Cross reactivities among some mammalian haptoglobins studied by a monoclonal antibody.

Mouse monoclonal antibody antihuman-Hp, product of clone 2.36.71.41 was found to recognize, however, with different affinities, the immunological determinant on haptoglobin of some mammals (goat, sheep, cow, horse, rabbit). The following differences in immunological cross reactivities were noticed: (i) haptoglobins present in sera of goat, sheep and cow (Artiodactyla, Bovidae) react in enzyme-linked immunosorbent assay (ELISA) and form precipitates in agarose gel; (ii) horse (Perissodactyla) and rabbit (Lagomorpha) haptoglobins react in ELISA, but they do not form precipitates; and (iii) haptoglobins of dog, fox, cat (Carnivora) and pig (Artiodactyla, Suidae) are not recognized by the tested monoclonal antibody either in ELISA or in agarose gel. This study suggests that monoclonal antibody, clone 2.36.71.41, recognizes the structure on haptoglobin around the disulphide bond linking two alpha chains. Antigenic structure of Bovidae haptoglobins is rather similar to human haptoglobin 2-2 (circular polymers) than to 2-1 type (linear polymers). Monoclonal antibody 2.36.71.41 could be used for classification of mammalian haptoglobins by epitope structure. It can distinguish polymeric haptoglobins similar to human type 2-2 from other mammalian haptoglobins.

Haptoglobin concentration in blood sera of hoofed mammals in captivity.

There were 151 serum samples of the zoological species of hoofed animals from the Bovidae, Cervidae and Equidae families examined for the presence of haptoglobin (Hp). In the majority of samples the Hp levels were close to the ones defined in the related species of domestic animals (86.49, 52.38 and 69.7% in particular families, respectively). In females (13.94% in the Bovidae family, 63.64% in the Cervidae, 15% in the Equidae) more often than in males (11.11, 26.32 and 0%, respectively) the increased Hp concentrations were found. What is striking it is the relatively high frequency of the raised Hp concentrations occurrence in the Cervidae sera, which may be connected with the higher responsiveness to the factors inducing Hp rise in this family. The contribution of infectious, parasitic, stress-inducing factors and injuries is taken into consideration as the main cause of the raised levels occurrence in the sera of animals kept in zoological gardens. The obtained results suggest the potential usefulness of the Hp estimation as a routine examination in the diagnostics of the inflammation in wild species of Bovidae and Cervidae as well as in monitoring the animal well being in the zoological gardens.

An improved ELISA for the determination of sialyl Lewis(x) structures on purified glycoconjugates.

The membrane carbohydrate antigen, sialyl Lewis x (sLe(x)), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLe(x) in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLe(x), many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLe(x) antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLe(x) in purified soluble glycoconjugates using the anti-sLe(x) antibody, CSLEX I. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6-12%, and it could detect less than a pmol of sLe(x). It could also distinguish between different densities of sLe(x) on the same amount of glycoconjugate. Determination of sLe(x) in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described will be useful for determining sLe(x) in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe(x) in the future.

Haptoglobin concentration in serum and other body fluids measured by comparison of its reactivity with hemoglobin and concanavalin A.

Haptoglobin (Hp) concentration in normal human sera was determined either by means of the reaction with hemoglobin (Hb) or by reaction with the lectin concanavalin A (Con A). The ratio of Hp-Con A (1.02 +/- 0.68 g/l) to Hp-Hb (1.10 +/- 0.58 g/l) equal 0.96 +/- 0.32 was found to be characteristic for sera from healthy subjects. Measurements were also carried out for some pathological body fluids. Values of the ratio Hp-Con A/Hp-Hb in ascitic fluid, pleural effusion and cerebrospinal fluid were 1.72 +/- 1.13, 0.42 +/- 0.23 and 0.15 +/- 0.18. respectively. Haptoglobin present in urine and saliva was not recognized by Con A, whereas could form a complex with hemoglobin. Hp concentration determined by this method reached 2 mg/l.

Measurements of haptoglobin by the reaction with concanavalin A in sera of patients with ovarian tumours.

The concentration of haptoglobin in sera of healthy women and patients with non-malignant and malignant ovarian tumours was measured by two methods, i.e. complex formation with haemoglobin and complex formation with concanavalin A. High correlation (r = 0.74) between both the methods was found in the group of healthy women, but correlation coefficients were much lower in the group of non-malignant and malignant tumours (r = 0.42 and 0.37, respectively). Direct determinations of haptoglobin, and calculation of the ratio of haptoglobin bound to concanavalin A to haptoglobin bound to haemoglobin revealed statistically significant differences among the examined groups. Comparison of two methods of haptoglobin quantitation suggest that processes connected with ovarian disorders may alter the glycosylation of haptoglobin oligosaccharide chains.

Quantitation of human haptoglobin by ELISA system based on streptococcal haptoglobin receptors.

Streptococcus pyogenes cells with binding properties for human haptoglobin were used for quantitative determination of the acute phase protein, haptoglobin in various biological fluids. The S. pyogenes cells with protein surface antigen T4 served as solid phase in a microtitre ELISA system. After binding to the bacteria the amount of haptoglobin could be quantified by polyclonal or monoclonal antibodies. The constructed ELISA proved to be sensitive and correlated well with a conventional peroxidase method and with an immunoassay based on hemoglobin binding to haptoglobin.

Immunoaffinity purification of human haptoglobin using monoclonal antibodies.

Two monoclonal antibodies, coupled to Sepharose 4B, were used for rapid isolation of the human haptoglobin in a single chromatographic step. The purity of haptoglobin was judged by SDS polyacrylamide gel electrophoresis, immunoblotting, immunoelectrophoresis and size exclusion high performance liquid chromatography. Moreover, the preparation showed ability to form active complexes with hemoglobin, Concanavalin A and specific antibody. Polyclonal goat antiserum produced with the obtained haptoglobin preparation formed one precipitin line with whole human serum. On the other hand, monoclonal antibodies can be purified by means of haptoglobin coupled to matrix.

Development of concanavalin A-enzyme immunosorbent assay for glycated haptoglobin using polyclonal and monoclonal antibodies.

Two-site lectin-haptoglobin-enzyme immunosorbent assay (L-Hp-ELISA), is described. Haptoglobin binding to Concanavalin A, immobilized to polystyrene microtiter plate, was estimated by anti-haptoglobin polyclonal and monoclonal antibodies conjugated with horse-radish peroxidase. The range of haptoglobin binding to Concanavalin A, measured by the L-Hp-ELISA was 25 to 300 ng/ml using polyclonal, and 50 to 600 ng/ml using monoclonal anti-haptoglobin antibodies, respectively.

Monoclonal antibody to human haptoglobin reacts with goat haptoglobin.

1. Monoclonal antibody 2.36.71.41 produced to human haptoglobin forms precipitates with goat haptoglobin in double immunodiffusion and electroimmunodiffusion. 2. Solid-phase immunoenzymatic assay (ELISA) based on the reaction of the monoclonal antibody 2.36.71.41 with goat haptoglobin can be used for quantitative estimation of haptoglobin content in goat sera. 3. The minimum detectable concentration of goat haptoglobin is 0.03 micrograms/ml.

Enzyme immunoassay to measure low levels of haptoglobin in biological fluids.

A novel immunoenzymatic assay is described for the quantitation of human haptoglobin (Hp). Two binding sites on the Hp molecule, namely for hemoglobin (Hb) and for the specific antibody, are involved in the reaction. Hb adsorbed onto the polystyrene microplate binds Hp present in any biological fluid. The formed Hp-Hb complex is detected with horse-radish peroxidase conjugated with anti-Hp antibody. By means of this ELISA, Hp may be measured in the range of 5 to 150 micrograms/L. Comparison of the Hp-ELISA with two other methods of Hp determination resulted in correlation coefficients of 0.97 to 0.99. Intra- and inter-assay coefficients of variation ranged from 4.7 to 6.7%. Hp levels were measured in urine, cord serum, cerebrospinal fluid, amniotic fluid and saliva.

Monoclonal antibodies against human haptoglobin.

Three monoclonal antibodies: 2.36.71.41, 7.60.66.55, and 18.4.40. 80 to human haptoglobin 2-1 were produced, purified and characterized. The affinity constants ranged within 0.3-2.4 x 10(8) M-1. The monoclonal antibodies 7.60.66.55 and 18.4.40.80 reacted with beta subunit of haptoglobin, showed similar epitope affinities and epitope densities on main haptoglobin types. However, the epitope on the haptoglobin molecule for the monoclonal antibody 18.4.40.80 occupied somewhat more surface than that for the antibody 7.60.66.55. The monoclonal antibody 2.36.71.41 was able to bind both alpha and beta chains of haptoglobin. In ELISA affinity reactions this antibody achieved with haptoglobin 2-2 the plateau phase at absorbance values 15% higher than with haptoglobin 2-1, and 60% higher than with haptoglobin 1-1. End-point titration of the monoclonal antibody 2.36.71.41 against three haptoglobin types showed differences in titer, indicating distinct epitope densities.

Microheterogeneity of alpha 1-acid glycoprotein in the sera of patients with cancer or inflammatory states of the ovaries.

alpha 1-Acid glycoprotein (AGP) and its concanavalin A-dependent four microheterogeneous fractions were measured in sera from women with ovarian carcinoma and those with an inflammatory ovarian disease. Total concentrations of AGP in inflammatory and cancerous sera were significantly higher than in control group, but did not differ from each other. In the fourth stage cancer the distribution of Con A-dependent fractions 1, 3 and 4 was significantly different as compared with control, second stage cancer and inflammatory groups.

Polyethylene glycol enzyme immunoassay for screening anti-haptoglobin monoclonal antibodies.

An enzyme immunoassay for the screening of anti-haptoglobin activities in supernatants of hybridoma cell cultures is described. The sandwich technique was greatly improved by the addition of 2% polyethylene glycol 6000 to all the reagents. It allowed a six-fold shortening in the incubation time compared with the standard method without sacrificing either sensitivity or accuracy.

Affino-immunoelectrophoresis of haptoglobin with wheat germ agglutinin. Diagnostic significance in ovarian carcinoma.

Affino-immunoelectrophoresis with wheat germ agglutinin (WGA) was applied to the analysis of microheterogenous fractions of human haptoglobin (Hp), present in cancerous ascitic fluids and in sera from patients either with ovarian carcinoma or with inflammatory ovarian disease. Normal and inflammatory sera contained two WGA-dependent Hp fractions: strongly- and weakly-retarded while cancer sera and ascitic fluids: non-retarded and weakly-retarded fractions. The content of WGA-dependent Hp fractions following curative surgery and chemotherapy of the patients was found to resemble the pattern of the control group.

Factors influencing the reaction of haptoglobin with concanavalin A in affino-immunoelectrophoresis.

In crossed affino-immunoelectrophoresis pure haptoglobin or that present in serum moved in the shape of either non-retarded or weakly-, or strongly-retarded fractions, related to varying Con A concentrations included into the first dimension gel. At low concentrations of Con A non-, weakly-, and strongly-retarded fractions were observed, whereas at higher concentrations only one, strongly-retarded fraction could be found. The number and percentage composition of haptoglobin fractions were dependent not only on the concentration of Con A, but also on other proteins added to the sample submitted to electrophoresis. Irregular distribution of haptoglobin Con A-dependent fractions made quantitative measurements unreliable.

Studies on haptoglobin binding to concanavalin A.

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.

Immunological comparison of glycopeptides obtained from haptoglobin types 1-1, 2-1 and 2-2.

Immunochemical properties of glycopeptides III and IV obtained from haptoglobin (Hp) types 1-1, 2-1, and 2-2, by trypsin digestion were studied by double immunodiffusion test, quantitative precipitation and crossed immunoelectrophoresis with antisera directed against Hp 1-1, Hp 2-1, and Hp 2-2, respectively. Trypsin digestion resulted in an exposition in the glycopeptide IV 1-1 of the antigenic determinant characteristic of Hp 1-1, which had been hidden inside the molecule of the native Hp 1-1. On the contrary, it was not possible to find an antigenic determinant characteristic of the product of alpha 2 gene.

The effects of limited proteolysis by trypsin on human haptoglobin.

Trypsin digestion of haptoglobin resulted i four glycopeptides. The glycopeptides were characterized by amino acid composition and molecular weight, as determined by thin-layer chromatography, and sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. Hemoglobin-binding capacity and immunological properties were investigated. glycopeptides I and II did not form an active complex with hemoglobin and they inhibited the reaction of haptoglobin with specific antiserum by over 70%. Glycopeptides III and IV showed 11 and 4% of the hemoglobin-binding capacity and 82 and 67% of antigenic reactivity of native haptoglobin, respectively. Glycopeptide IV contained three antigenic determinants, whereas glycopeptides III contained four, one of them being exposed by trypsin digestion. In crossed two-dimensional immunoelectrophoresis, glycopeptide III showed at least four components reacting with antihaptoglobin serum, and glycopeptide IV, two components.