GB2362211A - Detecting circulating immune complexes using protein G - Google Patents

Abstract

The method involves precipitating circulating immune complexes (CIC) from a body fluid sample, preferably using PEG and EDTA, diluting the precipitate and reacting it with immobilised protein G, preferably immobilised on microtitre wells. Bound CIC are detected, for example by ELISA.

Description

2362211 1 A sensible and reproducible assay for circulating immune

complexes using Protein G.

coated substrates.

2 Prior art and Background of the invention

Circulating immune complexes (CIC) are detectable in a variety of systemic disorders such as rheumatological and auto-immune diseases (1-4), allergic diseases (5), viral, bacterial and parasitic infections (1,6-8), and malignancies (2,9). A variety of tests that can detect CIC have been described (10-18). The assays that are commonly used in clinical laboratories are based on (i) precipitation of CIC by polyethylene glycol, (ii) interaction of CIC with complement (Clq assay), (iii) detection of CIC bound to C3 (Raji cell assay, conglutinin assay), and (iv) binding of CIC by autoantibodies such as the monoclonal rheumatoid factors, which are known to interact with IgG (mRF assay). In a comparative WHO study (10), only a few tests were described as sufficiently sensitive and reproducible. Reliable methods were the C I q solid-phase binding assay, the conglutinin assay, the monoclonal rheumatoid factor (mRF) assay and the Raji cell assay. No single assay is capable of detecting all CIC (10). Therefore, the WHO recommends the use of at least two assays that are sensitive, reliable, and based on different immunological properties. The combined use of C I q and mRF-SP RIA allows the detection of both complement-fixing and non-complement fixing immune complexes and a wide range of sizes of complexes (12,19). Commercial ELISA kits use complement factors or monoclonal anti-comPlement antibodies (Clq, maClq, CM or maC3d) for the detection of immune complexes. However, a good ELISA alternative for the mRF assay is not commercially available. This invention presents for the first time an immuno assay where Protein G, which like mRF is based on Fc recognition, is used as capture molecule for detection of CIC.

3 Summary of the invention

The present invention relates to a novel method and an apparatus for detection and quantification of circulating immune complexes (CIC) using Protein G coated substrates. More particularly the invention involves a simple and sensitive enzyme linked immuno-sorbent assay (ELISA) for the detection of circulating immune complexes in bodily fluids of said subject. It is an alternative for the monoclonal rheumatoid factor solidphase binding radio-immuno-assay. The method is based on precipitating the CIC, capturing the CIC on a surface coated with streptococcal protein, said protein G (Prot G), and the detection or quantification of the CIC by enzyme labelled anti-IgG immunoglobulin which binds to IgG of test subject.

The test kit for said CIC comprises strips or Prot G or microtiter plates coated with said Prot G, CIC positive control serum, CIC negative control serum, CIC standard, anti-IgG conjugated with a measurable label and preferably with conjugated labelling enzyme and further comprising a chromogenic enzyme substrate, and colour reaction stop solution. The compounds or conjugates of test kit, can be in separate packages in the form of solutions or a dry powder.

The method and test kit can be used as a screening or analysis method for detecting blood borne CIC caused by a variety of systemic disorders such as rheumatological and autoimmune diseases, allergic diseases, viral, bacterial and parasitic infections, and malignancies.

This invention, thus involves a method to detect or quantify circulating immune complexes (CIC) in the body fluid of specimen, comprising the steps of forming a coating of protein G on a substrate, treating said body fluid in a preselected manner to precipitate CIC present in fluid, isolating said immune complexes from the body fluid, mixing said isolated immune complexes with a suitable vehicle to produce a preselected dilution, permitting said isolated immune complexes to be captured on the protein G coated substrate, treating said affixed CIC in a preselected manner to produce an indication of the concentration of the CIC present in said specimen Furthermore this invention involves an ELISA diagnostic kit for the detection or quantification of circulating immune complexes (CIC) in the serum; comprising in separate packaging, at least one of the following: protein G, a positive control vial of serum of a same species as test subject, a 4 negative control vial of serum of a same species as test subject, a known quantity of isolated CIC of a same species as test subject for preparation of standard and a detectably labelled conjugate which binds to IgG of test subject Detailed embodiment of the invention The following definitions and abbreviations are supplied for the purpose of clarifying aspects of this invention.

Abbreviations:

RIA = radio-immuno-assay; PEG = polyethyleneglycol; IgG-Fab: Fab fragment of immunoglobulin G; IgG = immunoglobulin G; Fab = the antigen binding fragment of an immunoglobulin molecule consisting of both a light chain and a part of a heavy chain; Fc = the crystallisable fragment of an immunoglobulin molecule composed of part of a heavy chain and responsible for the binding to antibody receptors on cells and the Clq component of complement.; CIC = circulating immune complexes; BSA = bovine serum albumin; DMF = dimethylformamide; DMSO = dimethylsulfoxide; EDTA ethylene diamine tetraacetic acid; ELISA = enzyme-linked immunosorbent assay; PBS phosphate-buffered saline; PBST-Tween 20 = phosphate-buffered saline with Tween 20; HRP horse radish peroxidase; TMB = tetramethylbenzidine; OD = Optical Density Definitions:

Antibody: A member of the Immunoglobulin class of proteins. Mammalian antibody molecules comprise at least two Fab and one Fc region in their structures.

Immunoglobulin: Any member of the gammaglobulin fraction possessing chemical ability to bind to another agent. Such agents include proteins, carbohydrates, nucleic acids, complex lipids, simple organic compounds, or any other compound interacting with the immunoglobulin through topographically determined binding at the "Fab" region.

Antigen: Any compound against which antibody molecules may be directed provided binding occurs to the antibody at the binding site found in the Fab portion of the antibody molecule.

Immune complex: Any combination of antibodies and antigens in which the binding occurs through interaction between Fab sites on the antibody and topographic features of the antigen.

Serum: Is intended to mean the fluidic component of any bodily fluid remaining after cells and coagulable proteins such as fibrin, which may be present in such bodily fluidic components, have been removed by appropriate physical, chemical, or physicochemical means. Typically, this term refers to the residual watery fluid remaining after clotting of blood and removal of the clot, but in its broad sense is intended to include the fluidic component of cerebrospinal fluid, urine, interstitial fluid, cellular cytoplasm, and the like.

Phosphate buffered saline: Sodium chloride was added to water to the final concentration of 9 g/l. To this solution was added 0.01 moles of potassium phosphate (monobasic). The pH was adjusted to 7.4 and the solution volume brought to exactly 1.00 liter by the addition of dernineralised water.

Tween-20: A polyoxyethylene sorbitan monolaureate: In the description, this compound was added to phosphate buffered saline for some steps. When indicated,Tween-20 was used at a concentration

6 of 0.05% (v/v).

Non-ionic detergents such as Tween-20 are used to prohibit adventitious binding of proteins.

Conjugated antibodies: For the enzyme immunoassay portion of the determination, antibodies directed against human immunoglobulin fractions are used. These antibodies had been chemically conjugated with horseradish peroxidase to serve as a detection agent.

Substrate solution: To quantitate the horseradish peroxidase, a solution of TMB was used. It is clear for the man skilled in the art that any enzyme that is suitable to activate a chromogenic substrate into a pigment can be used to produce an indication of the concentration of CIC. A chromogenic substrate is a substance, itself without colour that may be transformed into a pigment.

Labelled Antibodies: Any antibody substance which has bound covalently or otherwise contained with a molecule or ion for the purpose of selectively identifying that group of antibodies. Such adduct molecules or ions include enzymes, fluorescent substances, radionucleotides, and the like.

Optical Density (OD) or Absorbance: A number, which refers to the colour absorbance of a sample. Optical density is related to the percent of light transmitted through the sample by the following formula: OD = 2 log(percent transmittance) Polyethylene glycol: is a condensation polymer of ethylene oxide and water, of the general formula HO(CH2CH20),H, where n equals the average number of oxyethylene groups (3006,000). They vary in consistency based on molecular weight.

Protein G: is a surface receptor from Streptococcus sp.. It is a highly stable monomeric protein, which is capable of binding the Fc portion of immunoglobulins, especially IgGs, from a large number of species. Recombinant Protein G is available.

7 CIC positive sera and CIC negative sera: are respectively sera from subjects, preferably mammals that have respectively a detectable or no detectable level of circulating immune complexes.

Concordance analysis: agreement between test results based on class. A difference of one class is considered concordant, a difference of more than one class is considered discordant.

Blocking agent: agent used in ELISA for blocking possible excess solid surface after coating with one immuno-reactant to avoid unspecific immobilization of succeeding reactants.

The assay of present invention is a solid-phase immunosorbent assay carried out on substrate, preferably microtiter plates, which have been coated with protein G. Protein G from group G Streptococcus is a cellsurface protein that binds the Fc portion of all subclasses of IgG antibody molecules (20, 21).

In the first stage, diluted CIC, which are obtainable from precipitation, preferably by PEG, and preincubated with EDTA in order to remove endogenous complement from the serum, are added to the wells coated with protein G and incubated for a specified length of time. If CIC are present they will attach to protein G. Unbound proteins are removed in the subsequent washing step.

In the second stage, horseradish peroxidase (HRP)-conjugated goat antihuman IgG-Fab 4n (conjugate) is added to each test well. The conjugate will bind to the immune complexes bound to protein G on the wells. A wash cycle removes unbound conjugate.

In the third stage, an enzyme substrate is added to each of these wells. The bound HRPconjugated antibody reacts with the chromogenic substrate resulting in a coloured end product. After incubation, a reagent is added to stop colour development. Absorbances are measured spectrophotometrically. The colour formed is proportional to the amount of CIC binding to the solid phase. The concentration of immune complexes present in the test specimen is determined by reference to 15 normal controls.

EXAMPLES

The following Examples are included solely to provide a more complete understanding of the invention. The Examples do not limit the invention described and claimed herein in any fashion.

EXAMPLE 1 Process of analysing serum for CIC Coating Hundred tl of protein G, at a concentration of 0.1 gg/ml in PBS, is added to the wells of a microtiter plate (Nunc Immuno TM plate, MaxiSorpTm Surface, Life Technologies) and incubated overnight at 4"C.

Sera Serum (50 gl) is incubated with 150 gl EDTA (Merck) 0,2 M for 30 min at 37C. Afterwards, I ml PEG 6% (polyethylene glycol 6000, BDH 29577) is added. The mixture is stored overnight at 4'C. The serum is centrifuged for 20 min at 1000g. One ml PEG 5% is added to the pellet. After mixing, serum is centrifuged again for 20 min at 1000g. Phosphate buffered saline (PBS) 200 d is added to the pellet and mixed for 20 min. Then the precipitate is diluted 1/30 with PBS containing 2% goat serum (Life Technologies).

ELISA Wells are washed 3 times with cold PBS and incubated for I h at room temperature with 100 gl 15% goat serum in PBS. After washing 4 times with PBS containing 0.05% (v/v) Tween 20, 100 gI of the diluted precipitate is added in duplicate to the wells and incubated for I h at room temperature. Wells are washed again with PBS -Tween 20. Afterwards, 100 gI of peroxidase conjugated goat anti-human IgG-Fab (Nordic Irnmunological Laboratories) diluted 1/5000 in PBS 2% goat serum is added to all wells and incubated for lh at room temperature After another wash cycle with PBS-Tween 20, wells are incubated for 15 min at 9 room temperature with 100 gl of TMB (DAKOR TMB One-step substrate system) substrate. The reaction is stopped by adding 100 gl of stop solution (I N HCI + 3 N H2SO4). Reactions are read immediately at 450 nm.

Calculation of results. After subtraction of the blank optical density (OD), the mean OD and the standard deviation (SD) of the 15 normal sera is calculated. Results (OD) of patient sera are then expressed as the percentage of this mean and graded negative when OD is less than the mean + 2 SD, equivocal when between the mean + 2 SD and 3 SD, positive + when between the mean + 3SD and 4 SD, positive ++ when between the mean + 4 SD and 5 SD, positive... when between the mean + 5 SD and 6 SID, and positive 1 l 1 + when more than 6 SD.

EXAMPLE2

Blocking of aspecific bing In order to prevent aspecific binding to the micotiter plate, we tested whether we could block free binding sites either with 1 % BSA or with 10% goat serum. As can be seen in the Table, no dose-dependent response could be observed when blocking was performed with 1% BSA. This was the case in the condition in which conjugate and serum were diluted in Tween 0.05% as well as in the condition in which the dilution was done with BSA. The aspecific binding, however, was higher in the condition in which serum was diluted with Tween than in the condition in which the serum was diluted with BSA. When blocking was performed with 10% goat serum, a dose- dependent response was observed and when serum and conjugate were diluted in 2% goat serum but not when serum and conjugate were diluted in I % BSA. From these data it is obvious that the use of goat serum as blocking agent and as diluent for conjugate and serum is necessary in order to prevent aspecific binding in the CIC assay.

Table: Inhibition of aspecific binding Blocking agent PBS + 1% BSA PBS + 10% goat serum Conjugate diluent PBS+TweenO.05% PBS+l%BSA PBS + 1% BSA PBS + 2% wat C C Serum diluent serum Serum 1/100 2832 OD 252 OD 246 OD 1271 OD Serum 11500 2830 266 231 1078 Serum 1/1000 2880 270 224 854 No serum 2852 469 223 441 EXAMPLE3

Evaluation of the analysis method Results Precision 10 different sera were assayed in duplicate on the same day (Table 1). The coefficient of variation within assay was 3%.

Table 1: Intra-assay variability Serum Mean Optical number Density (OD) 1 890 2 875 3 833 4 850 844 6 867 7 867 8 855 9 857 805 sera, 5 positive and 5 negative sera, were assayed on 4 different occasions. Results are shown in table 2. There is a great difference in variability between samples with low and high optical densities. Therefore, coefficient of variation is calculated separately for positive and negative sera. The mean variation coefficient was 10% for positive sera and 33% for negative sera.

12 Table 2: Intra-assay variability Serum Day 1 Day 2 Day 3 Day 4 % CV number 1 1638 1519 1411 1297 10 2 1302 1189 1094 1106 8 3 1350 1207 1060 1024 13 4 1248 1119 883 881 18 811 800 823 784 2 6 554 717 347 464 31 7 593 357 414 266 35 8 282 266 335 239 15 9 230 197 264 129 29 174 99 223 29 83 Linearity A twofold dilution series was prepared from a positive serum in ELISA Results are represented in Table 3 and in figure 1. The dilution curve is an S-shaped curve. The linear part of the curve is situated between dilution 1/4 to 1/512. The dilution used in our assay is 1/30 and is located in the middle of the linear part of the curve.

Table 3: Dilution of a positive serum in ELISA Dilution factor OD 1 OD2 Mean OD 1 2828 2850 2839 2 2750 2761 2756 4 2713 2618 2666 8 2537 2437 2487 16 2305 2241 2273 32 1877 1933 1905 64 1571 1446 1509 128 1239 1245 1242 256 949 967 958 512 816 883 850 1024 738 747 743 2048 793 764 779 4096 784 832 808 8192 734 736 735 16384 790 751 771 32768 703 742 723 14 Concordance analysis between ELISA and RIA The comparison between ELISA and mRF-RIA for the detection of circulating immune complexes is presented in Table 4.

Table 4: Test results of samples (n = 339) that were analysed for the presence of CIC by ELISA method and compared with mRF-RIA CONCORDANT RIA ELISA - 251 RIA +/- ELISA 6 RIA + ELISA + 15 RIA - ELISA +1 21 RIA +/ELISA - 7 RIA + ELISA +/- 3 RIA +/- ELISA + 8 DISCORDANT RIA - ELISA + 21 RIA + ELISA - 7 Complete agreement between ELISA and mRF-RIA was 80% (272/339). An equivocal result in the RIA and, either a positive or a negative result in ELISA or vice versa was found in 12% (39/339) of the samples. Thus, concordant results were obtained for 88% of the sera. The percentage of samples that was negative in RIA and positive in ELISA or positive in RIA and negative in ELISA was 8% (28/339).

Sensitivity and specificity For calculation of sensitivity and specificity, an equivalent result was considered negative. When the mRF-RIA was considered the reference method, sensitivity of the ELISA was 60%. Of the 25 samples that were positive in the RIA, 15 were also positive in ELISA. The specificity of the ELISA compared to the RIA was 91%. Of the 314 samples that were negative in the mRF-RIA, 285 were also negative in the ELISA.

Complementarity of mRF-RIA and ELISA compared to C1q-RIA For calculation of complernentarity, an equivocal result was considered negative. Of the 339 samples, 22 were positive in ELISA while negative in C1q-RIA (6%), and 10 were positive in mRF-RIA (3 %). This means that the complementarity of our ELISA with the C 1 q-RIA is the double of that of the mRF-RIA. mRF-RIA and ELISA were both positive in 5/339 samples while negative in C 1q-RIA.

16 References 1. Nydegger UE, Lambert PH, Gerber N, Miescher PA. Circulating immune complexes in the serum in systemic lupus erythernatosus and in carriers of hepatitis B antigen. Quantitation by binding to radiolabeled C I q. J Clin Invest 1974; 54: 297-309.

2. Nydegger UE, Davis JS. IV. Soluble immune complexes in human disease. CRC Crit Rev Clin Lab Sci 1980; 15: 123-70.

3. Report of an IUISIWHO working group. Use and abuse of laboratory tests in clinical immunology: critical considerations of eight widely used diagnostic procedures. Clin Exp Immunol 198 1; 46: 662-74.

4. Bernstein KA, Kahl LE, Balow JE, Lefkowith JB. Serologic markers of lupus nephritis in patients: use of a tissue-based ELISA and evidence for immunopathogenic heterogeneity. Clin Exp Immunol 1994; 98: 60-5.

5. Theofilopoulos AN, Dixon FJ. The biology and detection of immune complexes. Adv Immunol 1979; 28: 89-220.

6. Ritzmann SE, Daniels JC. Immune complexes: Characteristics, clinical correlations and interpretative approaches in the clinical laboratory. Clin Chem 1982; 28: 1259-7 1.

7. Sagnelli E, Felaco FM, Triolo G, Vernace SJ, Piccinino F, Behrens U et al. Circulating complement fixing immune complexes in chronic hepatitis. Use of anti-C3 enzyme immunoassay to define antibody class and nature of antigen. J Clin Lab Immunol 1983; 12: 11-5.

8. Cook L, Agnello V. Detection of immune complexes. In: Rose NR, De Macario EC, Fahey JL, Friedman H, Penn GM, eds. Manual of clinical laboratory immunology. Washington DC: ASM, 1992: 110-13.

9. Segal-Eiras A, Croce MV. Immune complexes in human malignant turnours. A review. Allergol et Immunopath 1984; 12: 225-32.

10. Lambert PH, Dixon FJ, Zubler RH, Agnello V, Cambiaso C, Casali P et al. A WHO collaborative study for the evaluation of eighteen methods for detecting, immune complexes in serum. J Clin Lab Immunol 1978; 1: 1-15.

17 11. Hay FC, Ninehma LJ, Roitt IM. Routine assay for the detection of immune complexes of known immunoglobulin class using solid phase Clq. Clin Exp Immunol 1976; 24: 396-400.

12. Gabriel A, Agnello V. Detection of immune complexes. The use of radioirnmunoassays with Clq and monoconal rheumatoid factor. J Clin Invest 1977; 59: 990-1001.

13. Pereira AB, Teofilopoulos AN, Dixon FJ. Detection and partial characterization of circulating immune complexes with solid-phase anti-C3. J Immunol 1980; 125: 763-70.

14. Glikmann G, Svehag SE. Detection and quantitation of circulating immune complexes by the C 1 q-protein A binding assay (C lq-PABA). Methods Enzymol 198 1; 74: 571-87.

15. McDougal JS, Hubbard M, Strobel PL, McDuffie FC. Comparison of five assays for immune complexes in the rheumatic diseases. Performance characteristics of the assays. J Lab Clin Med 1982; 100: 705-19.

16. Jones DB, Goulding NJ, Casey CR, Gallagher PJ. Clq binding and Raji immune complex assays: a comparison using defined inimunoglobulin aggregates. J Immunol Methods 1982; 53: 201-8.

17. Levinson SS, Goldman JO. Evaluation of anti-Clq capture assay for detecting circulating immune complexes and comparison with polyethylene glycolimmunoglobulin G, Clq binding, and Raji cell methods. J Clin Micobiol 1987; 25: 1567-9.

18. Theofilopoulos AN, Aduado MT. Assays for the detection of complementfixing immune complexes: Raji cells, conglutinin, and anti-C3 assays. In: Rose NR, Friedman H, Fahey JL, Eds. Manual of clinical laboratory immunology. Washington DC: ASM, 1992: 104-9 19. Vanham G, Bloemmen FJ, Ceuppens JL, Stevens EAM. Influence of complex size and antigen-antibody ratio on immune complex detection with monoclonal rheumatoid factor and C 1 q. J Clin Lab Immunol 1984; 15: 63-8.

20. Bjorck L, Kronvall G. Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol 1984; 133: 969-974.

21. Akerstrom B, Brodin T, reis K, Bjorck L. Protein G: A powerful tool for binding and detection of monoclonal and polyclonal antibodies. J Immunol 1985; 135: 2589-2592.

18

Claims (1)

1. Claims

   1) A method to detect or quantify circulating immune complexes (CIQ in the body fluid of specimen, comprising the steps of forming a coating of protein G on a substrate, treating said body fluid with in a preselected manner to precipitate CIC present in fluid, isolating said immune complexes from the body fluid, mixing said isolated immune complexes with a suitable vehicle to produce a preselected dilution, permitting said isolated immune complexes to be captured on the protein G coated substrate, treating said affixed CIC in a preselected manner to produce an indication of the concentration of the CIC present in said specimen 2) The method of claim 1, further comprising the step of separating the antibody fraction from isolated immune complexes for further analysis.

   3) A method according to the claims I or 2, wherein said affixed CIC is quantified by an appropriate enzyme-labelled antibody and an appropriate substrate.

   4) The method of any of the claims I to 3, wherein aspecific binding is blocked by mammalian serum.

   5) The method of claim 4, wherein aspecific binding is blocked by goat serum.

   6) An ELISA diagnostic kit for the detection or quantification of circulating immune complexes (CIC) in the serum; comprising in separate packaging, at least one of the following: protein G, a positive control vial of serum of a same species as test subject, a negative control vial of serum of a same species as test subject, a blocking agent 7) The kit of claim 6, with a vial with known quantity of isolated CIC of a same species as test subject for preparation of standard and a vial with a detectably labelled conjugate which binds to IgG of test subject 8) The kit of claim 7, wherein the protein G is bound to a solid support.

   19 9) The kit of claim 8, wherein said solid support is a dipstick or wetting plate.

   10) The kit of claim 8, wherein said solid support is microtiter plate 11) A kit of any of the claims 7 to 10, wherein any of said sera or CIC standards are lyophilised.

   12) A kit of any of the claims 7 to 11, which further comprises a substrate, which allows the visualisation of the detectably labelled conjugate.

   13) The kit of claims 12, wherein said detectably labelled conjugate comprises an enzyme label.

   14) The kit of claim 12, wherein said detectably labelled conjugate comprises anti-lgG immunoglobulins coupled to peroxidase.

   15) The kit of claim 14, further comprising in separate packaging, a colorimetric composition consisting of TMB, TMBLue TM or ABTS.

   16) A kit of any of the claims 6 to 15, wherein the blocking agent is a mammalian serum.

   17) A kit of any of the claims 6 to 15, wherein the blocking agent is goat serum.

   18) The use of method or apparatus of any of the claims 1 to 17 to detect or quantify circulating immune complexes.