat anti-mouse immunoglobulin tCappel Lab.) labe~edwith horseradish peroxidase as a secondary antibody, I further incubation was carried out at room temperature for I about 1 hr. TXe brown color produced by the addition of - .
.. . . . . , ~ .
:
-: , . . ..
.

lZ~37801 Il H2O2 and o-phenylenediamine as a substrate was recognized ¦! qualitatively by naked eyes or measured by way of spectro-¦l photometry for absorbance at 500 nm using a Corona double jl wave microplate spectrophotometer (MTP-22, Corona Electric).
5 ¦1 ( f) Cloning:
In order to obtain hybridomas capable of producing monoclonal antibodies it is necessary for cloning to treat cultures according to the limiting dilution method, since each well can develop more than two kinds of hybridomas.
i A cloning culture medium containing in the NS-l culture medium 107 feeder cells per ml prepared from mouse thymocytes I was prepared and placed in the three groups of 36, 36 and 24 ; ¦ wells of a 96-well microwell, with 5, 1 and 0. 5 hybridomas being added to the wells of the three groups, respectively.
I About 0.1 ml of the NS-1 culture medium was added to each ' ! well on the 5th and 12th day after incubation. About 14 to ¦1 15 days after the cloning when the sufficient growth of hybridoma could be recognized, the culture group in which more than 50% wells showed no colony formation was submitted to ELISA. When all of the tested wells showed positivity for antibody production, the colonies in antibody-positive wells were enumerated and out of them 4-6 wells exhibiting the formation of one colony were selected for recloning.
¦ Finally, 5, 22 and 5 clones were obtained for the type III, 25 1¦ IV and VI collagens.
¦ ~g) Production in vitro and in vivo of monoclonal antibody:
I Monoclonal antibodies can be obtained from the supernatant of a proper culture medium such as NS-l culture medium in ¦I which the clones are incubated (in vitro proliferation) (The 30 1I concentration of the monoclonal antibody protein 10-100 ~g/ml).
*Trade Mark 14 - ~ : .
.

~ 28~80~
!~ In case a large amount of antibody is needed, on the other Il hand, it is recom~ended to adopt an in vivo proliferation ¦l as described below. A carcinogenic excitant, Pristane*
~l (2,6,10,14-tetramethylpentadecane, Aldrich Chemical), was 5 I administered to animals (BALB/c) intraperitoneally in an ! amount of 0.5 ml per animal, which were of the same strain ¦ as those providing the Splenocytes and the myeloma cells.
!¦ One to 3 weeks after the administration, 1 x 107 cells of ¦¦ hybridoma were administered intraperitoneally as above, and 10 ¦1 ascites containing noclonal antibody protein in a concentra-¦, tion of 4-7 mg/ml were obtained after 1 to 2 weeks.
li (h) Isotypes of monoclonal antibodies with heavy and light ¦i chains:
! First of all, each of the resultant ascites was dispersed 15 ¦1 into a well coated with the human type III, IV or VI collagen ,I for binding according to the above-described ELISA method. I
After washing the well, different subclass speci~ic rabbit Il anti-mouse Ig antibodies (Zymed Lab.) were added thereto.
¦l After washing the well, affinity purified goat anti-rabbit 20 ¦1 IgG ( H~L ) antibody labeled with horseradish peroxidase, , was added thereto, and 2,2'-azino-di(3-ethylbenzthiazoline sulfate-6) as a substrate and hydrogen peroxide were then added to detect the resultant monoclonal antibodies. The ¦I results are shown in Tables 1, 2 and 3. Among the resultant 25 l¦ monoclonal antibodies against the human type III collagen, ¦I four antibodies contain immunoglobulin chain yl/~ and one antibody contains y2a/K (Table 1). Among those against the human type IV collagen, sixteen antibodies contain yl/K, !l il two antibodies y2b/~, one antibody a/~ and three antibodies 30 li ~/K (Table 2). Among those against the human type VI collagen ll ' I *Trade Mark . A 15 .:~ ,.. . . . . .

37~01 .~ i 1~ one antibody contains yl/~, another one antibody y2a/~ and Il three antibodies y2b/K (Table 3).
.l (i) Purification of monoclonal antibody:
Il Each of the ascites obtained in the foregoing (g), was 5 ,I fractionated with ammonium sulfate (40~ saturation) and IgG
class was subjected to chromatography on DEAE Sephacel I (Pharmacia) equilibrated with a 40-mM phosphate buffer solution il (pH 8.0) containing 0.06-M NaCl to separate an unabsorbed Ii fraction. This IgG fraction was gel-filtered on Sephacryl 10 !I S-300 superfine column (Pharmacia) equilibrated with a 50-mM
¦' phosphate buffer solution (pH 7.4) containing 0.42-M NaCl, whereby the fetal calf serum and a mouse-derived protein contained therein were separated and removed. In the purifica-ll tion of IgA and IgM fractions, these fractions wereobtained 15 ! separately by elution with a NaCl solution of 0.06-1.0 M
gradient on DEAE-Sephacel column chromatography. The subsequent gel-filtration was carried out under the same condition as used for the IgG class.

ll Example 2 20 Ij Preparation of antisera and polyclonal antibodies against the human type III, IV and VI collagens Rabbits were immunized, being injected subcutaneously 4-6 times at aninterval of 2 weeks with 1 mg of each of the I human type III, IV and VI collagens which had been purified 25 ~¦ from human placenta in the same manner as described in Example ¦¦ l(a), together with equivalent Freund's complete adjuvant.
Il The antisera obtained as final product were purified by ¦l, affinity chromatography using a column coupled with each type I of the human collagens. In order to assess the antibody titer , . 1, .
: 16 ,, . , . , . , , . :

~ 2~3~8C)~

¦l and the specificity of immunizing activity, the polyclonal ! antibodies were examined for cross-reaction with each type of l~ the human collagens according to ELISA method referred to in ¦, Example l(e). The result proved that the polyclonal antibodies 5 ¦I were highly specific; the polyclonal antibodies were cross-reacted only with the corresponding type of the human collagens.

' Example 3 I Measurement of the human type III, IV and VI collagen peptides ~l, in serum by using a combination of the monoclonal and polyclonal 10 li antibodies , The type III, IV and VI collagens in human serum were assayed, using a sandwich method as described in "Enzyme immunoassay" (eds., E. Ishikawa, T. Kawai and K. Miyai, ll Igakushoin, 2nd ed. (1982), pp. 30-49). Polystyrene 96-well 15 ' microtitration plates (Flow Lab.) were coated with the monoclonal antibodies to the human type III, IV or VI collagen obtained in a purified form in Example l(i), and the others were blocked I¦ with 1.0~ BSA. On the other hand, the purified human type III, ¦; IV or VI collagen obtained in Example l(a) was used as a 20 ¦¦ standard,and 10 ~1 of each of the sera collected from normal ,, subjects and from patients with chronic active hepatitis or , Il liver cirrhosis which was definitely diagnosed based on Il histological observation of the tissue biopsied was used as a human serum sample. The sample solutions were added to the 25 1 microtitration plates treated with the monoclonal antibodies and incubated at room temperature for 1 hour. ~fter the addition of the rabbit polyclonal antibodies obtained in Example 2 and i with goat anti-rabbit IgG (Miles-Yeda) labeled with horseradish peroxidase as a secondary antibody, the samples were incubated ~ , . ~ , ..
.. . -- . . . . .
:: , ' . ~ : - :
.. . . .

¦ at room temperature for about 1 hour. After the subsequent addition of hydrogen peroxide and o-phenylenediamine as a !~ substrate, the absorbance of the brownish red reactionproduct I, formed was measured using a Corona double wavelength microplate 5 ~ spectrophotometer (MTP-22). The standard curves for the human type III, IV and VI collagens were drawn in Fig. 1, I from which it was read out that the sensitivity was ca. 5 ng for all the human type collagens examined. According to this j sandwich method enabling the assay of collagen peptide 10 1, concentration in blood, it is recognized that the concentration in blood was significant~y increased in case of hepatic diseases (Figs. 2, 3 and 4). The concentration of the type III and IV
collagen peptides was increased especially in blood from I patients with chronic active hepatitis and liver cirrhosis.
15 I In this Example, the present invention is mainly explained with ! respect to the sandwich method which utilizes the monoclonal ¦i antibody as a solid phase, and the p~oxidase-labeled anti-rabbit IgG as a secondary antibody. However, the present I invention is also applicable as a means for determining 20 ! blood collagen peptides to the sandwich immunoassay wherein an I enzyme-labeled mouse monoclonal antibody or rabbit polyclonal ¦¦ antibody (IgG and Fab') to the human type III, IV and VI
collagen peptides is used, as described by Yoshitake in I "Men-eki Jikken Sosa-ho (Manual for Immunological Expe,riments)"
25 I vol. XI (1982), pp. 3497-3519,- and also to the radioimmunoassay ¦ disclosed in Japanese Laid-open Patent Appln. No. Sho. 61-202162 and Japanese Patent Appln. No. Sho. 60-179357 wherein ,~ isotope-labeled monoclonal and polyclonal antibodies are used.

Example 4 - !

- ~ .: .. - . . . .

Measurement of the human type III collagen peptide in serum ~ by the aid of monoclonal antibody ¦ The measurement was carried out according to the method of Ishikawa et al., J. Immunoassay 4, 209-327 (1983) wherein the monoclonal antibody was used for a dual function as a solid phase and a conjugate. At first, the purified monoclonal antibody IgGl (clone No. 12) to the human type III collagen, I which had been obtained in Example l(i), was dissolved in a ¦ 0.1-M phosphate buffer solution (pH 7.5) containing 0.1 10 I sodium azide, and adjusted at 0.1 mg/ml in concentration.
¦ A polystyrene ball (6.5 mm in diameter, Precision Plastic Ball i Co.) as a solid phase was soaked in the solution and coated ¦ with the antibody. After collecting the solution used for I the soa~ing, the polystyrene ball was washed with a 10-mM
15 ¦ phosphate buffer solution (pH 7.0) containing 0.1~ ~SA, 0.1 M
NaCl and 0.1~ sodium azide, to be ready for use. On the other ¦ hand, the purifiedhùman type III collagen obtained in Example l(a) was used as a standard sample and a 10 ~1 of each serum from ¦
¦ healthy subjects and from patients with chronic active hepatitis and liver cirrhosis which had been definitely diagnosed according to the histological observation of the tissue biopsied was used as a serum sample. A solution of these samples was added to the polystyrene ball coated with the monoclonal antibody, and incubated at 30C for 1 hour (the first reaction).
25 1 The solution was then incorporated with the peroxidase-labeled ¦ monoclonal antibody IgG (clone No. 53) or the fragment, Fab' I derived from clones different from those which provided the ¦I monoclonal antibody for the solid phase as a conjugate, and incubated at 30C for 1 hour (the second reaction). The solution was then incorporated with hydrogen peroxide and ' ~ 1, .
~,.,....
', 19 ' ~1 2~37~301 I! 1 3,3',5,5'-tetramethylbenzene (TMBZ) as a substrate and incubated for reaction at 30C for 1 hour (the third reaction).
This enzyme reaction was then stopped with 1.33-N H2S04.
j After the cease of the reaction, the absorbance of the reaction I product was measured at 450 nm wave length by means of a ! microflow ultraviolet-visible spectrophotometer (Shimazu, il W -730)*, using water as a reference and a difference between ¦ the absorbance of the blank and that of the reaction products ¦¦ was calculated. From the referential absorbance curve obtained 10 ¦~ from the standard samples, the content of the type III collagen ¦¦ peptide in 10 ~1 of the serum sample was read out, and multiplied by 100 to estimate the amount of the type III collagen contained in 1 ml of the serum sample.
. I
Example 5 15 ¦ The measurement of the human type IV collagen in serum ¦ by the aid of the mon~clonal antibody ¦i The condition for measurementwas identical with that for the human type III collagen peptide in serum described in Example 4, except that the monoclonal antibody IgGl (clone 4H12) was used as a solid phase and the monoclonal antibody ; ¦1 IgG or Fab'(clone lD3) labeled with a peroxidase was used as ¦l a conjugate. Thus, the assessment of serum type IV collagen peptide was undertaken, using sera from normal subjects and patients with chronic active hepatitis and liver cirrhosis.

11 , *Trade Mark ~............. , . . - -~ 2~780~ j Ii !

¦ Table 1 Monoclonal I antibody Isotype Chain !
¦ No.ll IgG2a y2a/K
No.12 IgGl yl/~
No.33 IgGl yl/~ i No.41 IgGl yl/K
No.53 IgGl yl/K

i I; Table 2 ¦~ Monoclonal 1, antibody Isotype Chain !
4C1 IgM ~/K
7All IgM
lD3 IgGl yl/~
lD6 IgGl yl/K
lE10 IgGl yl/~
2A7 IgM ~/ K
2DS IgG1 yl/~
2Hl IgA a/K
3A9 IgGl yl/~
4B1 IgGl yl/~
4H12 IgGl yl/~
SD10 IgGl yl/K
5F6 IgG1 yl/~
6BS IgGl yl/K
6Cll IgGl yl/K
6GS IgG2b y2 7C8 IgGl yl/~
7H2 IgGl yl/~
8B4: IgGl yl/K
8G12 IgGl yl/K
9A3 IgG2b y2b/
gc7 ~ IgG1 yl/~

~ 21 - ,: ,- .- , : . . .

~ 28~7801 Table 3 Monoclonal ¦ antibody Isotype Chain No. 14 IgG2b y2b/K
No. 17 IgG2b y2b/K
¦ No. 26 IgGl yl/K
No. 29 IgG2b y2b/K
No.38 I<~G2a y2a/K
i ¦! .
l! I
~ 1 ~

~ ~ I : .

~ `
. I
,, I ~ .
, 22 I

Claims (6)

1. A method for determining human type III, IV or VI
collagen peptides by way of enzyme sandwich immunoassay which comprises:
(a) contacting a serum sample suspected of containing human type III, IV or VI collagen peptides with a first monoclonal antibody specific for either of human type III, IV
or VI collagen peptides, said monoclonal antibody being bound to a solid phase under conditions which allow formation of a reaction product between said first monoclonal antibody and said corresponding collagen peptide;
(b) contacting said first reaction product with a second monoclonal antibody specific for either of said human type III, IV or VI collagen peptides and which is conjugated with an enzyme under conditions which allow formation of a second reaction product between said second monoclonal antibody and said first reaction product, said conjugated monoclonal antibody being different from said monoclonal antibody used in step (a):
(c) adding a substrate for said enzyme: and (d) assaying for reaction products of said substrate.

2. The method according to Claim 1, wherein said first monoclonal antibody bound to said solid phase and said second monoclonal antibody conjugated with said enzyme are each specific for type III collagen peptide.

3. The method according to Claim 1, wherein said first monoclonal antibody bound to said solid phase and said second monoclonal antibody conjugated with said enzyme are each specific for type IV collagen peptide.

4. The method according to Claim 1, wherein said first monoclonal antibody bound to said solid phase and said second monoclonal antibody conjugated with said enzyme are each specific for type VI collagen peptide.

5. The method according to Claim 1, wherein said monoclonal antibody is selected from the group consisting of IgG, IgM and IgA classes.

6. A method for diagnosing hepatic diseases by subjecting the serum of a patient to be diagnosed to the enzyme sandwich immunoassay according to claim 1.