Precipitation by antibodies

Many qualitative and quantitative methods for soluble antigens are based on their precipitation by antibodies. It is important that the conditions for each assay are carefully optimized in order to achieve the desired end. 

The presence of a specific FDPase in mammalian kidney was established by the early studies of Gomori (2). This enzyme may be identical with that found in mammalian liver since it is precipitated by antibody prepared against the purified liver enzyme and purified preparations... 

Lerner MR, Andrews NC, Miller G, Steitz JA. Two smaE RNAs encoded by Epstein-Barr virus and com-plexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc Natl Acad Sci U S A 1981 78 805-9. 

Because of the artificial nature of the buffer and pH used in the determination, the distribution of thyroxine in vivo may not be accurately reflected by the electrophoretic data. In particular, the physiological role of TBPA (C4) in binding thyroxine in vivo has been questioned, but changes in the concentration of TBPA have been associated with alterations in free thyroxine (Oil), thus suggesting that TBPA does bind some thyroxine in vivo. Some normal values for thyroxinebinding proteins are shown in Table 2. Woeber et al. (W3) have recently questioned the values listed in Table 2 for TBPA thyroxine binding. They used specific antibody for TBPA and reported that approximately 15% of labeled thyroxine was precipitable by antibody. This value is about one-half the previously reported values of approximately 30%. 

The acetyl-LDL receptor is distinct from the LDL receptor. Acetyl-LDL can be precipitated by antibody raised against LDL, yet is not taken up by the LDL receptor [64]. By contrast, LDL is taken up by macrophages only at a low rate without accumulation of cholesterol esters [19,61]. Unlike the LDL receptor, the acetyl-LDL receptor does not require divalent cations for uptake [19]. In addition, monocytes from normal subjects exhibit an acetyl-LDL receptor activity comparable to that of monocytes from patients with LDL receptor-negative homozygous FH. 

The genetic mutations are mainly deletions, but insertions or duphcations also occur, as well as missense and nonsense point mutations (see Fig. 34.20). Four classes of mutations have been identified. The first class involves null alleles that either direct the synthesis of no protein at all or a protein that cannot be precipitated by antibodies to the LDL receptor. In the second class, the alleles encode proteins, but they cannot be transported to the cell surface. The third class of mutant alleles encodes proteins that reach the cell surface but cannot bind LDL normally. Finally, the fourth class encodes proteins that reach the surface and bind LDL but fail to cluster and internalize the LDL particles. The result of each of these mutations is that blood levels of LDL are elevated because cells cannot take up these particles at a normal rate. 

The phenomenon of hapten inhibition has been explained by Landsteiner as resulting from combination of hapten and antibody to form a soluble complex, thus effectively neutralizing the antibody. The formation of soluble complex instead of a precipitate by antibody and hapten is explained by the framework theory as the result of the univalence of the hapten. It might be expected that as the maximum amount of precipitate which can be obtained from a serum is decreased by addition of hapten there would occur a corresponding decrease in the optimum antigen concentration, as was observed in the dilution experiments. 

A number of experiments other than those reported in Table II were carried out in which varying amounts of purified antibody were added to a constant amount of antigen in a constant volume of solution. A 5 to 10% decrease in precipitated protein was noted at the largest amounts of antibody in every experiment. This effect of inhibition of precipitation by antibody excess is similar to but not nearly so marked as the effect in serum, in which a four-fold increase in amount of antiserum above the optimum was found to decrease the amount of precipitate to a third of the maximum value. 

Isocitrate lyase has been purified from the glyoxysomes of cucumber (Cucumis sativus) cotyledons. The glycoproteinaceous nature of the enzyme was demonstrated by periodate oxidation studies, and by the in vivo incorporation of 2-amino-2-deoxy-D-[ H]glucose into a species which was precipitable by antibodies to isocitrate lyase. 

In 1967, Heidelberger, Stacey et al. reported the purification, some structural features, and the chemical modification of the capsular polysaccharide from Pneumococcus Type I. Difficulties of direct hydrolysis of the polysaccharide were overcome and it was possible to identify some of the fragments in the hy-drolyzate. At least six products resulted from nitrous acid deamination. Two were disaccharides, which were identified, and sequences of linked sugar units were proposed. As modification of the polysaccharide decreased the amounts of antibody precipitated by anti-pneumococcal Type I sera, the importance of the unmodified structural features in contributing to the specificity of the polysaccharide was indicated. 

Two methods are commonly employed in RIAs to separate antigen-antibody complexes. The first, the double-antibody technique, precipitates antigen-antibody complexes out of solution by utilizing a second antibody, which binds to the first... 

It is unclear why certain foreign proteins can also stimulate the B-cells to secrete IgE antibodies, to result in allergy or hypersensitivity. The terms are used interchangeably, although the latter is usually restricted to milder forms of the response. The term anaphylaxis is used to describe the severe response (Box 17.4). Both reactions arise in genetically susceptible individuals and they are precipitated by exposure to environmental antigens such as pollen, some organic compounds, tobacco smoke, animal hairs or even components of some common foods such as milk and cereals. 

Extensive studies have been done on a clearly defined asthma syndrome produced by exposure to western red cedar. ° Plicatic acid has been identified as the etiologic agent. The western red cedar asthma syndrome includes rhinitis, conjunctivitis, wheezing, cough, and nocturnal attacks of breathlessness characterized by a precipitous decline in FEVi. There is no apparent relation between skin sensitivity and respiratory changes. No precipitating IgG antibodies are found in the serum of sensitized individuals, and circulating IgE antibodies are present in about one-third of affected individuals. 

With individuals of blood group 0, injection of A and B substances gave a big increase in the amounts of precipitins. For example, the specific antibodies to the blood group A substance could be obtained from the specific precipitate by the use of 15% sodium chloride for dissociating... 

Antibody complexes are precipitated by relatively low concentrations of polyetyleneglycol 6000 (PEG 6000). 

If an antigen-antibody complex has to be precipitated by a secondary antibody (e.g., antigen bound by mAb precipitated by goat anti-(mouse-IgG) antibody), this range of equivalence must be known. For estimation of the range of equivalence the precipitin assay is used. 

Figure 5. Size analysis of Inhibitors I and 11 specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. Poly(A ) RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf polyfA)" RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly...

Immunoprecipitation (IP) is one of the most widely used immunochemical techniques. It involves the interaction between an antigen and its specific antibody. Antigen-antibody interactions may produce a network of many antigen molecules cross-linked by antibody molecules, which result in insolubilization and precipitation of the complex (Williams 2000). 

Antigens and their corresponding antibodies precipitate by cross-linking to form an insoluble network. Polysaccharides have multiple, repetitive immunodeterminants and virtually none have demonstrable tertiary structure in solution (except, perhaps, under viscous stress). The number of these immunodeterminant groupings on each macromolecule is large. In the case of dextran, for instance, there are several thousand of them (if the dextran has a molecular weight of several million), even if the determinant involves the hep-tasaccharide. There is, thus, ample opportunity to form a precipitating, crosslinked complex with divalent (or polyvalent) antibody molecules. 

However, the proportion of antibody precipitated by the Merck preparation was significantly greater after immunization of each of the three individuals with a different dextran. This finding suggests that the three dextrans and the Merck polyglucose possessed structural similarities, such as a-D- (1 — 6) -glucosidic linkages. 

In heterogeneous assays the bound and free label forms must be separated by different means, such as precipitation of antibody or coupling of the antibody to a solid support. Heterogeneous immunoassays are more versatile as inclusion of a separation step eliminates most of the interferences before quantification. However, these assays are also more labor-intensive and time-consuming. Any of these methods can be performed in either competitive or non-competitive format. 

FIGURE 8 Separation of rabbit polyclonal antibodies by ion-exchange chromatography on DEAE Trisacryl M. Column dimensions 16 mm i.d.X 100 mm initial buffer 50 mM Tris-HCI, 0.035 M sodium chloride, pH 8.8 load 5 mL of rabbit serum previously precipitated with ammonium sulfate at 50% saturation and redissolved in column buffer flow rate 50 mL/hr elution of adsorbed protein performed using I M sodium chloride solution in the initial Tris buffer. The first peak represents IgG the second peak is composed of all other serum proteins precipitated by ammonium sulfate. The straight line is absorbance at 280 nm, and the broken line represents the variation of ionic strength of the buffer. The purity of IgG estimated by gel electrophoresis was over 98% and the calculated yield was over 90%. 

Immunoprecipitation also is a powerful and effective means of identifying interactions among proteins. For example, many proteins normally exist in multimeric complexes with other proteins within the cell. Immunoprecipitation can be used to precipitate a protein that is targeted by the antibodies used. Proteins in the precipitate then can be analyzed to determine what other proteins were associated with the targeted protein and co-precipitated by the antibodies. 

The fractionation and purification of deteriorated proteins is undoubtedly one of the least successful techniques. This is simply because all of the methods that have been developed, with very few exceptions, are directed toward purifying the undeteriorated protein. The methods available are usually based on some particular biochemical activity of the protein, usually enzyme activity, and sometimes an affinity column or affinity adsorbent could be used to separate the native protein from the deteriorated one. Quite often a good affinity adsorbent is unavailable. This procedure, however, does not always work properly even when an adsorbent is available, because the deteriorated protein may possess some activity or an affinity for the adsorbent even though it has lost its natural enzyme activity (see Figure 24). The antigen-antibody reaction can also be used by means of precipitation with antibodies against the native proteins or adsorption on the immobilized antibodies. But here again, the specific antibody must be available, and the deteriorated protein may retain so much affinity for the antibody that differential separations will be impractical in some cases. 

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