CA2074345A1 - Method and agent for the turbidimetric or nephelometric determination of analytes - Google Patents
Method and agent for the turbidimetric or nephelometric determination of analytesInfo
- Publication number
- CA2074345A1 CA2074345A1 CA002074345A CA2074345A CA2074345A1 CA 2074345 A1 CA2074345 A1 CA 2074345A1 CA 002074345 A CA002074345 A CA 002074345A CA 2074345 A CA2074345 A CA 2074345A CA 2074345 A1 CA2074345 A1 CA 2074345A1
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- Canada
- Prior art keywords
- fragments
- fab
- solution
- analytes
- turbidimetric
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
- G01N33/6857—Antibody fragments
Abstract
MERCK PATENT GESELLSCHAFT
MIT BESCHR?NKTER HAFTUNG
DARMSTADT
ABSTRACT
The invention relates to methods and agents for the turbidimetric or nophelometric determination of analytes in liquids with the aid of an antibody-binding reaction. The method is characterised in that a poly-peptide is reacted with antibodies or fragments thereof, the resulting coupling product is incubated with the analyte, and the resulting turbidity is measured.
MIT BESCHR?NKTER HAFTUNG
DARMSTADT
ABSTRACT
The invention relates to methods and agents for the turbidimetric or nophelometric determination of analytes in liquids with the aid of an antibody-binding reaction. The method is characterised in that a poly-peptide is reacted with antibodies or fragments thereof, the resulting coupling product is incubated with the analyte, and the resulting turbidity is measured.
Description
2a~3~
Merck Patent Gesellschaft mit beschr~nkter ~aftung 6100 Darmstadt Method and agent for the turbidLmetric or nephelometric determination of analytes The invention relates to method and agent for the turbidimetric or nephelometric determination of analytes in liguids with the aid of an antibody-binding reacti~n.
Both immunoturbidimetry and immunonephelometry are based on the interaction between antibodies and the antigen to be detected. This interaction re~ults in the formation of high molecular weight aggregates which act as centres for scattering incident light. The scattering ~
of the light is recorded as an increase in extinction in immunoturbidimetry and a~ an incr~ase in the intensity of scattered light at an angle > 0 - s 90 relative to the incident light in immunonephelometry. Since the extent of the light scattering is approximately proportional to 1~4, the wavelength regions typically selected for immunoturbidimetry and immunonephelometry are of short wavelength (320-360 nm). The theoretical ba~es of immuno-precipitation are described, for example, in Immunology 32, 445-457 (1977).
The optimal conditions for carrying out such determinations, ~uch as buffers, detergents, neutral salts, accelerators, etc., are described ln the litera-ture and vary slightly depending on the antibodies used in each case and on the antigens to be detected tAnn.
Clin. Biochem. 20, 1-14 (1983)).
The great advantage of the immunoturbidimetry and immunonephelometry described to date i~ that the methods can be automated relatively simply. Immunoturbidimetry and immunonephelometry are described for many serum proteins (The Plasma Protein~, Vol. 2, Academic Press, New York, pages 375-425 (1975), Automated Immunoanalysis, Part 1+2, R.F. Ritchie (ed.), Marcel Dekker Inc., New York, Basel (1978)~.
The detection limits reported in the state of the art differ and vary depending on the antibody and anti-gen. The detection limits for immunoturbidimetry are at about 5 mg/l, and for immunonephelometry are at about 1 mg~l. The upper limits for the measurement range for immunotuxbidimetry and immunonephelometry are at about 200-500 mg/l, depending on the op~imisation of the assay.
It i5 not possible to determine lower concen-trations of analytes by immunoturbidimetry and immuno-nephelometry. Employed for thi~ is, for example, latex agglutination which use~ relatively large particles (diameter about 0.05-3 ~m) which are coated with antibodies or antigens depending on the principle of the assay. Detection limits < 1 ~g/l can be achieved with the latex agglutination technique. The typical upper limits for the measurement range are then, however, as much as 200-1000 ~g/l (0.2-1 mg/l).
The present invention i8 ba~ed on the ob~ect of providing method~ and agents for the turbidimetric or nephelometric determination of analytes which are more sensitive than the known methods and which permit detec-tion of antigens in the concentration range 0.1 > 100 mg/l.
The invention relates to a method for the tur-bidimetric or nephelometric determination of analytes in liquids with the aid of an antibody-binding reaction, which $~ characterised in that a polypeptide is reacted with~ antibodieEI or fragments thereof, the coupling product resulting therefrom is incubated with the ana~
lyte, and the resulting turbidity is measured.
The invention further relates to an agent for the turbidimetric or nephelometric determination of analytes in liquids, which contains a coupling product composed of a polypeptide and antibodies or fragments thereof, preferably a coupling product composed of a globular protain and Fab' fragments.
- -It has been found, surpri~ingly, that polymerised antibodies which have been obtained, for example, by acid precipitation from anti~erum provide a higher measu~ement signal than monomeric antibodies in the turbidimetric assay. However, there is variation in the degree of polymerisation of these antibodies and thus in the measurement signal during storage. To avoid this disad-vantage, a defined, ~table complex with multiple binding sites has been prepared by coupling natural IgG or antibody fragments, preferably Fab' fragments, to a molecular carrier.
Suitable molecular carriers are polypeptides such as protein~ with a molecular weight of at least 10,00~ D.
Preferably used are globular proteins such as albumin, thyroglobulin, transferrin, haemocyanin or else poly-peptides such as polylysine. It is likawise possible to employ the known avidin~biotin or streptavidin/biotin system, in which case biotinylated antibodies or frag-ments thereof form a stable complex with avidin or streptavidin.
The coupling to the antibodies or fragments thereof can be carried out in a variety of ways. Prefer-ably employed for this purpose are bifunctional hetero-crosslinkers, for example succinimidylmaleimide cross-linkers and related substances. The advantage of thesecrosslinkers is that unwanted coupling products are avoided, antibodi.es or antibody fragments are introduced stepwise, the entibodies or antibody fragment~ are coupled end-on, and the coupling products are easy to purify by chromatography.
The coupling products are generally prepared by the following stepss 1. cleavage of the antibodies, for example with pep6in, 2. purification of the F~ab')z fragments by chromatography, 3. reduction of the F(ab' )2 fxagments, 4. isolation of the Fab' fragments by chromatography, 5. coupliny of the crossli;nker onto the carrier, - 4 - ~7~3l~
Merck Patent Gesellschaft mit beschr~nkter ~aftung 6100 Darmstadt Method and agent for the turbidLmetric or nephelometric determination of analytes The invention relates to method and agent for the turbidimetric or nephelometric determination of analytes in liguids with the aid of an antibody-binding reacti~n.
Both immunoturbidimetry and immunonephelometry are based on the interaction between antibodies and the antigen to be detected. This interaction re~ults in the formation of high molecular weight aggregates which act as centres for scattering incident light. The scattering ~
of the light is recorded as an increase in extinction in immunoturbidimetry and a~ an incr~ase in the intensity of scattered light at an angle > 0 - s 90 relative to the incident light in immunonephelometry. Since the extent of the light scattering is approximately proportional to 1~4, the wavelength regions typically selected for immunoturbidimetry and immunonephelometry are of short wavelength (320-360 nm). The theoretical ba~es of immuno-precipitation are described, for example, in Immunology 32, 445-457 (1977).
The optimal conditions for carrying out such determinations, ~uch as buffers, detergents, neutral salts, accelerators, etc., are described ln the litera-ture and vary slightly depending on the antibodies used in each case and on the antigens to be detected tAnn.
Clin. Biochem. 20, 1-14 (1983)).
The great advantage of the immunoturbidimetry and immunonephelometry described to date i~ that the methods can be automated relatively simply. Immunoturbidimetry and immunonephelometry are described for many serum proteins (The Plasma Protein~, Vol. 2, Academic Press, New York, pages 375-425 (1975), Automated Immunoanalysis, Part 1+2, R.F. Ritchie (ed.), Marcel Dekker Inc., New York, Basel (1978)~.
The detection limits reported in the state of the art differ and vary depending on the antibody and anti-gen. The detection limits for immunoturbidimetry are at about 5 mg/l, and for immunonephelometry are at about 1 mg~l. The upper limits for the measurement range for immunotuxbidimetry and immunonephelometry are at about 200-500 mg/l, depending on the op~imisation of the assay.
It i5 not possible to determine lower concen-trations of analytes by immunoturbidimetry and immuno-nephelometry. Employed for thi~ is, for example, latex agglutination which use~ relatively large particles (diameter about 0.05-3 ~m) which are coated with antibodies or antigens depending on the principle of the assay. Detection limits < 1 ~g/l can be achieved with the latex agglutination technique. The typical upper limits for the measurement range are then, however, as much as 200-1000 ~g/l (0.2-1 mg/l).
The present invention i8 ba~ed on the ob~ect of providing method~ and agents for the turbidimetric or nephelometric determination of analytes which are more sensitive than the known methods and which permit detec-tion of antigens in the concentration range 0.1 > 100 mg/l.
The invention relates to a method for the tur-bidimetric or nephelometric determination of analytes in liquids with the aid of an antibody-binding reaction, which $~ characterised in that a polypeptide is reacted with~ antibodieEI or fragments thereof, the coupling product resulting therefrom is incubated with the ana~
lyte, and the resulting turbidity is measured.
The invention further relates to an agent for the turbidimetric or nephelometric determination of analytes in liquids, which contains a coupling product composed of a polypeptide and antibodies or fragments thereof, preferably a coupling product composed of a globular protain and Fab' fragments.
- -It has been found, surpri~ingly, that polymerised antibodies which have been obtained, for example, by acid precipitation from anti~erum provide a higher measu~ement signal than monomeric antibodies in the turbidimetric assay. However, there is variation in the degree of polymerisation of these antibodies and thus in the measurement signal during storage. To avoid this disad-vantage, a defined, ~table complex with multiple binding sites has been prepared by coupling natural IgG or antibody fragments, preferably Fab' fragments, to a molecular carrier.
Suitable molecular carriers are polypeptides such as protein~ with a molecular weight of at least 10,00~ D.
Preferably used are globular proteins such as albumin, thyroglobulin, transferrin, haemocyanin or else poly-peptides such as polylysine. It is likawise possible to employ the known avidin~biotin or streptavidin/biotin system, in which case biotinylated antibodies or frag-ments thereof form a stable complex with avidin or streptavidin.
The coupling to the antibodies or fragments thereof can be carried out in a variety of ways. Prefer-ably employed for this purpose are bifunctional hetero-crosslinkers, for example succinimidylmaleimide cross-linkers and related substances. The advantage of thesecrosslinkers is that unwanted coupling products are avoided, antibodi.es or antibody fragments are introduced stepwise, the entibodies or antibody fragment~ are coupled end-on, and the coupling products are easy to purify by chromatography.
The coupling products are generally prepared by the following stepss 1. cleavage of the antibodies, for example with pep6in, 2. purification of the F~ab')z fragments by chromatography, 3. reduction of the F(ab' )2 fxagments, 4. isolation of the Fab' fragments by chromatography, 5. coupliny of the crossli;nker onto the carrier, - 4 - ~7~3l~
6. purification of the carrier-crosslinker conjugate by chromatography, 7. coupling of the Fab~ fragments onto the carrier-crosslinker con~ugate, 8. purification of the coupling product by chromato~raphy.
The carrier-crosslinker con~ugate can be prepared without problems in a batch process and be stored frozen in solution or lyophilised. It is likewise possible for the F(ab' )2 fragments to be prepared in a batch process and to be ~tored as described above. Only steps 3, 4, 7 and 8 are then necessary for the actual preparation.
Compared with known reagents, in the turbidi-metric and nephelometric a~say the reagents prepared in thi~ way give distinctly higher measurement signals an~
thus allow the sensitivity of detection to be increased.
The sensitivity of detection can be further optimised by the presence of known accelerators (for example poly-ethylene glycols). The reagents prepared in this way are stable in solution, they can be employed without problems in automatic analysers, and they are thus also suitable for automated immunoturbidimetric determination of analyte~.
Both the immunoturbidimetric and the immuno-nephelometric del:ermination i8 carried out at 320-400 nm, preferably at about 340 nm. The determination can be carried out klnetically but preferably a~ endpoint reactlon.
The method and agents according to the present invention can be u~ed to determine all antigen~, that is to say substance~ which have a plurality of binding sites for antibodies. Examples of proteins of thi~ type are transferrin, CRP, ~l-microglobulin, RF, TBG, RBP, C3, C4, Igs etc. which occur in body fluids and are of diagnostic relevance. These proteins are measured in the concen-tration range of 0.05 - > 100 mg/l, preferably 0.5 -100 mg/l, without further prior dilution. Haptens which nave only one binding site are amenable to competitive - 5 ~ 3 ~ 3 determination using so-called polyhaptens.
Example 1 Preparation of an anti-transferrin Fab~-albumin conjugate 3.2 g of sheep anti-tran ferrin IgG are taken up in 150 ml of 0.9 % NaCl solution. The solution is adjusted to pH = 4.5 with 1 M acetic acid. 80 mg of pepsin are added to this ~olution, and the mixture is incubated at 37C for about 16 h. The reaction solution is ad~usted to pH = 7.0 with 1 M TRIS solution. The reaction ~olution is purified by affinity chromatography using a Sepharos~4~ column to which human transferrin is covalently bonded. The bound F(ab')2 fragments are eluted with 0.1 M glycine/HCl (pH = 2.8). The protein-containing fractions are pooled and ad~usted to pH = 7.0 with 1 M
TRIS solution. The solution is concentrated to 30 ml in an ultrafiltration cell. After photometric determination 376 mg of specific F(ab' )2 fragments are obtained.
16 ml of this solution are ad~usted to pH = 4.9 with 1 M acetic acid, deaerated and blanketed with argon and mixed with 0.9 ml of an 11% solution of cysteamine hydrochloride in water. After incubation at 37C for 2 h, the Fab' fragments are purified by gel chromatography on Sephadex G-25 (150 mM NaCl, 20 mM phosphate buffer, pH =
7.0).
The protein-containing fractions are pooled.
After photometric determination, 190 mg of Fab' fragments are obtained in 34 ml of ~olution. The ~olution i~ stored in ~he cold under argon gas.
40.5 mg of bovine ~erum albumin are dissolved in 7 ml of water, snd 1 ml of a ~olution of 30 mg of suc-cinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) in dioxane i~ added. The reaction solution is incubated at 30C for 1 h and then purified by gel chromatography on Sephadex~G-250 The protein-containing fractions are pooled. After photometric determination, 38 mg of the coupling product are obtained in 14.8 ml of solution. The Fab' solution is mixed under argon gas with - 6 _ s~7~
the solution of the coupling product and incubated at room temperature for 17 h.
The reaction mixture is purified by gel fil-tration chromatography (for exampls Superdex@~
Pharmacia/L~B, 150 mM KCl, 20 mM pho~phate buffer, 1 mM
EDTA, 0.02 ~ NaN3, pH = 7.0). The fractions which contain the required product are combined.
Example 2 TurbidLmetric Lmmunoassay with anti-tran~ferrin Fab~-albumin con~ugate.
Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 6~ polyethylene glycol 6000, 0.1~ CHAPS, O.1~ TRITON~ X-100, 0.02~ NaN3.
Fab' albumins Prepared as in Example 1.
Transferrin:
Human transferrin was dissolved in 0.1 M TRIS
HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l transferrin were prepared by serial dilution.
Apparatus ad~u~tments The aB8ay i8 carried out a4 endpoint assay in an EPOS 5060 automatic analyser for clinical chemical assay~. Firstly 40 ~1 of sample (tran~ferrin ~olution) and-200 ~1 of re~action bufer are pipetted, mixed and preincubated at 37~C for 64 sec. Then 30 ~1 of Pab'-albumin con~ugate are pipetted in, mixed and the re~ult-ing extinction i~ measured after 288 sec. The apparatus cycle is set at 16 sec. The extinction at the end of the preincubation time, before addition of the start reagent, i~ u8ed to determine the sample blank. The resulting calibration plot (extinction/concentration) i~ depicted in Fig. 1.
- 7 _ 2~43~
Example 3 Preparation of an anti-CRP Fab~-albumin conjugate 250 mg of lyophilised sheep anti-CRP IgG are dissolved in 20 ml of a 150 mM NaCl solution. The 601u-tion is mixed with 4 ml of 1 M sodium acetate buffer,pH - 4.9. The pH of the solution is ad~usted to pH = 4.35 with 1 M acetic acid. 6.3 mg of pep~in are added, and the mixture is incubated at 37~C for about 16 h. The result-ing F(ab' )2 fragments are purified by gel filtration chromatography (for example Superdex~ Pharmacia/LRB, 20 mM potassium phosphate buffer, pH = 7.0, 150 mM RCl).
The combined fractions contain 122 mg of F~ab' )2 frag-ments. The pool is concentrated to 12 ml in an ultrafil-tration cell (12.3 mg/ml F(ab') 2 ) ' 6 ml of this solution (73.3 mg F(ab' )2 are mixed ~
with 1 ml of 1 M sodium acetate buffer, pH = 4.9. The pH
is ad~usted to 5.0 with 1 M acetic acid. 0.35 ml of an aqueous 11~ cysteamine hydrochloride solution is added to this solution. The reaction mixture is blanketed with argon gas and incubated at 30C for 2 h. Tho resulting~
Fab' fragment~ are purified by gel filtration on Sephadex G-25. 53 mg of Fab' fragments are obtained.
14.6 mg of bovine serum albumin are dissolved in 1.8 ml of water. 7.2 mg of SMCC in 0.18 ml of dioxane are added to thi~ solution and incubated at 30C for 1 h. The resulting BSA-SMCC con~ugate is purified by gel chroma-tography on Sephadex~G-25. 14.3 mg of BSA-SMCC con~ugate are obtained. T~he solution of the Fab' fragments is com~ined under argon gas with the BSA-SMCC con~ugate solution and incubated at room temperature for 18 h.
0.25 ml of an 11% cysteamine hydrochloride solution i~
subsequently added. The reaction mixture is concentrated to 8 ml in an ultrafiltration cell. The reaction mixture i~ purified by gel filtration chromatography ~for example Superdex~ Pharmacia/LRB, 150 mM RCl, 20 mM phosphate buffer, 1 mM EDTA, 0.02~ NaN3, pH = 7.0). The fractions which contain the required product are combined ~21 ml).
6~7 ~3l~3 Example 4 TurbidLmetric immunoassay with anti-CRP Fab'-albumin conjugate Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 6% polyethylene glycol 6000, 0.1~ CHAPS, 0.1% TRITONP X-100, 0.02% NaN3 Fab'-albumin:
Prepared as in Example 3 CRP:
Human CRP is dissolved in 0.1 M TRIS HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l CRP are prepared by serial dilution.
Apparatus ad~ustments The assay is carried out as endpoint assay in an EPOS 5060 automatic analyser for clinical chemical assays. Firstly 20 ~l of sample (CRP solution) and 220 ~l of reaction buffer are pipetted, and the mixture is preincubated at 37C for 64 sec. Then 30 ~l of Fab'-albumin con~ugate are pipetted in, mixed and the result-ing extinction i3 measured after 288 sec. The apparatuCcycle i8 set at 16 sec. The extinction at the end of the preincubation time, before addition of the start reagent, iB used to determine the sample blank.
The resu:Lting calibration plot (extinction/con~
centration) is depicted in Fig. 2.
Example 5 Preparation of an anti-transferrin Fab~-thyroglobulin con~ugate Anti-transferrin IgG (sheep) i~ treated with pepsin in analogy to Example 1. The F(ab~ )2 fragments are purified by gel filtration chromatography. 1.3 ml of a ~olution of 41.5 mg of these F(ab~) 2 fragments in phosphate buffer (20 mM phosphate, lS0 mM KCl, pH = 7.0) ~7~
are mixed with 0.2 ml of 1 M acetate buffer, pH = 5.0, and blanketed with argon gas. 0.1 ml of an 11% cysteamine hydrochloride solution in degassed water is added and incubated at 37C for 2 h. The resulting Fab~ fragments are purified by gel chromatography on Sephade ~G-25. The yield is 34.6 mg of Fab' fragments in 2 ml of elution buffer.
25 mg of thyroglo]bulin (pig) are dissolved in 3 ml of phosphate buffer (20 mN phosphate, 150 m~ RCl, pH = 7.0). 0.1 ml of a solution of 27 mg/ml SMCC in dioxane i5 added, and the mixture is incubated at 30C
for 30 min. The reaction mixture i8 purified by gel chromatography on Sephadex~G-25. The solution of the Fab' fragments is added in portions to the coupling product and incubated at room temperature overnight.
The rasulting thyroglobulin-(Fab' )n con~ugates are purified by gel filtration chromatography (150 mM
KCl, 20 mM phosphate buffer, 1 mM EDTA, 0.02% NaN3, pH =
7.0). The fractions which contain the required product are combined (24 ml).
Example 6 Turbidimetric immunoassay with anti-transferrin Fab'-thyroglobulin con~ugate Reaction buffer:
0.1 M TRIS HCl ~buffer, pH = 7.6, 5% polyethylene glycol 6000, 0.1% CHAPS, 0.1% TRITON^, 0.02~ NMN3 -Fab'-thyroglobuli.ns Prephred as in Example 5 Tran~ferrins Human transferrin is dissolved in 0.1 M TRIS -HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l transferrin are prepared by serial dilution.
- 10 ~ J~ 3 Apparatus ad~ustment:
The assay is carried out as endpoint assay in an EPOS 5060 analy er. Firstly 15 ~1 of example (transferrin solution) and 235 ~1 of reaction buffex are pipetted, and the mixture is preincubated at 37C for 64 sec. Subse-quently 60 ~1 of Fab~-thyroglobulin con~u~ate are pipetted in, mixed and the resulting extinction is measured after 288 sec. The apparatus cycle is set at 16 sec. The extinction at the end of the preincubation time, before addition of the start reagent, is used to deter-mine the sample blank.
The resulting calibration plot (extinction/con-centration) is depicted in Fig. 3.
Example 7 Comparative experiment to determine transferrin Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 5~ polyethylene glycol 6000, 0.1% CHAPS, 0.1~ TRITONa X-100, 0.02% sodium azide Fab'-albumins Prepared as in Example 1 Anti-transferrin IgG:
Starting material of Example 1 Tran~ferrins ~ Human transferrin i8 dissolved in 0.1 M TRIS ~
HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l tran~ferrin are prepared by serial dilution.
Apparatus ad~ustments The assay i8 carried out as endpoint assay in an EPOS 5060 analy~er. Pirstly 20 ~1 of sample (transferrin solution) and 235 ~1 of reaction buffer are pipetted, and the mixture i8 preincubated at 37C for 64 sec. Sub~e-quently 50 ~1 of Fab'-albumin con~ugate or anti-~7 ~
transferrin IgG are pipetted in, mixed and the resultingextinction is measur~d after 288 sec. The apparatus cycle is set at 16 sec. The extinction at the end of the preincubation tLme, before addition of the start reagent, is used to determine the sample blank.
The resulting calibration plots (extinction/con-centration) with Fab'-albumin con~ugate and anti-trans-ferrin IgG are depicted in Fig. 4. Fig. 5 shows a section of these calibration plotR for low transferrin concen-trations. The measurement signals obtained with the Fab'-albumin conjugate are distinctly higher than with natural anti-transferrin IgG under conditions which are otherwise the same.
Example 8 Nephelometric immunoassay with anti-CRP Fab'-albumin con~ugate Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 5% polyethylene glycol fiO00, 0.1% CHAPS, 0.1% TRITON~ X-100, 0.02~ sodium azide.
Fab'-albumin:
Prepared as in Example 3 CRP:
Human C~ is dis~olved in 0.1 M TRIS HCl buffer, pH = 7.6. ConcentratLons in the range to 77.5 mg/l CRP are prepared by serial dilution.
Apparatu~ ad~ustments:
The assay i~ carried out as manual endpoint assay in a fluorescence spectrophotometer (HITACHI F 4000). ~he apparatus measures the intensities of scattered light at an angle of 90. The intensities are reported as arbi-trary units (a.u.~. The wavelength~ of excitation and emission are set at 340 nm. The slit width iR set at 3 mm. After preced.ing zero ad~ustment, an expansion of 12 ~7~3~
0-9999 a.u. is chosen.
200 ~1 of sample and 2500 ~1 of reaction buffer are pipetted into a 1 cm fluorescence cuvette, mixed and preincubated at room temperature for 90 sec. Subsequently 300 ~1 of Fab'-albumin con~ugate are pipetted in, mixed, and the increase in the scattered light inten~ity is followed for 10 min. The resulting measurements are plotted again3t the CRP concentrations.
The calihration plot (scattered light intensity as a.u./concentration) is depicted in Fig. 6.
The carrier-crosslinker con~ugate can be prepared without problems in a batch process and be stored frozen in solution or lyophilised. It is likewise possible for the F(ab' )2 fragments to be prepared in a batch process and to be ~tored as described above. Only steps 3, 4, 7 and 8 are then necessary for the actual preparation.
Compared with known reagents, in the turbidi-metric and nephelometric a~say the reagents prepared in thi~ way give distinctly higher measurement signals an~
thus allow the sensitivity of detection to be increased.
The sensitivity of detection can be further optimised by the presence of known accelerators (for example poly-ethylene glycols). The reagents prepared in this way are stable in solution, they can be employed without problems in automatic analysers, and they are thus also suitable for automated immunoturbidimetric determination of analyte~.
Both the immunoturbidimetric and the immuno-nephelometric del:ermination i8 carried out at 320-400 nm, preferably at about 340 nm. The determination can be carried out klnetically but preferably a~ endpoint reactlon.
The method and agents according to the present invention can be u~ed to determine all antigen~, that is to say substance~ which have a plurality of binding sites for antibodies. Examples of proteins of thi~ type are transferrin, CRP, ~l-microglobulin, RF, TBG, RBP, C3, C4, Igs etc. which occur in body fluids and are of diagnostic relevance. These proteins are measured in the concen-tration range of 0.05 - > 100 mg/l, preferably 0.5 -100 mg/l, without further prior dilution. Haptens which nave only one binding site are amenable to competitive - 5 ~ 3 ~ 3 determination using so-called polyhaptens.
Example 1 Preparation of an anti-transferrin Fab~-albumin conjugate 3.2 g of sheep anti-tran ferrin IgG are taken up in 150 ml of 0.9 % NaCl solution. The solution is adjusted to pH = 4.5 with 1 M acetic acid. 80 mg of pepsin are added to this ~olution, and the mixture is incubated at 37C for about 16 h. The reaction solution is ad~usted to pH = 7.0 with 1 M TRIS solution. The reaction ~olution is purified by affinity chromatography using a Sepharos~4~ column to which human transferrin is covalently bonded. The bound F(ab')2 fragments are eluted with 0.1 M glycine/HCl (pH = 2.8). The protein-containing fractions are pooled and ad~usted to pH = 7.0 with 1 M
TRIS solution. The solution is concentrated to 30 ml in an ultrafiltration cell. After photometric determination 376 mg of specific F(ab' )2 fragments are obtained.
16 ml of this solution are ad~usted to pH = 4.9 with 1 M acetic acid, deaerated and blanketed with argon and mixed with 0.9 ml of an 11% solution of cysteamine hydrochloride in water. After incubation at 37C for 2 h, the Fab' fragments are purified by gel chromatography on Sephadex G-25 (150 mM NaCl, 20 mM phosphate buffer, pH =
7.0).
The protein-containing fractions are pooled.
After photometric determination, 190 mg of Fab' fragments are obtained in 34 ml of ~olution. The ~olution i~ stored in ~he cold under argon gas.
40.5 mg of bovine ~erum albumin are dissolved in 7 ml of water, snd 1 ml of a ~olution of 30 mg of suc-cinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC) in dioxane i~ added. The reaction solution is incubated at 30C for 1 h and then purified by gel chromatography on Sephadex~G-250 The protein-containing fractions are pooled. After photometric determination, 38 mg of the coupling product are obtained in 14.8 ml of solution. The Fab' solution is mixed under argon gas with - 6 _ s~7~
the solution of the coupling product and incubated at room temperature for 17 h.
The reaction mixture is purified by gel fil-tration chromatography (for exampls Superdex@~
Pharmacia/L~B, 150 mM KCl, 20 mM pho~phate buffer, 1 mM
EDTA, 0.02 ~ NaN3, pH = 7.0). The fractions which contain the required product are combined.
Example 2 TurbidLmetric Lmmunoassay with anti-tran~ferrin Fab~-albumin con~ugate.
Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 6~ polyethylene glycol 6000, 0.1~ CHAPS, O.1~ TRITON~ X-100, 0.02~ NaN3.
Fab' albumins Prepared as in Example 1.
Transferrin:
Human transferrin was dissolved in 0.1 M TRIS
HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l transferrin were prepared by serial dilution.
Apparatus ad~u~tments The aB8ay i8 carried out a4 endpoint assay in an EPOS 5060 automatic analyser for clinical chemical assay~. Firstly 40 ~1 of sample (tran~ferrin ~olution) and-200 ~1 of re~action bufer are pipetted, mixed and preincubated at 37~C for 64 sec. Then 30 ~1 of Pab'-albumin con~ugate are pipetted in, mixed and the re~ult-ing extinction i~ measured after 288 sec. The apparatus cycle is set at 16 sec. The extinction at the end of the preincubation time, before addition of the start reagent, i~ u8ed to determine the sample blank. The resulting calibration plot (extinction/concentration) i~ depicted in Fig. 1.
- 7 _ 2~43~
Example 3 Preparation of an anti-CRP Fab~-albumin conjugate 250 mg of lyophilised sheep anti-CRP IgG are dissolved in 20 ml of a 150 mM NaCl solution. The 601u-tion is mixed with 4 ml of 1 M sodium acetate buffer,pH - 4.9. The pH of the solution is ad~usted to pH = 4.35 with 1 M acetic acid. 6.3 mg of pep~in are added, and the mixture is incubated at 37~C for about 16 h. The result-ing F(ab' )2 fragments are purified by gel filtration chromatography (for example Superdex~ Pharmacia/LRB, 20 mM potassium phosphate buffer, pH = 7.0, 150 mM RCl).
The combined fractions contain 122 mg of F~ab' )2 frag-ments. The pool is concentrated to 12 ml in an ultrafil-tration cell (12.3 mg/ml F(ab') 2 ) ' 6 ml of this solution (73.3 mg F(ab' )2 are mixed ~
with 1 ml of 1 M sodium acetate buffer, pH = 4.9. The pH
is ad~usted to 5.0 with 1 M acetic acid. 0.35 ml of an aqueous 11~ cysteamine hydrochloride solution is added to this solution. The reaction mixture is blanketed with argon gas and incubated at 30C for 2 h. Tho resulting~
Fab' fragment~ are purified by gel filtration on Sephadex G-25. 53 mg of Fab' fragments are obtained.
14.6 mg of bovine serum albumin are dissolved in 1.8 ml of water. 7.2 mg of SMCC in 0.18 ml of dioxane are added to thi~ solution and incubated at 30C for 1 h. The resulting BSA-SMCC con~ugate is purified by gel chroma-tography on Sephadex~G-25. 14.3 mg of BSA-SMCC con~ugate are obtained. T~he solution of the Fab' fragments is com~ined under argon gas with the BSA-SMCC con~ugate solution and incubated at room temperature for 18 h.
0.25 ml of an 11% cysteamine hydrochloride solution i~
subsequently added. The reaction mixture is concentrated to 8 ml in an ultrafiltration cell. The reaction mixture i~ purified by gel filtration chromatography ~for example Superdex~ Pharmacia/LRB, 150 mM RCl, 20 mM phosphate buffer, 1 mM EDTA, 0.02~ NaN3, pH = 7.0). The fractions which contain the required product are combined ~21 ml).
6~7 ~3l~3 Example 4 TurbidLmetric immunoassay with anti-CRP Fab'-albumin conjugate Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 6% polyethylene glycol 6000, 0.1~ CHAPS, 0.1% TRITONP X-100, 0.02% NaN3 Fab'-albumin:
Prepared as in Example 3 CRP:
Human CRP is dissolved in 0.1 M TRIS HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l CRP are prepared by serial dilution.
Apparatus ad~ustments The assay is carried out as endpoint assay in an EPOS 5060 automatic analyser for clinical chemical assays. Firstly 20 ~l of sample (CRP solution) and 220 ~l of reaction buffer are pipetted, and the mixture is preincubated at 37C for 64 sec. Then 30 ~l of Fab'-albumin con~ugate are pipetted in, mixed and the result-ing extinction i3 measured after 288 sec. The apparatuCcycle i8 set at 16 sec. The extinction at the end of the preincubation time, before addition of the start reagent, iB used to determine the sample blank.
The resu:Lting calibration plot (extinction/con~
centration) is depicted in Fig. 2.
Example 5 Preparation of an anti-transferrin Fab~-thyroglobulin con~ugate Anti-transferrin IgG (sheep) i~ treated with pepsin in analogy to Example 1. The F(ab~ )2 fragments are purified by gel filtration chromatography. 1.3 ml of a ~olution of 41.5 mg of these F(ab~) 2 fragments in phosphate buffer (20 mM phosphate, lS0 mM KCl, pH = 7.0) ~7~
are mixed with 0.2 ml of 1 M acetate buffer, pH = 5.0, and blanketed with argon gas. 0.1 ml of an 11% cysteamine hydrochloride solution in degassed water is added and incubated at 37C for 2 h. The resulting Fab~ fragments are purified by gel chromatography on Sephade ~G-25. The yield is 34.6 mg of Fab' fragments in 2 ml of elution buffer.
25 mg of thyroglo]bulin (pig) are dissolved in 3 ml of phosphate buffer (20 mN phosphate, 150 m~ RCl, pH = 7.0). 0.1 ml of a solution of 27 mg/ml SMCC in dioxane i5 added, and the mixture is incubated at 30C
for 30 min. The reaction mixture i8 purified by gel chromatography on Sephadex~G-25. The solution of the Fab' fragments is added in portions to the coupling product and incubated at room temperature overnight.
The rasulting thyroglobulin-(Fab' )n con~ugates are purified by gel filtration chromatography (150 mM
KCl, 20 mM phosphate buffer, 1 mM EDTA, 0.02% NaN3, pH =
7.0). The fractions which contain the required product are combined (24 ml).
Example 6 Turbidimetric immunoassay with anti-transferrin Fab'-thyroglobulin con~ugate Reaction buffer:
0.1 M TRIS HCl ~buffer, pH = 7.6, 5% polyethylene glycol 6000, 0.1% CHAPS, 0.1% TRITON^, 0.02~ NMN3 -Fab'-thyroglobuli.ns Prephred as in Example 5 Tran~ferrins Human transferrin is dissolved in 0.1 M TRIS -HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l transferrin are prepared by serial dilution.
- 10 ~ J~ 3 Apparatus ad~ustment:
The assay is carried out as endpoint assay in an EPOS 5060 analy er. Firstly 15 ~1 of example (transferrin solution) and 235 ~1 of reaction buffex are pipetted, and the mixture is preincubated at 37C for 64 sec. Subse-quently 60 ~1 of Fab~-thyroglobulin con~u~ate are pipetted in, mixed and the resulting extinction is measured after 288 sec. The apparatus cycle is set at 16 sec. The extinction at the end of the preincubation time, before addition of the start reagent, is used to deter-mine the sample blank.
The resulting calibration plot (extinction/con-centration) is depicted in Fig. 3.
Example 7 Comparative experiment to determine transferrin Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 5~ polyethylene glycol 6000, 0.1% CHAPS, 0.1~ TRITONa X-100, 0.02% sodium azide Fab'-albumins Prepared as in Example 1 Anti-transferrin IgG:
Starting material of Example 1 Tran~ferrins ~ Human transferrin i8 dissolved in 0.1 M TRIS ~
HCl buffer, pH = 7.6. Concentrations in the range to 100 mg/l tran~ferrin are prepared by serial dilution.
Apparatus ad~ustments The assay i8 carried out as endpoint assay in an EPOS 5060 analy~er. Pirstly 20 ~1 of sample (transferrin solution) and 235 ~1 of reaction buffer are pipetted, and the mixture i8 preincubated at 37C for 64 sec. Sub~e-quently 50 ~1 of Fab'-albumin con~ugate or anti-~7 ~
transferrin IgG are pipetted in, mixed and the resultingextinction is measur~d after 288 sec. The apparatus cycle is set at 16 sec. The extinction at the end of the preincubation tLme, before addition of the start reagent, is used to determine the sample blank.
The resulting calibration plots (extinction/con-centration) with Fab'-albumin con~ugate and anti-trans-ferrin IgG are depicted in Fig. 4. Fig. 5 shows a section of these calibration plotR for low transferrin concen-trations. The measurement signals obtained with the Fab'-albumin conjugate are distinctly higher than with natural anti-transferrin IgG under conditions which are otherwise the same.
Example 8 Nephelometric immunoassay with anti-CRP Fab'-albumin con~ugate Reaction buffer:
0.1 M TRIS HCl buffer, pH = 7.6, 5% polyethylene glycol fiO00, 0.1% CHAPS, 0.1% TRITON~ X-100, 0.02~ sodium azide.
Fab'-albumin:
Prepared as in Example 3 CRP:
Human C~ is dis~olved in 0.1 M TRIS HCl buffer, pH = 7.6. ConcentratLons in the range to 77.5 mg/l CRP are prepared by serial dilution.
Apparatu~ ad~ustments:
The assay i~ carried out as manual endpoint assay in a fluorescence spectrophotometer (HITACHI F 4000). ~he apparatus measures the intensities of scattered light at an angle of 90. The intensities are reported as arbi-trary units (a.u.~. The wavelength~ of excitation and emission are set at 340 nm. The slit width iR set at 3 mm. After preced.ing zero ad~ustment, an expansion of 12 ~7~3~
0-9999 a.u. is chosen.
200 ~1 of sample and 2500 ~1 of reaction buffer are pipetted into a 1 cm fluorescence cuvette, mixed and preincubated at room temperature for 90 sec. Subsequently 300 ~1 of Fab'-albumin con~ugate are pipetted in, mixed, and the increase in the scattered light inten~ity is followed for 10 min. The resulting measurements are plotted again3t the CRP concentrations.
The calihration plot (scattered light intensity as a.u./concentration) is depicted in Fig. 6.
Claims (7)
1. Method for the turbidimetric or nephelometric determination of analytes in liquids with the aid of an antibody-binding reaction, which is characterised in that a polypeptide is reacted with antibodies or fragments thereof, the coupling product resulting therefrom is incubated with the analyte, and the resulting turbidity is measured.
2. Method according to Claim 1, characterised in that a protein with a molecular weight of at least 10,000 D is used as polypeptide.
3. Method according to Claims 1 to 2, characterised in that a globular protein is used as polypeptide.
4. Method according to Claims 1 to 3, characterised in that albumin, thyroglobulin, transferrin, haemocyanin is used as polypeptide.
5. Method according to Claims 1 to 4, characterised in that Fab' fragments are used as fragments.
6. Agent for the turbidimetric or nephelometric determination of analytes in liquids, containing a coupling product composed of a polypeptide and antibodies or fragments thereof.
7. Agent according to Claim 6, containing a coupling product composed of a globular protein and Fab' fragments.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4124324.2 | 1991-07-23 | ||
| DE4124324A DE4124324A1 (en) | 1991-07-23 | 1991-07-23 | METHOD AND MEANS FOR TURBIDIMETRIC OR NEPHELOMETRIC DETERMINATION OF ANALYZES |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2074345A1 true CA2074345A1 (en) | 1993-01-24 |
Family
ID=6436778
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002074345A Abandoned CA2074345A1 (en) | 1991-07-23 | 1992-07-21 | Method and agent for the turbidimetric or nephelometric determination of analytes |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0524583B1 (en) |
| JP (1) | JPH05203579A (en) |
| CA (1) | CA2074345A1 (en) |
| DE (2) | DE4124324A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053838A (en) * | 2016-07-12 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining transferrin and preparation method thereof |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5728589A (en) * | 1993-08-24 | 1998-03-17 | Wako Pure Chemical Industries, Ltd. | Immunoassay method |
| GB9713559D0 (en) * | 1997-06-26 | 1997-09-03 | Axis Biochemicals Asa | Assay |
| WO2002046755A1 (en) * | 2000-12-04 | 2002-06-13 | Matsushita Electric Industrial Co., Ltd. | Method of determining solution concentration and method of examining urine |
| WO2004036214A2 (en) * | 2002-10-18 | 2004-04-29 | Pfizer Products Inc. | Automated kinetci solubility assay apparatus and method |
| CN104407154B (en) * | 2014-12-05 | 2016-03-30 | 重庆中元生物技术有限公司 | A kind of high sensitivity latex enhancing immune turbidimetry kit |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4657853A (en) * | 1984-09-14 | 1987-04-14 | E. I. Du Pont De Nemours And Company | Immunoassays utilizing covalent conjugates of polymerized enzyme and antibody |
| EP0324015A4 (en) * | 1987-07-21 | 1990-11-07 | Coulter Electronics Inc. | Improved turbidimetric rate inhibition assay for haptens |
| US5051356A (en) * | 1988-06-13 | 1991-09-24 | Eastman Kodak Company | Specific binding composition comprising a low pI protein or carbohydrate and a diagnostic test kit and method of use |
-
1991
- 1991-07-23 DE DE4124324A patent/DE4124324A1/en not_active Withdrawn
-
1992
- 1992-07-20 DE DE59209232T patent/DE59209232D1/en not_active Expired - Fee Related
- 1992-07-20 EP EP92112384A patent/EP0524583B1/en not_active Expired - Lifetime
- 1992-07-21 JP JP4194017A patent/JPH05203579A/en active Pending
- 1992-07-21 CA CA002074345A patent/CA2074345A1/en not_active Abandoned
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053838A (en) * | 2016-07-12 | 2016-10-26 | 安徽伊普诺康生物技术股份有限公司 | Kit for determining transferrin and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| EP0524583A1 (en) | 1993-01-27 |
| DE4124324A1 (en) | 1993-01-28 |
| DE59209232D1 (en) | 1998-04-23 |
| EP0524583B1 (en) | 1998-03-18 |
| JPH05203579A (en) | 1993-08-10 |
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